# Notch3 is a genetic modifier of NODAL signalling for patterning asymmetry during mouse heart looping

Tobias Holm Bønnelykke, Marie-Amandine Chabry, Emeline Perthame, Audrey Desgrange, Sigolène M Meilhac

► To cite this version:

Tobias Holm Bønnelykke, Marie-Amandine Chabry, Emeline Perthame, Audrey Desgrange, Sigolène M Meilhac. Notch3 is a genetic modifier of NODAL signalling for patterning asymmetry during mouse heart looping. 2024. pasteur-04614415

HAL Id: pasteur-04614415

https://pasteur.hal.science/pasteur-04614415

Preprint submitted on 17 Jun 2024

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# Notch3 is a genetic modifier of NODAL signalling for patterning asymmetry during mouse heart looping

Tobias Holm Bønnelykke, Marie-Amandine Chabry, Emeline Perthame, Audrey Desgrange, Sigolène M Meilhac

► To cite this version:

Tobias Holm Bønnelykke, Marie-Amandine Chabry, Emeline Perthame, Audrey Desgrange, Sigolène M Meilhac. Notch3 is a genetic modifier of NODAL signalling for patterning asymmetry during mouse heart looping. 2024. pasteur-04614415

HAL Id: pasteur-04614415

https://pasteur.hal.science/pasteur-04614415v1

Preprint submitted on 17 Jun 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 **Notch3 is a genetic modifier of NODAL signalling for patterning asymmetry during mouse heart looping**

2 **heart looping**

3

4

5 Tobias Holm Bønnelykke¹,², Marie-Amandine Chabry¹, Emeline Perthame¹,³, Audrey Desgrange¹* and Sigolène M. Meilhac¹**

6 Desgrange¹* and Sigolène M. Meilhac¹**

7

8 ¹Université Paris Cité, Imagine - Institut Pasteur, Unit of Heart Morphogenesis, INSERM UMR1163, F-75015, Paris, France

9 Paris, France

10 ²Sorbonne Université, Collège Doctoral, 75005 Paris, France

11 ³Institut Pasteur, Université Paris Cité, Bioinformatics and Biostatistics Hub, F-75015, Paris, France

12 *co-senior authors

13 *correspondence to Sigolène Meilhac, sigolene.meilhac@institutimagine.org

14

15 Author ORCIDs

16 Tobias Holm Bønnelykke 0009-0008-8060-2808

17 Marie-Amandine Chabry 0009-0007-3882-758X

18 Emeline Perthame 0000-0002-8266-5908

19 Audrey Desgrange 0000-0001-9716-755X

20 Sigolène M. Meilhac 0000-0003-4080-2617

21

22

23 Short title: Notch3 role in left-right asymmetry

24
1 **Notch3 is a genetic modifier of NODAL signalling for patterning asymmetry during mouse heart looping**

2 **heart looping**

3

4

5 Tobias Holm Bønnelykke¹,², Marie-Amandine Chabry¹, Emeline Perthame¹,³, Audrey Desgrange¹* and Sigolène M. Meilhac¹**

6 Desgrange¹* and Sigolène M. Meilhac¹**

7

8 ¹Université Paris Cité, Imagine - Institut Pasteur, Unit of Heart Morphogenesis, INSERM UMR1163, F-75015, Paris, France

9 Paris, France

10 ²Sorbonne Université, Collège Doctoral, 75005 Paris, France

11 ³Institut Pasteur, Université Paris Cité, Bioinformatics and Biostatistics Hub, F-75015, Paris, France

12 *co-senior authors

13 *correspondence to Sigolène Meilhac, sigolene.meilhac@institutimagine.org

14

15 Author ORCIDs

16 Tobias Holm Bønnelykke 0009-0008-8060-2808

17 Marie-Amandine Chabry 0009-0007-3882-758X

18 Emeline Perthame 0000-0002-8266-5908

19 Audrey Desgrange 0000-0001-9716-755X

20 Sigolène M. Meilhac 0000-0003-4080-2617

21

22

23 Short title: Notch3 role in left-right asymmetry

24
25 **Abstract**

The TGFβ secreted factor NODAL is a major left determinant required for the asymmetric
morphogenesis of visceral organs, including the heart. Yet, when this signalling is absent,
shape asymmetry, for example of the embryonic heart loop, is not fully abrogated, indicating
that there are other factors regulating left-right patterning. Here, we used a tailored
transcriptomic approach to screen for genes asymmetrically expressed in the field of heart
progenitors. We thus identify *Notch3* as a novel left-enriched gene and validate, by
quantitative in situ hybridization, its transient asymmetry in the lateral plate mesoderm and
node crown, overlapping with *Nodal*. In mutant embryos, we analysed the regulatory hierarchy
and demonstrate that *Nodal* in the lateral plate mesoderm amplifies *Notch3* asymmetric
expression. The function of *Notch3* was uncovered in an allelic series of mutants. In single
neonate mutants, we observe that *Notch3* is required with partial penetrance for the
development of ventricles, in addition to its known role in coronary arteries. In compound
mutants, we reveal that *Notch3* acts as a genetic modifier of *Nodal*, able to modulate heart
looping direction and the curvature of the outflow tract. Whereas *Notch3* was previously
associated with the CADASIL syndrome, its contribution to asymmetric organogenesis is now
relevant to severe laterality defects such as the heterotaxy syndrome.

42 **Abbreviations**

43 Ao = Aorta

44 AVC = Atrioventricular canal

45 BH = Benjamini-Hochberg

46 BMP = bone morphogenetic protein

47 E = Embryonic day
25 Abstract

The TGFβ secreted factor NODAL is a major left determinant required for the asymmetric
morphogenesis of visceral organs, including the heart. Yet, when this signalling is absent,
shape asymmetry, for example of the embryonic heart loop, is not fully abrogated, indicating
that there are other factors regulating left-right patterning. Here, we used a tailored
transcriptomic approach to screen for genes asymmetrically expressed in the field of heart
progenitors. We thus identify *Notch3* as a novel left-enriched gene and validate, by
quantitative in situ hybridization, its transient asymmetry in the lateral plate mesoderm and
node crown, overlapping with *Nodal*. In mutant embryos, we analysed the regulatory hierarchy
and demonstrate that *Nodal* in the lateral plate mesoderm amplifies *Notch3* asymmetric
expression. The function of *Notch3* was uncovered in an allelic series of mutants. In single
neonate mutants, we observe that *Notch3* is required with partial penetrance for the
development of ventricles, in addition to its known role in coronary arteries. In compound
mutants, we reveal that *Notch3* acts as a genetic modifier of *Nodal*, able to modulate heart
looping direction and the curvature of the outflow tract. Whereas *Notch3* was previously
associated with the CADASIL syndrome, its contribution to asymmetric organogenesis is now
relevant to severe laterality defects such as the heterotaxy syndrome.

42 Abbreviations

43 Ao = Aorta

44 AVC = Atrioventricular canal

45 BH = Benjamini-Hochberg

46 BMP = bone morphogenetic protein

47 E = Embryonic day
48 HREM = High Resolution Episcopic Microscopy

49 HT = Heart tube

50 ISH = In situ hybridisation

51 IPCCC ICD = International Paediatric and Congenital Cardiac Code and International

52 Classification of Diseases

53 L = Left

54 LA = Left atrium

55 LPM = Lateral plate mesoderm

56 LV = Left ventricle

57 Myo = Myocardium

58 No = Node

59 n.s. = non-significant

60 NT = Neural tube

61 P = Postnatal day

62 R = Right

63 RA = Right atrium

64 RT-qPCR = Reverse transcription quantitative polymerase chain reaction

65 RV = Right ventricle

66 SHF = Second Heart Field

67 TGFβ = Transforming growth factor-β

68 VSD = Ventricular Septal Defect

69
48 HREM = High Resolution Episcopic Microscopy

49 HT = Heart tube

50 ISH = In situ hybridisation

51 IPCCC ICD = International Paediatric and Congenital Cardiac Code and International

52 Classification of Diseases

53 L = Left

54 LA = Left atrium

55 LPM = Lateral plate mesoderm

56 LV = Left ventricle

57 Myo = Myocardium

58 No = Node

59 n.s. = non-significant

60 NT = Neural tube

61 P = Postnatal day

62 R = Right

63 RA = Right atrium

64 RT-qPCR = Reverse transcription quantitative polymerase chain reaction

65 RV = Right ventricle

66 SHF = Second Heart Field

67 TGFβ = Transforming growth factor-β

68 VSD = Ventricular Septal Defect

69
70 **Introduction**

71 Left-right patterning of the embryo is essential for the formation of visceral organs (1,2).

72 Anomalies in this process lead to the heterotaxy syndrome, a severe condition including
73 complex congenital heart defects which determine the prognosis of patients (3). The
74 mechanisms of symmetry-breaking are now well established in the mouse (4). Chirality of
75 tubulin underlies the rotational movement of motile cilia in the pit of the left-right organiser,
76 also referred to as the node. This generates a leftward fluid flow sensed by crown cells,
77 resulting in the asymmetric expression of the left determinant NODAL in the lateral plate
78 mesoderm. Genetic alterations to the formation of the node, ciliogenesis or NODAL signalling
79 are associated with heterotaxy in both mice (2,5–8) and humans (9,10). NODAL signalling is
80 then sensed by organ precursors to modulate morphogenesis, as shown in the intestine (11)
81 and heart (12). In the absence of *Nodal*, some features are symmetrical (spleen, bronchi, lung
82 lobes, atrial appendages), but many others (e.g heart looping, stomach, gut) retain some level
83 of asymmetry, although abnormal (6,12–15). This indicates that *Nodal* is not always required
84 to initiate asymmetry and that there are other factors of asymmetry in addition to NODAL
85 signalling.

86 The heart is the first organ to undergo asymmetric morphogenesis in the embryo. This
87 manifests as a rightward looping of the heart tube primordium, at E8.5 in the mouse (16). We
88 have previously reconstructed the dynamics of this process and defined morphological and
89 quantitative staging criteria from E8.5c to E8.5j, including the repositioning of ventricles from
90 cranio-caudal to left-right, and the left displacement of the venous pole (17). We have shown
91 that heart looping is primarily a buckling mechanism, when the heart tube elongates between
92 fixed poles (17). We found that *Nodal* is not required for buckling and thus to initiate looping.
93 In contrast, *Nodal* biases buckling, by amplifying and coordinating asymmetries independently
70 **Introduction**

71 Left-right patterning of the embryo is essential for the formation of visceral organs (1,2).

72 Anomalies in this process lead to the heterotaxy syndrome, a severe condition including
73 complex congenital heart defects which determine the prognosis of patients (3). The
74 mechanisms of symmetry-breaking are now well established in the mouse (4). Chirality of
75 tubulin underlies the rotational movement of motile cilia in the pit of the left-right organiser,
76 also referred to as the node. This generates a leftward fluid flow sensed by crown cells,
77 resulting in the asymmetric expression of the left determinant NODAL in the lateral plate
78 mesoderm. Genetic alterations to the formation of the node, ciliogenesis or NODAL signalling
79 are associated with heterotaxy in both mice (2,5–8) and humans (9,10). NODAL signalling is
80 then sensed by organ precursors to modulate morphogenesis, as shown in the intestine (11)
81 and heart (12). In the absence of *Nodal*, some features are symmetrical (spleen, bronchi, lung
82 lobes, atrial appendages), but many others (e.g heart looping, stomach, gut) retain some level
83 of asymmetry, although abnormal (6,12–15). This indicates that *Nodal* is not always required
84 to initiate asymmetry and that there are other factors of asymmetry in addition to NODAL
85 signalling.

86 The heart is the first organ to undergo asymmetric morphogenesis in the embryo. This
87 manifests as a rightward looping of the heart tube primordium, at E8.5 in the mouse (16). We
88 have previously reconstructed the dynamics of this process and defined morphological and
89 quantitative staging criteria from E8.5c to E8.5j, including the repositioning of ventricles from
90 cranio-caudal to left-right, and the left displacement of the venous pole (17). We have shown
91 that heart looping is primarily a buckling mechanism, when the heart tube elongates between
92 fixed poles (17). We found that *Nodal* is not required for buckling and thus to initiate looping.
93 In contrast, *Nodal* biases buckling, by amplifying and coordinating asymmetries independently
at the two poles of the heart tube (12). Thus, in the absence of *Nodal* in the lateral plate mesoderm, asymmetries are reduced and the helical shape of the looped heart tube is abnormal. Because two asymmetries, at the arterial and venous poles, have a randomised orientation, *Nodal* mutants display 4 classes of abnormal heart loop. We observed that NODAL signalling is transient, before the formation of the heart tube (E8.5c-d), in precursor cells of the heart tube poles. Cardiac precursor cells are progressively incorporated into the heart tube, as a main driver of its elongation. Patterning of the field of cardiac precursors has been studied in terms of molecular profiling (18) and fate (19). However, the left-right patterning of cardiac precursors has remained centered on the *Nodal* pathway.

Other players of asymmetry have been identified, including BMP (20–22), HH (23), NOTCH (24–26) and WNT (27–29) signalling. Few of these are reported to be asymmetric: *Wnt3* in the left side of the node (28), and BMP signalling in the right lateral plate mesoderm (21). They were all shown to be required for *Nodal* asymmetry. Thus, the factors which can provide asymmetry besides *Nodal* have remained enigmatic.

NOTCH signalling is an example of a pathway required both for left-right asymmetry and for heart morphogenesis (30), but playing multiple roles. Among the four paralogues, *Notch2* is expressed in the node, and *Notch1* around the node in the caudal epiblast and presomitic mesoderm (24,31). *Notch1;Notch2* double mutants disrupt heart looping asymmetry, in keeping with a role in the left-right organiser (25). NOTCH1 and the transcriptional NOTCH effector RBPJ are required in the heart field for cardiomyocyte differentiation (32–34). *Notch1* is later expressed in the endocardium, where it is important to regulate myocardial trabeculation (35) and valve formation (36,37). *Notch3* mutant mice are viable and fertile (38), but display defects in the maturation of the smooth muscle wall of coronary arteries (39). *Notch3* is a broad marker of pericytes and controls the formation of smooth muscles in non-
at the two poles of the heart tube (12). Thus, in the absence of *Nodal* in the lateral plate mesoderm, asymmetries are reduced and the helical shape of the looped heart tube is abnormal. Because two asymmetries, at the arterial and venous poles, have a randomised orientation, *Nodal* mutants display 4 classes of abnormal heart loop. We observed that NODAL signalling is transient, before the formation of the heart tube (E8.5c-d), in precursor cells of the heart tube poles. Cardiac precursor cells are progressively incorporated into the heart tube, as a main driver of its elongation. Patterning of the field of cardiac precursors has been studied in terms of molecular profiling (18) and fate (19). However, the left-right patterning of cardiac precursors has remained centered on the *Nodal* pathway.

Other players of asymmetry have been identified, including BMP (20–22), HH (23), NOTCH (24–26) and WNT (27–29) signalling. Few of these are reported to be asymmetric: *Wnt3* in the left side of the node (28), and BMP signalling in the right lateral plate mesoderm (21). They were all shown to be required for *Nodal* asymmetry. Thus, the factors which can provide asymmetry besides *Nodal* have remained enigmatic.

NOTCH signalling is an example of a pathway required both for left-right asymmetry and for heart morphogenesis (30), but playing multiple roles. Among the four paralogues, *Notch2* is expressed in the node, and *Notch1* around the node in the caudal epiblast and presomitic mesoderm (24,31). *Notch1;Notch2* double mutants disrupt heart looping asymmetry, in keeping with a role in the left-right organiser (25). NOTCH1 and the transcriptional NOTCH effector RBPJ are required in the heart field for cardiomyocyte differentiation (32–34). *Notch1* is later expressed in the endocardium, where it is important to regulate myocardial trabeculation (35) and valve formation (36,37). *Notch3* mutant mice are viable and fertile (38), but display defects in the maturation of the smooth muscle wall of coronary arteries (39). *Notch3* is a broad marker of pericytes and controls the formation of smooth muscles in non-
118 cardiac arteries and intestinal lacteals (40-42). Gain-of-function of NOTCH3 in CADASIL
119 syndrome is responsible for smooth muscle degeneration in the brain (40,43,44). *Notch3* is
120 also involved in the regulation of quiescence and stemness of neural, skeletal muscle and
121 mammary stem cells (45-48). Finally, *Notch4* is expressed together with *Notch1* in endothelial
122 cells to regulate vascular remodelling (49). Thus *Notch1/2* but not *Notch3/4* have been
123 previously associated with left-right patterning, at the level of the left-right organiser.

124 To uncover asymmetric factors in organ specific precursors, rather than the left-right
125 organiser, recent transcriptomic screens were performed on micro-dissected tissues excluding
126 the node (50,51). Whereas NODAL signalling is transient (12,52), these screens were based on
127 pooled embryos, which limits their resolution. The asymmetric candidate genes that have
128 been identified have not yet been analysed for functional significance.

129 Here, we focused on asymmetric expression in the field of heart progenitors, at the time of
130 heart looping. We compared paired left/right samples in single embryos to avoid confounding
131 individual or stage variations. Our screen and quantitative in situ validations reveal *Notch3* as
132 a novel gene enriched in the left lateral plate mesoderm. *Notch3* is co-expressed with *Nodal*,
133 although with a slight time-delay and partial spatial overlap. In mutants, we show that *Notch3*
134 asymmetry is amplified by NODAL signalling. Single *Notch3* mutants do not develop
135 heterotaxy, but show ventricle and coronary artery defects after birth. Analysis of compound
136 mutants indicate that *Notch3* is a genetic modifier of NODAL signalling during heart looping.
137 Overall, we identify not only a novel asymmetric gene, but also a novel player in the left-right
138 patterning of the lateral plate mesoderm, relevant to congenital heart defects.
118 cardiac arteries and intestinal lacteals (40-42). Gain-of-function of NOTCH3 in CADASIL
119 syndrome is responsible for smooth muscle degeneration in the brain (40,43,44). *Notch3* is
120 also involved in the regulation of quiescence and stemness of neural, skeletal muscle and
121 mammary stem cells (45-48). Finally, *Notch4* is expressed together with *Notch1* in endothelial
122 cells to regulate vascular remodelling (49). Thus *Notch1/2* but not *Notch3/4* have been
123 previously associated with left-right patterning, at the level of the left-right organiser.

124 To uncover asymmetric factors in organ specific precursors, rather than the left-right
125 organiser, recent transcriptomic screens were performed on micro-dissected tissues excluding
126 the node (50,51). Whereas NODAL signalling is transient (12,52), these screens were based on
127 pooled embryos, which limits their resolution. The asymmetric candidate genes that have
128 been identified have not yet been analysed for functional significance.

129 Here, we focused on asymmetric expression in the field of heart progenitors, at the time of
130 heart looping. We compared paired left/right samples in single embryos to avoid confounding
131 individual or stage variations. Our screen and quantitative in situ validations reveal *Notch3* as
132 a novel gene enriched in the left lateral plate mesoderm. *Notch3* is co-expressed with *Nodal*,
133 although with a slight time-delay and partial spatial overlap. In mutants, we show that *Notch3*
134 asymmetry is amplified by NODAL signalling. Single *Notch3* mutants do not develop
135 heterotaxy, but show ventricle and coronary artery defects after birth. Analysis of compound
136 mutants indicate that *Notch3* is a genetic modifier of NODAL signalling during heart looping.
137 Overall, we identify not only a novel asymmetric gene, but also a novel player in the left-right
138 patterning of the lateral plate mesoderm, relevant to congenital heart defects.

139
140 **Results**

141 **Identification of Notch3 as a novel asymmetric marker**

142 To screen for left-right asymmetric gene expression in cardiac cells, aside the *Nodal*
143 pathway, we used a transcriptomic approach in the mouse embryo. To reduce confounding
144 factors, the approach was targeted in space and time and left/right comparisons were
145 conducted on single rather than pooled embryos. We focused on the heart field, given that it
146 is a tissue which can be bisected at the midline and is patterned by NODAL signalling (12). In
147 contrast, the heart tube changes its position during looping (17) and has a left-right origin
148 difficult to dissect (19). We have previously shown that NODAL signalling is transient in heart
149 precursors (12), highlighting the importance of specifically staging embryos. We focused on
150 the E8.5f stage, when we have detected morphological asymmetry at the arterial pole of the
151 heart tube (17). Thus, we micro-dissected the heart field of E8.5f wild-type embryos (Fig. 1A
152 and S1A-D), in the dorsal pericardial wall and down to the second somite, in agreement with
153 landmarks of fate mapping experiments (19) and profiling of single cardiac cells (18). Bulk RNA
154 sequencing of paired left and right samples were compared to identify differential asymmetric
155 expression in the heart field. As expected, targets of NODAL signalling, *Pitx2* and *Lefty2*, were
156 significantly enriched on the left side (Fig. 1B). A total set of 597 genes were predicted to be
differentially expressed (Table S1). Ingenuity Pathway analysis showed NOTCH signalling as the
157 strongest asymmetric pathway and left-sided (Fig. 1C). We controlled the transcriptomic levels
158 of individual genes associated with NOTCH in this differential analysis and their cardiac
159 expression in a published single cell transcriptomic dataset at the same stage (18): whereas
160 *Dtx4* was found to be expressed only in ectodermal cells (Fig. S1E), *Notch3*, *Hes1*, *Jag1*, *Ncstn*
161 were identified as asymmetric candidate genes in the heart field (Fig. 1D).
140 **Results**

141 **Identification of Notch3 as a novel asymmetric marker**

142 To screen for left-right asymmetric gene expression in cardiac cells, aside the *Nodal*
143 pathway, we used a transcriptomic approach in the mouse embryo. To reduce confounding
144 factors, the approach was targeted in space and time and left/right comparisons were
145 conducted on single rather than pooled embryos. We focused on the heart field, given that it
146 is a tissue which can be bisected at the midline and is patterned by NODAL signalling (12). In
147 contrast, the heart tube changes its position during looping (17) and has a left-right origin
148 difficult to dissect (19). We have previously shown that NODAL signalling is transient in heart
149 precursors (12), highlighting the importance of specifically staging embryos. We focused on
150 the E8.5f stage, when we have detected morphological asymmetry at the arterial pole of the
151 heart tube (17). Thus, we micro-dissected the heart field of E8.5f wild-type embryos (Fig. 1A
152 and S1A-D), in the dorsal pericardial wall and down to the second somite, in agreement with
153 landmarks of fate mapping experiments (19) and profiling of single cardiac cells (18). Bulk RNA
154 sequencing of paired left and right samples were compared to identify differential asymmetric
155 expression in the heart field. As expected, targets of NODAL signalling, *Pitx2* and *Lefty2*, were
156 significantly enriched on the left side (Fig. 1B). A total set of 597 genes were predicted to be
differentially expressed (Table S1). Ingenuity Pathway analysis showed NOTCH signalling as the
157 strongest asymmetric pathway and left-sided (Fig. 1C). We controlled the transcriptomic levels
158 of individual genes associated with NOTCH in this differential analysis and their cardiac
159 expression in a published single cell transcriptomic dataset at the same stage (18): whereas
160 *Dtx4* was found to be expressed only in ectodermal cells (Fig. S1E), *Notch3*, *Hes1*, *Jag1*, *Ncstn*
161 were identified as asymmetric candidate genes in the heart field (Fig. 1D).
We selected *Notch3* for further validation, because it is the gene with highest expression, it encodes a receptor and transcription co-factor central to NOTCH signalling. This *Notch* paralogue has not previously been associated with left-right patterning. We mapped *Notch3* expression in whole mount E8.5 wild-type embryos using sensitive RNAscope in situ hybridization. *Notch3* was detected throughout the lateral plate mesoderm, as well as in the crown of the node, with higher expression on the left side (Fig. 1E-F). *Notch3* was not expressed in the myocardium of the forming cardiac tube. We then assessed the dynamics of *Notch3* expression within the heart field (Fig. 1G). Our quantification shows that *Notch3* is transiently asymmetric, reaching 4.5-fold left-sided enrichment in the heart field at E8.5d, a stage when the myocardium bulges out to form a tube (Fig. 1H). Although *Nodal* asymmetry starts one stage earlier (E8.5c, (12)), *Notch3* and *Nodal* overlapped in the left lateral plate mesoderm at E8.5d (Fig. 1I, Video S1), and were found co-expressed in single cells (Fig. S1F). We have thus identified *Notch3* as a novel left-sided gene in the heart field.

<caption>**Fig 1. RNA-sequencing of paired samples identifies a left-sided enrichment of *Notch3* at E8.5.**</caption>

(A) Outline of the left (light blue) and right (light red) heart field micro-dissected for RNA-sequencing at the indicated stage of mouse heart looping. (B) MA-plot representing relative gene expression between paired left and right heart field at E8.5f. Non-significant differential expression is represented in red, differential expression in green (p-value<0.05), and blue (Benjamini-Hochberg (BH) corrected p-value<0.05, LimmaVoom, n=4 embryos). (C) Ingenuity Pathway Analysis on the gene list shown in Table S1 ordered according to significance and colour-coded for the activation state (z-score: blue, active pathway on the left; red, on the right). (D) Normalised read counts of genes involved in the *Notch* pathway in the left (blue) and right (red) heart field. The dotted line indicates the threshold of background expression. Whisker plots show the median, 25th- and 75th quartiles (boxes), and the extreme data points (whiskers). \*p-value < 0.05, \*\*Benjamini-Hochberg corrected p-value < 0.05 (LimmaVoom, n=4). (E-F) Expression of *Notch3* (white) detected by whole mount RNAscope ISH in E8.5d wild-type embryos, shown in frontal views (e1, cranial part of the embryo, f1, caudal part of the embryo) and transverse sections (e2-e3, f2, at the levels indicated in e1-f1) (n=6). Filled and empty arrowheads point to high and low expression, respectively. (G) Segmentation of the cardiac region in 3D images, to quantify gene expression in the left (white) or right (yellow) heart field. The heart tube is coloured in red. Somites are outlined by white dotted lines. Expression of *Notch3* within the segmented second heart field is extracted in the right panel. (H) Quantification of normalised *Notch3* asymmetric expression in the heart field at sequential stages. The means are shown on a red dotted line and standard deviations are in blue (n=3 at
We selected *Notch3* for further validation, because it is the gene with highest expression, it encodes a receptor and transcription co-factor central to NOTCH signalling. This *Notch* paralogue has not previously been associated with left-right patterning. We mapped *Notch3* expression in whole mount E8.5 wild-type embryos using sensitive RNAscope in situ hybridization. *Notch3* was detected throughout the lateral plate mesoderm, as well as in the crown of the node, with higher expression on the left side (Fig. 1E-F). *Notch3* was not expressed in the myocardium of the forming cardiac tube. We then assessed the dynamics of *Notch3* expression within the heart field (Fig. 1G). Our quantification shows that *Notch3* is transiently asymmetric, reaching 4.5-fold left-sided enrichment in the heart field at E8.5d, a stage when the myocardium bulges out to form a tube (Fig. 1H). Although *Nodal* asymmetry starts one stage earlier (E8.5c, (12)), *Notch3* and *Nodal* overlapped in the left lateral plate mesoderm at E8.5d (Fig. 1I, Video S1), and were found co-expressed in single cells (Fig. S1F). We have thus identified *Notch3* as a novel left-sided gene in the heart field.

<caption>**Fig 1. RNA-sequencing of paired samples identifies a left-sided enrichment of *Notch3* at E8.5.**</caption>

(A) Outline of the left (light blue) and right (light red) heart field micro-dissected for RNA-sequencing at the indicated stage of mouse heart looping. (B) MA-plot representing relative gene expression between paired left and right heart field at E8.5f. Non-significant differential expression is represented in red, differential expression in green (p-value<0.05), and blue (Benjamini-Hochberg (BH) corrected p-value<0.05, LimmaVoom, n=4 embryos). (C) Ingenuity Pathway Analysis on the gene list shown in Table S1 ordered according to significance and colour-coded for the activation state (z-score: blue, active pathway on the left; red, on the right). (D) Normalised read counts of genes involved in the *Notch* pathway in the left (blue) and right (red) heart field. The dotted line indicates the threshold of background expression. Whisker plots show the median, 25th- and 75th quartiles (boxes), and the extreme data points (whiskers). \*p-value < 0.05, \*\*Benjamini-Hochberg corrected p-value < 0.05 (LimmaVoom, n=4). (E-F) Expression of *Notch3* (white) detected by whole mount RNAscope ISH in E8.5d wild-type embryos, shown in frontal views (e1, cranial part of the embryo, f1, caudal part of the embryo) and transverse sections (e2-e3, f2, at the levels indicated in e1-f1) (n=6). Filled and empty arrowheads point to high and low expression, respectively. (G) Segmentation of the cardiac region in 3D images, to quantify gene expression in the left (white) or right (yellow) heart field. The heart tube is coloured in red. Somites are outlined by white dotted lines. Expression of *Notch3* within the segmented second heart field is extracted in the right panel. (H) Quantification of normalised *Notch3* asymmetric expression in the heart field at sequential stages. The means are shown on a red dotted line and standard deviations are in blue (n=3 at
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E8.5c, 6 at E8.5d, 6 at E8.5e, 3 at E8.5f). (I) *Notch3* (white) and *Nodal* (magenta) co-expression detected by double whole mount RNAscope ISH in E8.5d wild-type embryos, shown in a frontal view (i1) and transverse section (i2) (n=5). The midline is indicated by a yellow dotted line. HT, heart tube; L, left; LPM, lateral plate mesoderm (green dotted outline); Myo, myocardium (red dotted outline); No, node; R, right; SHF, second heart field. See also Video S1.

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Given that *Notch3* asymmetry overlaps with that of *Nodal*, we investigated whether *Notch3* and *Nodal* depend on each other. NOTCH signalling has been shown to directly activate *Nodal* in the node, in *Dll1* and *Rbpj* mutants (26). In contrast, we found that *Nodal* was correctly patterned in the node and left lateral plate mesoderm of *Notch3*⁻/⁻ mutants (Fig. S2A-B). In reverse, in mutants in which *Nodal* is inactivated in the lateral plate mesoderm but not in the node (12), *Notch3* expression was impacted (Fig. 2A-B). Quantification of *Notch3* expression at sequential stages in the heart field shows that *Notch3* asymmetry is severely decreased in *Nodal* mutants compared to controls (Fig. 2C). Yet, some significant asymmetry is still detectable. In keeping with a partial dependency of *Notch3* on *Nodal*, we observed that the expression domain of *Notch3* partially overlaps with that of *Nodal*, extending more medially and less laterally (Fig. S2C-D). Thus, *Nodal* is required in the left lateral plate mesoderm to amplify, rather than initiate, *Notch3* asymmetric expression.

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**Fig 2. Decreased Notch3 asymmetry in *Nodal* mutants.**

(A-B) Whole mount RNAscope ISH of *Notch3* in control (A) and *Nodal* mutants (B) at E8.5d in frontal views. Expression of *Notch3* within the segmented heart field is extracted in right panels. The midline of the embryo is indicated by a yellow dotted line. (C) Corresponding quantification of normalised *Notch3* asymmetric expression in the heart field at sequential stages in littermate controls (n= 3 at E8.5c, 5 at E8.5d, 3 at E8.5e) and *Nodal* mutants (n= 5 at E8.5c, 3 at E8.5d, 6 at E8.5e). Means and standard deviations are shown. \*p-value<0.05 between controls and mutants, \*p-value<0.05 to compare mutant levels with a symmetry hypothesis (Log2 ratio =0) (Pairwise Mann-Whitney Wilcoxon tests). L, left; R, right; SHF, second heart field.

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E8.5c, 6 at E8.5d, 6 at E8.5e, 3 at E8.5f). (I) *Notch3* (white) and *Nodal* (magenta) co-expression detected by double whole mount RNAscope ISH in E8.5d wild-type embryos, shown in a frontal view (i1) and transverse section (i2) (n=5). The midline is indicated by a yellow dotted line. HT, heart tube; L, left; LPM, lateral plate mesoderm (green dotted outline); Myo, myocardium (red dotted outline); No, node; R, right; SHF, second heart field. See also Video S1.

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Given that *Notch3* asymmetry overlaps with that of *Nodal*, we investigated whether *Notch3* and *Nodal* depend on each other. NOTCH signalling has been shown to directly activate *Nodal* in the node, in *Dll1* and *Rbpj* mutants (26). In contrast, we found that *Nodal* was correctly patterned in the node and left lateral plate mesoderm of *Notch3*⁻/⁻ mutants (Fig. S2A-B). In reverse, in mutants in which *Nodal* is inactivated in the lateral plate mesoderm but not in the node (12), *Notch3* expression was impacted (Fig. 2A-B). Quantification of *Notch3* expression at sequential stages in the heart field shows that *Notch3* asymmetry is severely decreased in *Nodal* mutants compared to controls (Fig. 2C). Yet, some significant asymmetry is still detectable. In keeping with a partial dependency of *Notch3* on *Nodal*, we observed that the expression domain of *Notch3* partially overlaps with that of *Nodal*, extending more medially and less laterally (Fig. S2C-D). Thus, *Nodal* is required in the left lateral plate mesoderm to amplify, rather than initiate, *Notch3* asymmetric expression.

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**Fig 2. Decreased Notch3 asymmetry in *Nodal* mutants.**

(A-B) Whole mount RNAscope ISH of *Notch3* in control (A) and *Nodal* mutants (B) at E8.5d in frontal views. Expression of *Notch3* within the segmented heart field is extracted in right panels. The midline of the embryo is indicated by a yellow dotted line. (C) Corresponding quantification of normalised *Notch3* asymmetric expression in the heart field at sequential stages in littermate controls (n= 3 at E8.5c, 5 at E8.5d, 3 at E8.5e) and *Nodal* mutants (n= 5 at E8.5c, 3 at E8.5d, 6 at E8.5e). Means and standard deviations are shown. \*p-value<0.05 between controls and mutants, \*p-value<0.05 to compare mutant levels with a symmetry hypothesis (Log2 ratio =0) (Pairwise Mann-Whitney Wilcoxon tests). L, left; R, right; SHF, second heart field.

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We next investigated the role of *Notch3* in heart development, in mutants inactivated for it (Fig. S3). Given its asymmetric expression when the cardiac tube forms, we looked for a potential effect on the rightward looping of the heart tube. E9.5 mutant embryos were collected at Mendelian ratio (Fig. 3A) and no heart looping defect was detected, both qualitatively (Fig. 3B-D) and quantitatively (Fig. 3E-F). *Notch3*⁻/⁻ mutant embryos were indistinguishable from controls, with a normal orientation of the right ventricle-left ventricle axis relative to the midline, and a normal left displacement of the venous pole.

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**Fig 3. Normal heart looping in single *Notch3* mutants.**

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(A) Histogram showing the percentage of genotypes recovered in E9.5 litters of *Notch3*⁺/⁻ x *Notch3*⁺/⁻ crosses. The observed frequency is not significantly different from the expected Mendelian ratio (dotted lines) (p-value = 0.30, chi-square test, n as indicated). (B-D) Brightfield ventral views (b1-d1), and 3D segmented heart tubes shown in ventral (b2-d2) and cranial (b3-d3) views, of littermate *Notch3*⁺/⁺ (B), *Notch3*⁺/⁻ (C) and *Notch3*⁻/⁻ (D) E9.5 embryos. Cardiac regions are colour coded: outflow tract in green, right ventricle in red, left ventricle in blue, atrioventricular canal and left atrium in yellow, right atrium in magenta. The notochord (cyan) is used as a reference axis to align samples. (E) Quantification of the orientation of the RV/LV axis relative to the notochord, as schematised in b2. (F) Quantification of the displacement of the venous pole relative to the notochord, normalised by the tube length, as schematised in b3. Means and standard deviations are shown (Pairwise Mann-Whitney Wilcoxon tests with Benjamini-Hochberg correction, n=11 *Notch3*⁺/⁺, 10 *Notch3*⁺/⁻ and 16 *Notch3*⁻/⁻). LV, left ventricle; n.s., non-significant; RV, right ventricle.

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We then collected *Notch3* mutants after birth. In agreement with previous reports (38), they were collected at Mendelian ratio (Fig. 4A). *Notch3*⁻/⁻ mutants did not show heterotaxy, unlike *Nodal* mutants, but had congenital heart defects with partial penetrance (67%), including peri-membranous and muscular ventricular septal defects, thinning of the right ventricular wall and coronary artery dilatation (Fig. 4B-C). Defects of coronary arteries are in keeping with fetal expression of *Notch3* in pericytes and smooth muscle cells (Fig. S4). Overall, our results show that *Notch3* is required for ventricle and coronary artery development.
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We next investigated the role of *Notch3* in heart development, in mutants inactivated for it (Fig. S3). Given its asymmetric expression when the cardiac tube forms, we looked for a potential effect on the rightward looping of the heart tube. E9.5 mutant embryos were collected at Mendelian ratio (Fig. 3A) and no heart looping defect was detected, both qualitatively (Fig. 3B-D) and quantitatively (Fig. 3E-F). *Notch3*⁻/⁻ mutant embryos were indistinguishable from controls, with a normal orientation of the right ventricle-left ventricle axis relative to the midline, and a normal left displacement of the venous pole.

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**Fig 3. Normal heart looping in single *Notch3* mutants.**

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(A) Histogram showing the percentage of genotypes recovered in E9.5 litters of *Notch3*⁺/⁻ x *Notch3*⁺/⁻ crosses. The observed frequency is not significantly different from the expected Mendelian ratio (dotted lines) (p-value = 0.30, chi-square test, n as indicated). (B-D) Brightfield ventral views (b1-d1), and 3D segmented heart tubes shown in ventral (b2-d2) and cranial (b3-d3) views, of littermate *Notch3*⁺/⁺ (B), *Notch3*⁺/⁻ (C) and *Notch3*⁻/⁻ (D) E9.5 embryos. Cardiac regions are colour coded: outflow tract in green, right ventricle in red, left ventricle in blue, atrioventricular canal and left atrium in yellow, right atrium in magenta. The notochord (cyan) is used as a reference axis to align samples. (E) Quantification of the orientation of the RV/LV axis relative to the notochord, as schematised in b2. (F) Quantification of the displacement of the venous pole relative to the notochord, normalised by the tube length, as schematised in b3. Means and standard deviations are shown (Pairwise Mann-Whitney Wilcoxon tests with Benjamini-Hochberg correction, n=11 *Notch3*⁺/⁺, 10 *Notch3*⁺/⁻ and 16 *Notch3*⁻/⁻). LV, left ventricle; n.s., non-significant; RV, right ventricle.

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We then collected *Notch3* mutants after birth. In agreement with previous reports (38), they were collected at Mendelian ratio (Fig. 4A). *Notch3*⁻/⁻ mutants did not show heterotaxy, unlike *Nodal* mutants, but had congenital heart defects with partial penetrance (67%), including peri-membranous and muscular ventricular septal defects, thinning of the right ventricular wall and coronary artery dilatation (Fig. 4B-C). Defects of coronary arteries are in keeping with fetal expression of *Notch3* in pericytes and smooth muscle cells (Fig. S4). Overall, our results show that *Notch3* is required for ventricle and coronary artery development.
259 However, removal of *Notch3* alone does not uncover a role in left-right asymmetric organogenesis.
260

261 **Fig 4. Congenital heart defects in *Notch3* mutants.**
262 (A) Histogram showing the percentage of genotypes recovered in P14 litters of *Notch3+/−* x *Notch3+/−* crosses. The observed frequency is not significantly different from the expected Mendelian ratio (dotted lines) (p-value = 0.64, chi-square tests, n as indicated). (B-C) Sections of *Notch3+/−* mutant hearts at P0, imaged by HREM. An example of a mutant heart with no phenotype is shown (B, n=4/12). Defects (C) include peri-membranous ventricular septal defect (VSD) (yellow arrowhead, n=1/12), muscular VSD (red arrowheads, n=4/12), dilatation of the septal coronary artery (pink arrowhead, n=1/12) and thin RV wall (green arrow, n=3/12).
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269 AVC, atrioventricular canal; Ao, aorta; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle.
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272 **Notch3 is a genetic modifier of *Nodal* in heart looping**
273 Since *Notch3* overlaps with and is amplified by *Nodal*, we investigated their interaction in
274 the context of left-right asymmetry. We generated double heterozygote mutants and
275 quantified heart looping. Compared to single heterozygotes (Fig. 5A-B), *Notch3+/−*; *Hoxb1Cre+/*;
276 *Nodal flox/+* double heterozygotes (Fig. 5C) did not show any heart looping defects, including
277 normal orientation of the right ventricle-left ventricle axis relative to the midline, and normal
278 left displacement of the venous pole (Fig. 5D-E). Thus, we did not detect a genetic interaction
279 between *Notch3* and *Nodal*.

280 We then analysed the role of *Notch3* in a sensitized background. We generated a large
281 cohort of mutants, with a knock-down of *Notch3* within the context of *Nodal* inactivation in
282 the left lateral plate mesoderm. *Notch3+/−; Hoxb1Cre+/*; *Nodal flox/null* mutants lost the randomized
283 direction of heart looping, typical of *Nodal* mutants (Fig. 5F-G). Rightward looping was more
284 frequent when *Notch3* was reduced in *Nodal* mutants. However, this did not correspond to a
285 rescue, since all rightward mutant heart loops were abnormal, compared to controls.
286 Compared to *Nodal* mutants, additional defects in the outflow tract were observed with partial
259 However, removal of *Notch3* alone does not uncover a role in left-right asymmetric
260 organogenesis.

261 **Fig 4. Congenital heart defects in *Notch3* mutants.**

262 (A) Histogram showing the percentage of genotypes recovered in P14 litters of *Notch3+/−* x
263 *Notch3+/−* crosses. The observed frequency is not significantly different from the expected
264 Mendelian ratio (dotted lines) (p-value = 0.64, chi-square tests, n as indicated). (B-C) Sections
265 of *Notch3+/−* mutant hearts at P0, imaged by HREM. An example of a mutant heart with no
266 phenotype is shown (B, n=4/12). Defects (C) include peri-membranous ventricular septal
267 defect (VSD) (yellow arrowhead, n=1/12), muscular VSD (red arrowheads, n=4/12), dilatation
268 of the septal coronary artery (pink arrowhead, n=1/12) and thin RV wall (green arrow, n=3/12).
269 AVC, atrioventricular canal; Ao, aorta; LA, left atrium; LV, left ventricle; RA, right atrium; RV,
270 right ventricle.
271

272 **Notch3 is a genetic modifier of *Nodal* in heart looping**

273 Since *Notch3* overlaps with and is amplified by *Nodal*, we investigated their interaction in
274 the context of left-right asymmetry. We generated double heterozygote mutants and
275 quantified heart looping. Compared to single heterozygotes (Fig. 5A-B), *Notch3+/−*; *Hoxb1Cre+/*;
276 *Nodal flox/+* double heterozygotes (Fig. 5C) did not show any heart looping defects, including
277 normal orientation of the right ventricle-left ventricle axis relative to the midline, and normal
278 left displacement of the venous pole (Fig. 5D-E). Thus, we did not detect a genetic interaction
279 between *Notch3* and *Nodal*.

280 We then analysed the role of *Notch3* in a sensitized background. We generated a large
281 cohort of mutants, with a knock-down of *Notch3* within the context of *Nodal* inactivation in
282 the left lateral plate mesoderm. *Notch3+/−*; *Hoxb1Cre+/*; *Nodal flox/null* mutants lost the randomized
283 direction of heart looping, typical of *Nodal* mutants (Fig. 5F-G). Rightward looping was more
284 frequent when *Notch3* was reduced in *Nodal* mutants. However, this did not correspond to a
285 rescue, since all rightward mutant heart loops were abnormal, compared to controls.
286 Compared to *Nodal* mutants, additional defects in the outflow tract were observed with partial
287 penetrance. The outflow tract was straighter in 40% (7/18) of *Notch3*<sup>+/-</sup>; *Hoxb1*<sup>Cre/+</sup>; *Nodal*<sup>flox/null</sup> mutants within looping Class 4 (Fig. 5H-J), which is the less severe class. These genetic analyses 288 thus uncover a role for *Notch3* as a genetic modifier of *Nodal* during left-right asymmetric 289 organogenesis. 290

291 **Fig 5. Heart looping variations in *Notch3* and *Nodal* compound mutants.**
292 (A-C) 3D segmented heart tubes shown in ventral (a1-c1) and cranial views (a2-c2) of single 293 *Nodal* heterozygote (A), single *Notch3* heterozygote (B) and double heterozygote (C) embryos 294 at E9.5. Cardiac regions are colour coded as in Fig. 3. The notochord (cyan) is used as a 295 reference axis to align samples. (D) Quantification of the orientation of the RV/LV axis relative 296 to the notochord. (E) Quantification of the displacement of the venous pole relative to the 297 notochord, normalised by the tube length. Means and standard deviations are shown. 298 (Pairwise Mann-Whitney Wilcoxon tests with Benjamini-Hochberg correction, n=8 *Hoxb1*<sup>Cre/+</sup>; 299 *Nodal*<sup>flox/+</sup>, 14 *Notch3*<sup>+/-</sup>; *Nodal*<sup>flox/+</sup>, 12 *Notch3*<sup>+/-</sup>; *Hoxb1*<sup>Cre/+</sup>; *Nodal*<sup>flox/+</sup>). (F) Brightfield images 300 of homozygote *Nodal* mutants with decreased *Notch3* (*Notch3*<sup>+/-</sup>; *Hoxb1*<sup>Cre/+</sup>; *Nodal*<sup>flox/null</sup>) at 301 E9.5, in a ventral view. (G) Corresponding quantification of their looping direction frequency 302 (right bar), which, in contrast to *Nodal* mutants with normal *Notch3* levels (left bar), differs 303 from a randomised looping direction (dotted line). \*p-value<0.05 (Chi-square test with Yates' 304 continuity correction, n=76 *Hoxb1*<sup>Cre/+</sup>; *Nodal*<sup>flox/null</sup> and 47 *Notch3*<sup>+/-</sup>; *Hoxb1*<sup>Cre/+</sup>; *Nodal*<sup>flox/null</sup>). 305 (H-J) Comparison of Class 4 abnormal heart loops in homozygote *Nodal* (I), *Nodal*; *Notch3* 306 compound (J) mutants compared to control *Notch3*<sup>+/-</sup> embryos (H), seen in brightfield right- 307 sided views. Filled and empty arrowheads point to curved and straight outflow tract, 308 respectively. L, Left; LV, left ventricle; R, Right; RV, right ventricle. 309
287 penetrance. The outflow tract was straighter in 40% (7/18) of *Notch3*<sup>+/-</sup>; *Hoxb1*<sup>Cre/+</sup>; *Nodal*<sup>flox/null</sup> mutants within looping Class 4 (Fig. 5H-J), which is the less severe class. These genetic analyses 288 thus uncover a role for *Notch3* as a genetic modifier of *Nodal* during left-right asymmetric 289 organogenesis. 290

291 **Fig 5. Heart looping variations in *Notch3* and *Nodal* compound mutants.**
292 (A-C) 3D segmented heart tubes shown in ventral (a1-c1) and cranial views (a2-c2) of single 293 *Nodal* heterozygote (A), single *Notch3* heterozygote (B) and double heterozygote (C) embryos 294 at E9.5. Cardiac regions are colour coded as in Fig. 3. The notochord (cyan) is used as a 295 reference axis to align samples. (D) Quantification of the orientation of the RV/LV axis relative 296 to the notochord. (E) Quantification of the displacement of the venous pole relative to the 297 notochord, normalised by the tube length. Means and standard deviations are shown. 298 (Pairwise Mann-Whitney Wilcoxon tests with Benjamini-Hochberg correction, n=8 *Hoxb1*<sup>Cre/+</sup>; 299 *Nodal*<sup>flox/+</sup>, 14 *Notch3*<sup>+/-</sup>; *Nodal*<sup>flox/+</sup>, 12 *Notch3*<sup>+/-</sup>; *Hoxb1*<sup>Cre/+</sup>; *Nodal*<sup>flox/+</sup>). (F) Brightfield images 300 of homozygote *Nodal* mutants with decreased *Notch3* (*Notch3*<sup>+/-</sup>; *Hoxb1*<sup>Cre/+</sup>; *Nodal*<sup>flox/null</sup>) at 301 E9.5, in a ventral view. (G) Corresponding quantification of their looping direction frequency 302 (right bar), which, in contrast to *Nodal* mutants with normal *Notch3* levels (left bar), differs 303 from a randomised looping direction (dotted line). \*p-value<0.05 (Chi-square test with Yates' 304 continuity correction, n=76 *Hoxb1*<sup>Cre/+</sup>; *Nodal*<sup>flox/null</sup> and 47 *Notch3*<sup>+/-</sup>; *Hoxb1*<sup>Cre/+</sup>; *Nodal*<sup>flox/null</sup>). 305 (H-J) Comparison of Class 4 abnormal heart loops in homozygote *Nodal* (I), *Nodal*; *Notch3* 306 compound (J) mutants compared to control *Notch3*<sup>+/-</sup> embryos (H), seen in brightfield right- 307 sided views. Filled and empty arrowheads point to curved and straight outflow tract, 308 respectively. L, Left; LV, left ventricle; R, Right; RV, right ventricle. 309
310 **Discussion**

311 We have identified *Notch3* as a novel left asymmetric factor. We show that it is amplified by
312 NODAL signalling. We do not detect a genetic interaction between *Nodal* and *Notch3*, but
313 rather show that *Notch3* acts as a genetic modifier of *Nodal*. Knock-down of *Notch3*
314 exacerbates looping defects observed in *Nodal* mutants. Single *Notch3* mutant neonates have
315 other cardiac defects in coronary arteries, transmural growth and septation of ventricles.

316 Left-right asymmetry is mainly analysed in terms of patterning of the major left
317 determinant, *Nodal*, and its downstream pathway including *Lefty2* and *Pitx2* (4,53,54).
318 However, in the absence of *Nodal*, organ asymmetry is not fully abrogated (6,13–15). Heart
319 looping, which is the first asymmetric morphogenesis, still occurs to some extent in *Nodal*
320 mutants, producing helical heart tubes, even if abnormal (12). This indicates that there are
321 other factors than NODAL signalling required for heart asymmetry. BMP (20–22), HH (23),
322 NOTCH (24–26) and WNT (27–29) signalling have been shown to be required upstream of
323 *Nodal* for asymmetry. Transcriptomic screens have been performed to identify asymmetric
324 genes in embryos outside the node or in cardiac precursors (50,51). Yet, asymmetric candidate
325 genes were either not validated or not analysed functionally, leaving open the question of
326 which are genuine asymmetry factors. Here, we have used a transcriptomic screen tailored
327 spatially in the heart field, temporally at the stage of the first morphological asymmetry, and
328 specifically on left-right asymmetry based on paired comparison in single embryos. This has
329 successfully identified a novel left factor, *Notch3*. We have characterised by quantitative and
330 whole mount sensitive in situ hybridisation its dynamic spatio-temporal expression pattern
331 relative to *Nodal*. And we have demonstrated its role in left-right asymmetry based on an allelic
332 series of mutants. Similarly to *Nodal*, *Notch3* is transiently asymmetric, highlighting the
333 importance of careful staging to monitor left-right patterning. In contrast to *Nodal*, *Lefty2* and
310 **Discussion**

311 We have identified *Notch3* as a novel left asymmetric factor. We show that it is amplified by
312 NODAL signalling. We do not detect a genetic interaction between *Nodal* and *Notch3*, but
313 rather show that *Notch3* acts as a genetic modifier of *Nodal*. Knock-down of *Notch3*
314 exacerbates looping defects observed in *Nodal* mutants. Single *Notch3* mutant neonates have
315 other cardiac defects in coronary arteries, transmural growth and septation of ventricles.

316 Left-right asymmetry is mainly analysed in terms of patterning of the major left
317 determinant, *Nodal*, and its downstream pathway including *Lefty2* and *Pitx2* (4,53,54).
318 However, in the absence of *Nodal*, organ asymmetry is not fully abrogated (6,13–15). Heart
319 looping, which is the first asymmetric morphogenesis, still occurs to some extent in *Nodal*
320 mutants, producing helical heart tubes, even if abnormal (12). This indicates that there are
321 other factors than NODAL signalling required for heart asymmetry. BMP (20–22), HH (23),
322 NOTCH (24–26) and WNT (27–29) signalling have been shown to be required upstream of
323 *Nodal* for asymmetry. Transcriptomic screens have been performed to identify asymmetric
324 genes in embryos outside the node or in cardiac precursors (50,51). Yet, asymmetric candidate
325 genes were either not validated or not analysed functionally, leaving open the question of
326 which are genuine asymmetry factors. Here, we have used a transcriptomic screen tailored
327 spatially in the heart field, temporally at the stage of the first morphological asymmetry, and
328 specifically on left-right asymmetry based on paired comparison in single embryos. This has
329 successfully identified a novel left factor, *Notch3*. We have characterised by quantitative and
330 whole mount sensitive in situ hybridisation its dynamic spatio-temporal expression pattern
331 relative to *Nodal*. And we have demonstrated its role in left-right asymmetry based on an allelic
332 series of mutants. Similarly to *Nodal*, *Notch3* is transiently asymmetric, highlighting the
333 importance of careful staging to monitor left-right patterning. In contrast to *Nodal*, *Lefty2* and
Pitx2, which are exclusively expressed on the left of the lateral plate mesoderm, *Notch3* is also expressed on the right, with a 4.5-fold enrichment on the left side. Thus, quantitative and sensitive approaches are critical to uncover factors which have lower asymmetry levels compared to the *Nodal* pathway.

*Nodal* plays multiple roles in asymmetry. *Nodal* is well known to act as a bias, able to set the laterality of asymmetry, so that its absence leads to a randomized orientation. This is the case for the direction of heart looping (12) or the stomach position (14). In addition, *Nodal*, upstream of *Pitx2*, acts as a regulator of identity, able to confer a left anatomical structure to the atria and lungs or to induce the formation of the spleen (14,55,56). Finally, we had shown that *Nodal* can also act as an amplifier of asymmetry (12). This had been detected at a morphological level, with a reduced rotation of the arterial pole during heart looping in *Nodal* mutants. We now show this at a molecular level, with the amplification of *Notch3* asymmetry by *Nodal*.

NOTCH signalling is required at different levels for left-right asymmetry. *Notch1/2* were shown previously to play a role in the formation of the left-right organiser and the induction of *Nodal* (24–26). We now identify the role of another NOTCH paralogue, NOTCH3. Whereas *Notch1* is expressed around the node in the caudal epiblast and presomitic mesoderm, and *Notch2* in the node pit (24,31), we have detected *Notch3* in the node crown and lateral plate mesoderm. Our conditional mutants permit to dissect a role of *Notch3* in the lateral plate mesoderm for left-right asymmetry. How *Notch3* regulates heart looping direction and the curvature of the outflow tract remains an open question. *Notch3* regulates muscle differentiation and maturation in other tissues, such as the smooth muscles of arteries, coronary arteries and intestinal lacteals, or skeletal muscles, and is expressed in precursors cells such as pericytes or satellite stem cells (39–42,45). It will be interesting to investigate
Pitx2, which are exclusively expressed on the left of the lateral plate mesoderm, *Notch3* is also expressed on the right, with a 4.5-fold enrichment on the left side. Thus, quantitative and sensitive approaches are critical to uncover factors which have lower asymmetry levels compared to the *Nodal* pathway.

*Nodal* plays multiple roles in asymmetry. *Nodal* is well known to act as a bias, able to set the laterality of asymmetry, so that its absence leads to a randomized orientation. This is the case for the direction of heart looping (12) or the stomach position (14). In addition, *Nodal*, upstream of *Pitx2*, acts as a regulator of identity, able to confer a left anatomical structure to the atria and lungs or to induce the formation of the spleen (14,55,56). Finally, we had shown that *Nodal* can also act as an amplifier of asymmetry (12). This had been detected at a morphological level, with a reduced rotation of the arterial pole during heart looping in *Nodal* mutants. We now show this at a molecular level, with the amplification of *Notch3* asymmetry by *Nodal*.

NOTCH signalling is required at different levels for left-right asymmetry. *Notch1/2* were shown previously to play a role in the formation of the left-right organiser and the induction of *Nodal* (24–26). We now identify the role of another NOTCH paralogue, NOTCH3. Whereas *Notch1* is expressed around the node in the caudal epiblast and presomitic mesoderm, and *Notch2* in the node pit (24,31), we have detected *Notch3* in the node crown and lateral plate mesoderm. Our conditional mutants permit to dissect a role of *Notch3* in the lateral plate mesoderm for left-right asymmetry. How *Notch3* regulates heart looping direction and the curvature of the outflow tract remains an open question. *Notch3* regulates muscle differentiation and maturation in other tissues, such as the smooth muscles of arteries, coronary arteries and intestinal lacteals, or skeletal muscles, and is expressed in precursors cells such as pericytes or satellite stem cells (39–42,45). It will be interesting to investigate
whether *Notch3* in the heart field similarly regulates the differentiation and maturation of the
358
cardiac muscle to impact heart looping. Elongation of the heart tube is important for heart
359
looping, as a driver of a buckling mechanism (17). Thus, asymmetries in myocardial cell
360
differentiation, when precursor cells incorporate into the heart tube, can provide a left-right
361
bias to orient looping. We have shown previously that *Nodal* modulates a large set of genes
362
involved in myocardial cell differentiation, some of which like *Tnnt1* are asymmetrically
363
expressed within the wild-type heart tube (12). Whether *Notch3* acts similarly remains to be
364
investigated. For later transmural growth and septation of ventricles, it is possible that a
365
potential role of *Notch3* on muscle differentiation and maturation intervenes. However, since
366
*Notch3* is expressed in cardiac precursors and not cardiomyocytes, that would imply a long
367
delay from expression to phenotype. Another possibility is that ventricle growth in *Notch3*
368
mutants is a secondary consequence of defective coronary vascularisation (57).
369

Although *Notch3* expression is sensitive to *Nodal*, our observations support the idea that
370
*Notch3* is not a component of the *Nodal* pathway. The expression pattern of *Notch3* differs
371
from *Nodal*, with a right sided and more medial left sided localization. In contrast to *Pitx2* or
372
*Lefty2* (12), *Notch3* expression is not erased in *Nodal* mutants. Functionally, we do not detect
373
a genetic interaction between *Notch3* and *Nodal*, and show that knock-down of *Notch3* can
374
exacerbate the phenotype of *Nodal* mutants. Thus, *Notch3* comes out as a novel genetic
375
modifier of *Nodal*. The underlying mechanism is still unknown. A link between the two
376
pathways has been detected based on the direct binding of NOTCH3 to the NODAL co-receptor
377
TDGF1, which enhances NOTCH proteolytic maturation and sensitization to ligand-induced
378
NOTCH signalling (58). Given that the disease associated with *Nodal* dysfunction, heterotaxy,
379
has a heterogenous phenotypic spectrum, the identification of genetic modifiers of known
380
whether *Notch3* in the heart field similarly regulates the differentiation and maturation of the
358
cardiac muscle to impact heart looping. Elongation of the heart tube is important for heart
359
looping, as a driver of a buckling mechanism (17). Thus, asymmetries in myocardial cell
360
differentiation, when precursor cells incorporate into the heart tube, can provide a left-right
361
bias to orient looping. We have shown previously that *Nodal* modulates a large set of genes
362
involved in myocardial cell differentiation, some of which like *Tnnt1* are asymmetrically
363
expressed within the wild-type heart tube (12). Whether *Notch3* acts similarly remains to be
364
investigated. For later transmural growth and septation of ventricles, it is possible that a
365
potential role of *Notch3* on muscle differentiation and maturation intervenes. However, since
366
*Notch3* is expressed in cardiac precursors and not cardiomyocytes, that would imply a long
367
delay from expression to phenotype. Another possibility is that ventricle growth in *Notch3*
368
mutants is a secondary consequence of defective coronary vascularisation (57).
369

Although *Notch3* expression is sensitive to *Nodal*, our observations support the idea that
370
*Notch3* is not a component of the *Nodal* pathway. The expression pattern of *Notch3* differs
371
from *Nodal*, with a right sided and more medial left sided localization. In contrast to *Pitx2* or
372
*Lefty2* (12), *Notch3* expression is not erased in *Nodal* mutants. Functionally, we do not detect
373
a genetic interaction between *Notch3* and *Nodal*, and show that knock-down of *Notch3* can
374
exacerbate the phenotype of *Nodal* mutants. Thus, *Notch3* comes out as a novel genetic
375
modifier of *Nodal*. The underlying mechanism is still unknown. A link between the two
376
pathways has been detected based on the direct binding of NOTCH3 to the NODAL co-receptor
377
TDGF1, which enhances NOTCH proteolytic maturation and sensitization to ligand-induced
378
NOTCH signalling (58). Given that the disease associated with *Nodal* dysfunction, heterotaxy,
379
has a heterogenous phenotypic spectrum, the identification of genetic modifiers of known
380
381 genes involved in heterotaxy opens novel perspectives to fill gaps in the genetic diagnosis of

382 60% of patients (59).
381 genes involved in heterotaxy opens novel perspectives to fill gaps in the genetic diagnosis of

382 60% of patients (59).
383 **Methods**

384 EXPERIMENTAL MODEL AND SUBJECT DETAILS

385 C56BI6J mice were used as wild-type embryos. *Notch3*<sup>tm1Grid/tm1Grid</sup> mutants (abbreviated
386 *Notch3*<sup>-/-</sup>, (38)) were kept in a C56BI6J background; they are viable and fertile as homozygotes.
387 *Nodal*<sup>null/+</sup>; *Hoxb1*<sup>Cre/+</sup> males (12) were maintained in a mixed genetic background and crossed
388 with *Nodal*<sup>flox/flox</sup> females (60) to generate *Nodal* conditional mutants. Both male and female
389 embryos were collected and used randomly for experiments, except for RNA sequencing, in
390 which only male embryos were used to reduce variability. Embryonic day (E) 0.5 was defined
391 as noon on the day of vaginal plug detection. Embryonic stages were determined based on the
392 morphology of the heart according to (17) and (12). Somite numbers were evaluated from
393 brightfield images; samples with less than 18 somites were excluded from heart looping
394 quantifications. All embryos were genotyped by PCR, using primers listed in Table S2. Animals
395 were housed in individually ventilated cages containing tube shelters and nesting material,
396 maintained at 21°C and 50% humidity, under a 12h light/dark cycle, with food and water ad
397 libitum, in the Laboratory of Animal Experimentation and Transgenesis of the SFR Necker,
398 Imagine Campus, Paris. Animal procedures were approved by the ethical committees of the
399 Institut Pasteur, Université Paris Cité and the French Ministry of Research.

400 METHOD DETAILS

401 RNA isolation and sequencing

402 Paired left and right heart fields of E8.5f embryos were micro-dissected, from below the
403 headfolds to the end of the second somite (19), after removal of the heart and the back. The
404 tissue was flash frozen in liquid nitrogen. All samples were collected within 1h30 of sacrificing
383 **Methods**

384 EXPERIMENTAL MODEL AND SUBJECT DETAILS

385 C56BI6J mice were used as wild-type embryos. *Notch3*<sup>tm1Grid/tm1Grid</sup> mutants (abbreviated 386 *Notch3*<sup>-/-</sup>, (38)) were kept in a C56BI6J background; they are viable and fertile as homozygotes. 387 *Nodal*<sup>null/+</sup>; *Hoxb1*<sup>Cre/+</sup> males (12) were maintained in a mixed genetic background and crossed 388 with *Nodal*<sup>flox/flox</sup> females (60) to generate *Nodal* conditional mutants. Both male and female 389 embryos were collected and used randomly for experiments, except for RNA sequencing, in 390 which only male embryos were used to reduce variability. Embryonic day (E) 0.5 was defined 391 as noon on the day of vaginal plug detection. Embryonic stages were determined based on the 392 morphology of the heart according to (17) and (12). Somite numbers were evaluated from 393 brightfield images; samples with less than 18 somites were excluded from heart looping 394 quantifications. All embryos were genotyped by PCR, using primers listed in Table S2. Animals 395 were housed in individually ventilated cages containing tube shelters and nesting material, 396 maintained at 21°C and 50% humidity, under a 12h light/dark cycle, with food and water ad 397 libitum, in the Laboratory of Animal Experimentation and Transgenesis of the SFR Necker, 398 Imagine Campus, Paris. Animal procedures were approved by the ethical committees of the 399 Institut Pasteur, Université Paris Cité and the French Ministry of Research.

400 METHOD DETAILS

401 RNA isolation and sequencing

402 Paired left and right heart fields of E8.5f embryos were micro-dissected, from below the 403 headfolds to the end of the second somite (19), after removal of the heart and the back. The 404 tissue was flash frozen in liquid nitrogen. All samples were collected within 1h30 of sacrificing
405 the mother. Total RNA was extracted in TRIzol-Chloroform and purified using the RNeasy micro
406 kit (QIAGEN) including DNAse treatment. RNA quality and quantity were measured on
407 Fragment Analyzer (Agilent Technologies). All RQN were >9.7. The libraries were established
408 using the Nugen Universal Plus mRNA-Seq kit, using 15ng of total RNA per sample. The
409 oriented cDNAs produced from the poly-A+ fraction were PCR amplified (15-18 cycles). An
410 equimolar pool of the final indexed RNA-Seq libraries was sequenced on an Illumina
411 NovaSeq6000, with paired-end reads of 130 bases and a mean sequencing depth of 37.15
412 million reads per sample.

413 Embryo dissection

414 Embryos were dissected in 1xDPBS and fixed in 4% paraformaldehyde either for 6h at room
415 temperature or 24h at 4°C. Yolk sac or tail pieces were collected for genotyping. For embryos
416 dissected at E9.5, hearts were arrested in diastole by treatment with cold 250mM KCl for 5
417 minutes. Fixed embryos were gradually dehydrated into methanol and stored at -20°C. For
418 pups collected at P0, they were euthanised, immersed in a cardioplegia solution (110mM NaCl,
419 16mM KCl,16mM MgCl2, 1.5mM CaCl2, 10mM NaHCO3) for 5 minutes and fixed in 4% PFA 24h
420 at 4°C.

421 RNA in situ hybridization (ISH)

422 ISH was performed whole mount as in (12). *Wnt11* and *Bmp2* antisense riboprobes were
423 transcribed from plasmids. Signals were detected by alkaline phosphatase (AP)-conjugated
424 anti-DIG antibodies (1/2000), which were revealed with the BM purple (magenta) substrate.
425 The samples were washed in 1x DPBS, post-fixed and imaged by HREM.
405 the mother. Total RNA was extracted in TRIzol-Chloroform and purified using the RNeasy micro
406 kit (QIAGEN) including DNAse treatment. RNA quality and quantity were measured on
407 Fragment Analyzer (Agilent Technologies). All RQN were >9.7. The libraries were established
408 using the Nugen Universal Plus mRNA-Seq kit, using 15ng of total RNA per sample. The
409 oriented cDNAs produced from the poly-A+ fraction were PCR amplified (15-18 cycles). An
410 equimolar pool of the final indexed RNA-Seq libraries was sequenced on an Illumina
411 NovaSeq6000, with paired-end reads of 130 bases and a mean sequencing depth of 37.15
412 million reads per sample.

413 Embryo dissection

414 Embryos were dissected in 1xDPBS and fixed in 4% paraformaldehyde either for 6h at room
415 temperature or 24h at 4°C. Yolk sac or tail pieces were collected for genotyping. For embryos
416 dissected at E9.5, hearts were arrested in diastole by treatment with cold 250mM KCl for 5
417 minutes. Fixed embryos were gradually dehydrated into methanol and stored at -20°C. For
418 pups collected at P0, they were euthanised, immersed in a cardioplegia solution (110mM NaCl,
419 16mM KCl,16mM MgCl2, 1.5mM CaCl2, 10mM NaHCO3) for 5 minutes and fixed in 4% PFA 24h
420 at 4°C.

421 RNA in situ hybridization (ISH)

422 ISH was performed whole mount as in (12). *Wnt11* and *Bmp2* antisense riboprobes were
423 transcribed from plasmids. Signals were detected by alkaline phosphatase (AP)-conjugated
424 anti-DIG antibodies (1/2000), which were revealed with the BM purple (magenta) substrate.
425 The samples were washed in 1x DPBS, post-fixed and imaged by HREM.
426 Wholemount RNAscope ISH were performed using the Mutliplex Fluorescent v2 Assay
427 (Biotechne) and the protocol of (61). *Notch3* (425171-C1, 425171-C2) and *Nodal* (436321-C1)
428 probes were used. Amplification steps were performed using the TSA cyanine5 and cyanine3
429 amplification kit. Hoechst (1/1000) was used as a nuclear counterstain. Samples were then
430 transferred in R2 CUBIC clearing reagents and embedded in R2 reagent containing agarose
431 (62). Multi-channel 16-bit images were acquired with a Z.1 lightsheet microscope and a
432 20X/1.0 objective.

433 RT-qPCR

434 Reverse transcription was performed on RNA isolated from entire (left and right) micro-
435 dissected heart fields using a Reverse Transcription kit (QuantiTect, Qiagen). Quantitative PCR
436 was carried out using the ViiA7 real-time PCR system. mRNA expression levels were measured
437 relatively to *Polr2b* and normalised with a reference cDNA sample, taken as a pool of 7 whole
438 embryos at stage E8.5c-g, using the standard ΔΔCt method. Primers are listed in Table S2.

439 High Resolution Episcopic Microscopy (HREM)

440 E9.5 embryos or P0 hearts were imaged in 3D by HREM after embedding in methacrylate
441 resin (JB4) containing eosin and acridine orange as contrast agents (12). Two channel images
442 of the surface of the resin block were acquired using the optical high-resolution episcopic
443 microscope and a 1X Apo objective repeatedly after removal of on average 1.75 µm or 2.63
444 µm thick sections. The datasets comprise 742-1869 images of 0.90-4.48 µm resolution in x and
445 y depending on the stage. Icy (63) and Fiji (ImageJ) softwares were used to crop and scale the
446 datasets. 3D reconstructions and analysis were performed using Imaris.

447
426 Wholemount RNAscope ISH were performed using the Mutliplex Fluorescent v2 Assay
427 (Biotechne) and the protocol of (61). *Notch3* (425171-C1, 425171-C2) and *Nodal* (436321-C1)
428 probes were used. Amplification steps were performed using the TSA cyanine5 and cyanine3
429 amplification kit. Hoechst (1/1000) was used as a nuclear counterstain. Samples were then
430 transferred in R2 CUBIC clearing reagents and embedded in R2 reagent containing agarose
431 (62). Multi-channel 16-bit images were acquired with a Z.1 lightsheet microscope and a
432 20X/1.0 objective.

433 RT-qPCR

434 Reverse transcription was performed on RNA isolated from entire (left and right) micro-
435 dissected heart fields using a Reverse Transcription kit (QuantiTect, Qiagen). Quantitative PCR
436 was carried out using the ViiA7 real-time PCR system. mRNA expression levels were measured
437 relatively to *Polr2b* and normalised with a reference cDNA sample, taken as a pool of 7 whole
438 embryos at stage E8.5c-g, using the standard ΔΔCt method. Primers are listed in Table S2.

439 High Resolution Episcopic Microscopy (HREM)

440 E9.5 embryos or P0 hearts were imaged in 3D by HREM after embedding in methacrylate
441 resin (JB4) containing eosin and acridine orange as contrast agents (12). Two channel images
442 of the surface of the resin block were acquired using the optical high-resolution episcopic
443 microscope and a 1X Apo objective repeatedly after removal of on average 1.75 µm or 2.63
444 µm thick sections. The datasets comprise 742-1869 images of 0.90-4.48 µm resolution in x and
445 y depending on the stage. Icy (63) and Fiji (ImageJ) softwares were used to crop and scale the
446 datasets. 3D reconstructions and analysis were performed using Imaris.

447
448 QUANTIFICATION AND STATISTICAL ANALYSIS

449 Bioinformatics Analyses of bulk RNA sequences

450 FASTQ files were mapped to the ENSEMBL Mouse GRCm38/mm10 reference using Hisat2
451 and counted by featureCounts from the Subread R package. Due to a high number of
452 duplicates (on average 89%), duplicates were excluded. Flags were computed from counts
453 normalized to the mean coverage. All normalized counts < 20 were considered as background
454 (flag 0) and ≥ 20 as signal (flag = 1). P50 lists used for the statistical analysis gather genes
455 showing flag=1 for at least half of the samples. For the analysis of gene expression, normalized
456 read counts below 250 were considered as background, based on known markers included or
457 excluded from the micro-dissected tissue (Fig. S1B-D). Differential gene expression analysis
458 was performed using three independent methods (DESeq2, edgeR and LimmaVoom) followed
459 by thresholds on absolute fold change ≥1.2 and p-value ≤0.05, leading to 597 genes (Table S1).
460 Differential gene expression with LimmaVoom is plotted, and grouped in three categories
461 depending on p-value and corrected p-value (Benjamini-Hochberg procedure). For each
462 differential analysis method, functional analyses were carried out using the Ingenuity Pathway
463 analysis on the list of differentially expressed genes, using delta between conditions and p-
464 value of differential analysis. Transcriptomic data have been deposited in NCBI Gene
465 Expression Omnibus (GEO) with the accession number GSE237126.

466 Bioinformatics Analyses of published single cell RNA sequences

467 Data from single cardiac wild-type cells at E8.5 (18) were downloaded from
468 https://crukci.shinyapps.io/heartAtlas/ analysed using scran (64) and visualized using Seurat
469 v3.1.2 (65). Data from single cardiac wild-type cells at E11.5-P9 (66) were downloaded from
470 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE193346, and visualized similarly.
448 QUANTIFICATION AND STATISTICAL ANALYSIS

449 Bioinformatics Analyses of bulk RNA sequences

450 FASTQ files were mapped to the ENSEMBL Mouse GRCm38/mm10 reference using Hisat2
451 and counted by featureCounts from the Subread R package. Due to a high number of
452 duplicates (on average 89%), duplicates were excluded. Flags were computed from counts
453 normalized to the mean coverage. All normalized counts < 20 were considered as background
454 (flag 0) and ≥ 20 as signal (flag = 1). P50 lists used for the statistical analysis gather genes
455 showing flag=1 for at least half of the samples. For the analysis of gene expression, normalized
456 read counts below 250 were considered as background, based on known markers included or
457 excluded from the micro-dissected tissue (Fig. S1B-D). Differential gene expression analysis
458 was performed using three independent methods (DESeq2, edgeR and LimmaVoom) followed
459 by thresholds on absolute fold change ≥1.2 and p-value ≤0.05, leading to 597 genes (Table S1).
460 Differential gene expression with LimmaVoom is plotted, and grouped in three categories
461 depending on p-value and corrected p-value (Benjamini-Hochberg procedure). For each
462 differential analysis method, functional analyses were carried out using the Ingenuity Pathway
463 analysis on the list of differentially expressed genes, using delta between conditions and p-
464 value of differential analysis. Transcriptomic data have been deposited in NCBI Gene
465 Expression Omnibus (GEO) with the accession number GSE237126.

466 Bioinformatics Analyses of published single cell RNA sequences

467 Data from single cardiac wild-type cells at E8.5 (18) were downloaded from
468 https://crukci.shinyapps.io/heartAtlas/ analysed using scran (64) and visualized using Seurat
469 v3.1.2 (65). Data from single cardiac wild-type cells at E11.5-P9 (66) were downloaded from
470 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE193346, and visualized similarly.
471 Quantification of RNAscope ISH signal

472 3D images were analyzed using Imaris. For quantification of left-right asymmetry in the
473 second heart field, the second heart field was manually segmented in the lateral plate
474 mesoderm layer, using headfolds and the second somite pair as cranial and caudal boundaries,
475 respectively, and separated into left and right following the midline (using neural tube closure
476 as a landmark). Both Hoechst signal and the signal of the gene of interest were extracted. To
477 account for potential biases between the left and right during imaging, the spot detector
478 function was used on the Hoechst channel to generate a normalisation score between the two
479 sides, using as a threshold a coverage of 50% of spots on each side. To calculate the left/right
480 ratio of the gene of interest, its number of spots were measured on the left and right sides,
481 normalised to Hoechst signal and to the volume of the segmented region.

482 Quantification of the geometry of the heart tube at E9.5

483 HREM images were used to segment the different compartments of the heart tube in
484 Imaris. Eight landmarks along the tube were extracted and used for quantifications as
485 described previously (12) : one at the exit of the outflow tract, one at the sulcus between the
486 two ventricles, and one at the bifurcation of the two atria (taken as the venous pole). The five
487 other landmarks are centroids of cardiac regions: outflow tract, right ventricle, left ventricle,
488 atrioventricular canal and left atrium, and right atrium, segmented according to the expression
489 patterns of Wnt11 and Bmp2 and anatomical landmarks such as cushion boundaries or the
490 interventricular or interatrial sulcus. Heart shapes were aligned in 3D using an in-house
491 MATLAB code so that the Z-axis corresponds to the notochord axis and the x-axis to a
492 perpendicular dorso-ventral axis. The orientation of the axis between the left and right
471 **Quantification of RNAscope ISH signal**

472 3D images were analyzed using Imaris. For quantification of left-right asymmetry in the
473 second heart field, the second heart field was manually segmented in the lateral plate
474 mesoderm layer, using headfolds and the second somite pair as cranial and caudal boundaries,
475 respectively, and separated into left and right following the midline (using neural tube closure
476 as a landmark). Both Hoechst signal and the signal of the gene of interest were extracted. To
477 account for potential biases between the left and right during imaging, the spot detector
478 function was used on the Hoechst channel to generate a normalisation score between the two
479 sides, using as a threshold a coverage of 50% of spots on each side. To calculate the left/right
480 ratio of the gene of interest, its number of spots were measured on the left and right sides,
481 normalised to Hoechst signal and to the volume of the segmented region.

482 **Quantification of the geometry of the heart tube at E9.5**

483 HREM images were used to segment the different compartments of the heart tube in
484 Imaris. Eight landmarks along the tube were extracted and used for quantifications as
485 described previously (12) : one at the exit of the outflow tract, one at the sulcus between the
486 two ventricles, and one at the bifurcation of the two atria (taken as the venous pole). The five
487 other landmarks are centroids of cardiac regions: outflow tract, right ventricle, left ventricle,
488 atrioventricular canal and left atrium, and right atrium, segmented according to the expression
489 patterns of *Wnt11* and *Bmp2* and anatomical landmarks such as cushion boundaries or the
490 interventricular or interatrial sulcus. Heart shapes were aligned in 3D using an in-house
491 MATLAB code so that the Z-axis corresponds to the notochord axis and the x-axis to a
492 perpendicular dorso-ventral axis. The orientation of the axis between the left and right
493 ventricles, the distance of the venous pole to the notochord and the tube length were
494 measured as in (17).

495 **Phenotyping of congenital heart defects**

496 Hearts of *Notch3* mouse mutants were phenotyped at P0 in 3D images acquired by HREM,
497 based on the segmental approach (67) and IPCCC ICD-11 clinical code.

498 **Statistical Analyses**

499 Statistical tests and p-values are described in figure legends and Table S3. Group allocation
500 was based on PCR genotyping. All sample numbers (n) indicated in the text refer to biological
501 replicates, i.e. different embryos or different cells. Investigators were blinded to allocation
502 during imaging and phenotypic analysis, but not during quantifications. Pairwise Mann-
503 Whitney-Wilcoxon tests were used to compare experimental groups, with a Benjamini-
504 Hochberg correction applied on p-values when more than 2 populations are compared. A chi-
505 square test was used to compare percentage distributions, or a chi-square test with Yates'
506 continuity correction for comparison with an expected value. Tests were performed with R and
507 Excel. Embryos which had been damaged during experiments were excluded for
508 quantification.

509 **Acknowledgements**

510 We thank V. Benhamo, L. Guillemot, M. Bertrand, M. Cavaignac, M. Franco, J. Terret, N. Agueeff
511 for generous technical assistance; L. Bally-Cuif, H. Marlow, P. Seymour, N. Kurpios and S. Fre for
512 insightful discussions; A. Joutel for providing *Notch3*<sup>tm1Grid</sup> mice; D. Conrozet, S. Ameur and the
513 histology platform of the SFR Necker; the cell imaging platform; C. Bole-Feysot, M. Parisot and
493 ventricles, the distance of the venous pole to the notochord and the tube length were
494 measured as in (17).

495 **Phenotyping of congenital heart defects**

496 Hearts of *Notch3* mouse mutants were phenotyped at P0 in 3D images acquired by HREM,
497 based on the segmental approach (67) and IPCCC ICD-11 clinical code.

498 **Statistical Analyses**

499 Statistical tests and p-values are described in figure legends and Table S3. Group allocation
500 was based on PCR genotyping. All sample numbers (n) indicated in the text refer to biological
501 replicates, i.e. different embryos or different cells. Investigators were blinded to allocation
502 during imaging and phenotypic analysis, but not during quantifications. Pairwise Mann-
503 Whitney-Wilcoxon tests were used to compare experimental groups, with a Benjamini-
504 Hochberg correction applied on p-values when more than 2 populations are compared. A chi-
505 square test was used to compare percentage distributions, or a chi-square test with Yates'
506 continuity correction for comparison with an expected value. Tests were performed with R and
507 Excel. Embryos which had been damaged during experiments were excluded for
508 quantification.

509 **Acknowledgements**

510 We thank V. Benhamo, L. Guillemot, M. Bertrand, M. Cavaignac, M. Franco, J. Terret, N. Agueeff
511 for generous technical assistance; L. Bally-Cuif, H. Marlow, P. Seymour, N. Kurpios and S. Fre for
512 insightful discussions; A. Joutel for providing *Notch3*<sup>tm1Grid</sup> mice; D. Conrozet, S. Ameur and the
513 histology platform of the SFR Necker; the cell imaging platform; C. Bole-Feysot, M. Parisot and
514 M. Zarhrate of the genomics platform; N. Cagnard of the bioinformatics platform; the LEAT animal facility and H. Varet of the Pasteur Bioinformatics and Biostatistics Hub.

516

517

518 Author Contributions

519 Conceptualization, S.M.M., A.D., T.H.B.; Formal Analysis, T.H.B., A.D., M-A.C., E.P.; Funding

520 Acquisition, S.M.M.; Investigation, T.H.B., A.D., M-A.C.; Methodology, T.H.B., A.D., E.P., S.M.M.;

521 Project Administration, S.M.M.; Software, T.H.B., E.P. ; Supervision, S.M.M., A.D., T.H.B.;

522 Visualization T.H.B., A.D., M-A.C., E.P.; Writing – Original Draft, S.M.M. ; Writing – Review & Editing, all authors;

524

525 Additional information

526 Funding. This work was supported by core funding from the Institut Pasteur and INSERM, state

527 funding from the Agence Nationale de la Recherche under ‘‘Investissements d’avenir’’ program

528 (ANR-10-IAHU-01, ANR-10-LABX-73-01 REVIVE), the Philanthropy Department of Mutuelles

529 AXA through the Head and Heart Chair, a grant from the ANR (ANR-18-CE15-0025-03) and the

530 MSD-Avenir fund (Devo-Decode project) to S.M.M. T.H.B. was supported by the Pasteur - Paris

531 University (PPU) International PhD Program and the FRM, M-A.C. is a student from the FIRE

532 PhD program funded by the Bettencourt Schueller foundation and the EURIP graduate

533 program (ANR-17-EURE-0012). A.D. is an INSERM researcher and S.M.M. an INSERM research director.

535
514 M. Zarhrate of the genomics platform; N. Cagnard of the bioinformatics platform; the LEAT animal facility and H. Varet of the Pasteur Bioinformatics and Biostatistics Hub.

516

517

518 Author Contributions

519 Conceptualization, S.M.M., A.D., T.H.B.; Formal Analysis, T.H.B., A.D., M-A.C., E.P.; Funding

520 Acquisition, S.M.M.; Investigation, T.H.B., A.D., M-A.C.; Methodology, T.H.B., A.D., E.P., S.M.M.;

521 Project Administration, S.M.M.; Software, T.H.B., E.P. ; Supervision, S.M.M., A.D., T.H.B.;

522 Visualization T.H.B., A.D., M-A.C., E.P.; Writing – Original Draft, S.M.M. ; Writing – Review & Editing, all authors;

524

525 Additional information

526 Funding. This work was supported by core funding from the Institut Pasteur and INSERM, state

527 funding from the Agence Nationale de la Recherche under ‘‘Investissements d’avenir’’ program

528 (ANR-10-IAHU-01, ANR-10-LABX-73-01 REVIVE), the Philanthropy Department of Mutuelles

529 AXA through the Head and Heart Chair, a grant from the ANR (ANR-18-CE15-0025-03) and the

530 MSD-Avenir fund (Devo-Decode project) to S.M.M. T.H.B. was supported by the Pasteur - Paris

531 University (PPU) International PhD Program and the FRM, M-A.C. is a student from the FIRE

532 PhD program funded by the Bettencourt Schueller foundation and the EURIP graduate

533 program (ANR-17-EURE-0012). A.D. is an INSERM researcher and S.M.M. an INSERM research director.

535
536 **References**

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539 2. Meno C, Saijoh Y, Fujii H, Ikeda M, Yokoyama T, Yokoyama M, et al. Left-right asymmetric expression of the TGFβ-family member lefty in mouse embryos. Nature. 1996 May;381(6578):151-5.

542 3. Lin AE, Krikov S, Riehle-Colarusso T, Frías JL, Belmont J, Anderka M, et al. Laterality defects in the national birth defects prevention study (1998-2007): Birth prevalence and descriptive epidemiology. Am J Med Genet A. 2014;164(10):2581-91.

545 4. Hamada H. Molecular and cellular basis of left-right asymmetry in vertebrates. Proc Jpn Acad Ser B Phys Biol Sci. 2020;96(7):273-96.

547 5. Nonaka S, Tanaka Y, Okada Y, Takeda S, Harada A, Kanai Y, et al. Randomization of Left-Right Asymmetry due to Loss of Nodal Cilia Generating Leftward Flow of Extraembryonic Fluid in Mice Lacking KIF3B Motor Protein. Cell. 1998 Dec 11;95(6):829-37.

550 6. Brennan J, Norris DP, Robertson EJ. Nodal activity in the node governs left-right asymmetry. Genes Dev. 2002 Sep 15;16(18):2339-44.

552 7. McGrath J, Somlo S, Makova S, Tian X, Brueckner M. Two Populations of Node Monocilia Initiate Left-Right Asymmetry in the Mouse. Cell. 2003 Jul 11;114(1):61-73.

554 8. Katoh TA, Omori T, Mizuno K, Sai X, Minegishi K, Ikawa Y, et al. Immotile cilia mechanically sense the direction of fluid flow for left-right determination. Science. 2023 Jan 6;379(6627):66-71.

557 9. Guimier A, Gabriel GC, Bajolle F, Tsang M, Liu H, Noll A, et al. MMP21 is mutated in human heterotaxy and is required for normal left-right asymmetry in vertebrates. Nat Genet. 2015 Nov;47(11):1260-3.

560 10. Szenker-Ravi E, Ott T, Khatoo M, Moreau de Bellaing A, Goh WX, Chong YL, et al. Discovery of a genetic module essential for assigning left-right asymmetry in humans and ancestral vertebrates. Nat Genet. 2022 Jan;54(1):62-72.

563 11. Sanketi BD, Zuela-Sopilniak N, Bundschuh E, Gopal S, Hu S, Long J, et al. Pitx2 patterns an accelerator-brake mechanical feedback through latent TGFβ to rotate the gut. Science. 2022 Sep 23;377(6613):eabl3921.

566 12. Desgrange A, Le Garrec JF, Bernheim S, Bønnelykke TH, Meilhac SM. Transient Nodal Signaling in Left Precursors Coordinates Opposed Asymmetries Shaping the Heart Loop. Dev Cell. 2020 Nov 23;55(4):413-431.e6.

569 13. Lowe LA, Yamada S, Kuehn MR. Genetic dissection of nodal function in patterning the mouse embryo. Development. 2001 May 15;128(10):1831-43.

571 14. Saijoh Y, Oki S, Ohishi S, Hamada H. Left-right patterning of the mouse lateral plate requires nodal produced in the node. Dev Biol. 2003 Apr 1;256(1):161-73.
536 References

537 1. Hummel KP, Chapman DB. Visceral Inversion and Associated Anomalies in the Mouse. J Hered. 1959 Jan 1;50(1):9-13.

539 2. Meno C, Saijoh Y, Fujii H, Ikeda M, Yokoyama T, Yokoyama M, et al. Left-right asymmetric expression of the TGFβ-family member lefty in mouse embryos. Nature. 1996 May;381(6578):151-5.

542 3. Lin AE, Krikov S, Riehle-Colarusso T, Frías JL, Belmont J, Anderka M, et al. Laterality defects in the national birth defects prevention study (1998-2007): Birth prevalence and descriptive epidemiology. Am J Med Genet A. 2014;164(10):2581-91.

545 4. Hamada H. Molecular and cellular basis of left-right asymmetry in vertebrates. Proc Jpn Acad Ser B Phys Biol Sci. 2020;96(7):273-96.

547 5. Nonaka S, Tanaka Y, Okada Y, Takeda S, Harada A, Kanai Y, et al. Randomization of Left-Right Asymmetry due to Loss of Nodal Cilia Generating Leftward Flow of Extraembryonic Fluid in Mice Lacking KIF3B Motor Protein. Cell. 1998 Dec 11;95(6):829-37.

550 6. Brennan J, Norris DP, Robertson EJ. Nodal activity in the node governs left-right asymmetry. Genes Dev. 2002 Sep 15;16(18):2339-44.

552 7. McGrath J, Somlo S, Makova S, Tian X, Brueckner M. Two Populations of Node Monocilia Initiate Left-Right Asymmetry in the Mouse. Cell. 2003 Jul 11;114(1):61-73.

554 8. Katoh TA, Omori T, Mizuno K, Sai X, Minegishi K, Ikawa Y, et al. Immotile cilia mechanically sense the direction of fluid flow for left-right determination. Science. 2023 Jan 6;379(6627):66-71.

557 9. Guimier A, Gabriel GC, Bajolle F, Tsang M, Liu H, Noll A, et al. MMP21 is mutated in human heterotaxy and is required for normal left-right asymmetry in vertebrates. Nat Genet. 2015 Nov;47(11):1260-3.

560 10. Szenker-Ravi E, Ott T, Khatoo M, Moreau de Bellaing A, Goh WX, Chong YL, et al. Discovery of a genetic module essential for assigning left-right asymmetry in humans and ancestral vertebrates. Nat Genet. 2022 Jan;54(1):62-72.

563 11. Sanketi BD, Zuela-Sopilniak N, Bundschuh E, Gopal S, Hu S, Long J, et al. Pitx2 patterns an accelerator-brake mechanical feedback through latent TGFβ to rotate the gut. Science. 2022 Sep 23;377(6613):eabl3921.

566 12. Desgrange A, Le Garrec JF, Bernheim S, Bønnelykke TH, Meilhac SM. Transient Nodal Signaling in Left Precursors Coordinates Opposed Asymmetries Shaping the Heart Loop. Dev Cell. 2020 Nov 23;55(4):413-431.e6.

569 13. Lowe LA, Yamada S, Kuehn MR. Genetic dissection of nodal function in patterning the mouse embryo. Development. 2001 May 15;128(10):1831-43.

571 14. Saijoh Y, Oki S, Ohishi S, Hamada H. Left-right patterning of the mouse lateral plate requires nodal produced in the node. Dev Biol. 2003 Apr 1;256(1):161-73.
573

574

575

15. Kumar A, Lualdi M, Lewandoski M, Kuehn MR. Broad mesodermal and endodermal deletion of Nodal at postgastrulation stages results solely in left/right axial defects. *Dev Dyn.* 2008;**237**(12):3591-601.

576

577

16. Desgrange A, Le Garrec JF, Meilhac SM. Left-right asymmetry in heart development and disease: forming the right loop. *Dev Camb Engl. 2018 Nov 22;145(22):dev162776.*

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579

580

17. Le Garrec JF, Domínguez JN, Desgrange A, Ivanovitch KD, Raphaël E, Bangham JA, et al. A predictive model of asymmetric morphogenesis from 3D reconstructions of mouse heart looping dynamics. *eLife.* 2017 Nov 28;6:e28951.

581

582

583

18. Tyser RCV, Ibarra-Soria X, McDole K, Arcot Jayaram S, Godwin J, van den Brand TAH, et al. Characterization of a common progenitor pool of the epicardium and myocardium. *Science.* 2021 Mar 5;**371**(6533):eabb2986.

584

585

586

19. Domínguez JN, Meilhac SM, Bland YS, Buckingham ME, Brown NA. Asymmetric fate of the posterior part of the second heart field results in unexpected left/right contributions to both poles of the heart. *Circ Res.* 2012 Oct 26;111(10):1323-35.

587

588

20. Chang H, Zwijsen A, Vogel H, Huylebroeck D, Matzuk MM. Smad5 Is Essential for Left-Right Asymmetry in Mice. *Dev Biol.* 2000 Mar 1;**219**(1):71-8.

589

590

591

21. Mine N, Anderson RM, Klingensmith J. BMP antagonism is required in both the node and lateral plate mesoderm for mammalian left-right axis establishment. *Development.* 2008 Jul 15;**135**(14):2425-34.

592

593

594

22. Furtado MB, Solloway MJ, Jones VJ, Costa MW, Biben C, Wolstein O, et al. BMP/SMAD1 signaling sets a threshold for the left/right pathway in lateral plate mesoderm and limits availability of SMAD4. *Genes Dev.* 2008 Nov 1;**22**(21):3037-49.

595

596

23. Tsiairis CD, McMahon AP. An Hh-Dependent Pathway in Lateral Plate Mesoderm Enables the Generation of Left/Right Asymmetry. *Curr Biol.* 2009 Dec 1;**19**(22):1912-7.

597

598

599

24. Przemeck GKH, Heinzmann U, Beckers J, Hrabé de Angelis M. Node and midline defects are associated with left-right development in *Delta1* mutant embryos. *Development.* 2003 Jan 1;**130**(1):3-13.

600

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602

25. Krebs LT, Iwai N, Nonaka S, Welsh IC, Lan Y, Jiang R, et al. Notch signaling regulates left-right asymmetry determination by inducing Nodal expression. *Genes Dev.* 2003 May 15;**17**(10):1207-12.

603

604

605

26. Raya Á, Kawakami Y, Rodríguez-Esteban C, Büscher D, Koth CM, Itoh T, et al. Notch activity induces Nodal expression and mediates the establishment of left-right asymmetry in vertebrate embryos. *Genes Dev.* 2003 May 15;**17**(10):1213-8.

606

607

608

27. Nakaya M aki, Biris K, Tsukiyama T, Jaime S, Rawls JA, Yamaguchi TP. Wnt3alinks left-right determination with segmentation and anteroposterior axis elongation. *Development.* 2005 Dec 15;**132**(24):5425-36.

609

610

28. Kitajima K, Oki S, Ohkawa Y, Sumi T, Meno C. Wnt signaling regulates left-right axis formation in the node of mouse embryos. *Dev Biol.* 2013 Aug 15;**380**(2):222-32.
573

15. Kumar A, Lualdi M, Lewandoski M, Kuehn MR. Broad mesodermal and endodermal

574

deletion of Nodal at postgastrulation stages results solely in left/right axial defects. Dev

575

Dyn. 2008;237(12):3591-601.

576

16. Desgrange A, Le Garrec JF, Meilhac SM. Left-right asymmetry in heart development and

577

disease: forming the right loop. Dev Camb Engl. 2018 Nov 22;145(22):dev162776.

578

17. Le Garrec JF, Domínguez JN, Desgrange A, Ivanovitch KD, Raphaël E, Bangham JA, et al. A

579

predictive model of asymmetric morphogenesis from 3D reconstructions of mouse heart

580

looping dynamics. eLife. 2017 Nov 28;6:e28951.

581

18. Tyser RCV, Ibarra-Soria X, McDole K, Arcot Jayaram S, Godwin J, van den Brand TAH, et al.

582

Characterization of a common progenitor pool of the epicardium and myocardium.

583

Science. 2021 Mar 5;371(6533):eabb2986.

584

19. Domínguez JN, Meilhac SM, Bland YS, Buckingham ME, Brown NA. Asymmetric fate of

585

the posterior part of the second heart field results in unexpected left/right contributions

586

to both poles of the heart. Circ Res. 2012 Oct 26;111(10):1323-35.

587

20. Chang H, Zwijsen A, Vogel H, Huylebroeck D, Matzuk MM. Smad5 Is Essential for Left-

588

Right Asymmetry in Mice. Dev Biol. 2000 Mar 1;219(1):71-8.

589

21. Mine N, Anderson RM, Klingensmith J. BMP antagonism is required in both the node and

590

lateral plate mesoderm for mammalian left-right axis establishment. Development. 2008

591

Jul 15;135(14):2425-34.

592

22. Furtado MB, Solloway MJ, Jones VJ, Costa MW, Biben C, Wolstein O, et al. BMP/SMAD1

593

signaling sets a threshold for the left/right pathway in lateral plate mesoderm and limits

594

availability of SMAD4. Genes Dev. 2008 Nov 1;22(21):3037-49.

595

23. Tsiairis CD, McMahon AP. An Hh-Dependent Pathway in Lateral Plate Mesoderm Enables

596

the Generation of Left/Right Asymmetry. Curr Biol. 2009 Dec 1;19(22):1912-7.

597

24. Przemeck GKH, Heinzmann U, Beckers J, Hrabé de Angelis M. Node and midline defects

598

are associated with left-right development in Delta1 mutant embryos. Development.

599

2003 Jan 1;130(1):3-13.

600

25. Krebs LT, Iwai N, Nonaka S, Welsh IC, Lan Y, Jiang R, et al. Notch signaling regulates left-

601

right asymmetry determination by inducing Nodal expression. Genes Dev. 2003 May

602

15;17(10):1207-12.

603

26. Raya Á, Kawakami Y, Rodríguez-Esteban C, Büscher D, Koth CM, Itoh T, et al. Notch

604

activity induces Nodal expression and mediates the establishment of left-right

605

asymmetry in vertebrate embryos. Genes Dev. 2003 May 15;17(10):1213-8.

606

27. Nakaya M aki, Biris K, Tsukiyama T, Jaime S, Rawls JA, Yamaguchi TP. Wnt3alinks left-right

607

determination with segmentation and anteroposterior axis elongation. Development.

608

2005 Dec 15;132(24):5425-36.

609

28. Kitajima K, Oki S, Ohkawa Y, Sumi T, Meno C. Wnt signaling regulates left-right axis

610

formation in the node of mouse embryos. Dev Biol. 2013 Aug 15;380(2):222-32.
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614 30. MacGrogan D, Münch J, de la Pompa JL. Notch and interacting signalling pathways in cardiac development, disease, and regeneration. Nat Rev Cardiol. 2018 Nov;15(11):685-704.

617 31. Williams R, Lendahl U, Lardelli M. Complementary and combinatorial patterns of *Notch* gene family expression during early mouse development. Mech Dev. 1995 Nov 1;53(3):357-68.

620 32. Klaus A, Müller M, Schulz H, Saga Y, Martin JF, Birchmeier W. Wnt/β-catenin and Bmp signals control distinct sets of transcription factors in cardiac progenitor cells. Proc Natl Acad Sci. 2012 Jul 3;109(27):10921-6.

623 33. Kwon C, Qian L, Cheng P, Nigam V, Arnold J, Srivastava D. A Regulatory Pathway Involving Notch1/β-Catenin/Isl1 Determines Cardiac Progenitor Cell Fate. Nat Cell Biol. 2009 Aug;11(8):951-7.

626 34. Bardot ES, Jadhav B, Wickramasinghe N, Rezza A, Rendl M, Sharp AJ, et al. Notch Signaling Commits Mesoderm to the Cardiac Lineage [Internet]. bioRxiv; 2020 [cited 2024 Feb 21]. p. 2020.02.20.958348. Available from: https://www.biorxiv.org/content/10.1101/2020.02.20.958348v1

630 35. Del Monte-Nieto G, Ramialison M, Adam AAS, Wu B, Aharonov A, D'Uva G, et al. Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation. Nature. 2018 May;557(7705):439-45.

633 36. Timmerman LA, Grego-Bessa J, Raya A, Bertrán E, Pérez-Pomares JM, Díez J, et al. Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation. Genes Dev. 2004 Jan 1;18(1):99-115.

636 37. MacGrogan D, D'Amato G, Travisano S, Martinez-Poveda B, Luxán G, del Monte-Nieto G, et al. Sequential Ligand-Dependent Notch Signaling Activation Regulates Valve Primordium Formation and Morphogenesis. Circ Res. 2016 May 13;118(10):1480-97.

639 38. Krebs LT, Xue Y, Norton CR, Sundberg JP, Beatus P, Lendahl U, et al. Characterization of Notch3-deficient mice: normal embryonic development and absence of genetic interactions with a Notch1 mutation. Genesis. 2003 Nov;37(3):139-43.

642 39. Volz KS, Jacobs AH, Chen HI, Poduri A, McKay AS, Riordan DP, et al. Pericytes are progenitors for coronary artery smooth muscle. eLife. 2015 Oct 19;4:e10036.

644 40. Domenga V, Fardoux P, Lacombe P, Monet M, Maciazek J, Krebs LT, et al. Notch3 is required for arterial identity and maturation of vascular smooth muscle cells. Genes Dev. 2004 Nov 15;18(22):2730-5.

647 41. Wang Q, Zhao N, Kennard S, Lilly B. Notch2 and Notch3 function together to regulate vascular smooth muscle development. PloS One. 2012;7(5):e37365.
611 29. Minegishi K, Hashimoto M, Ajima R, Takaoka K, Shinohara K, Ikawa Y, et al. A Wnt5 Activity Asymmetry and Intercellular Signaling via PCP Proteins Polarize Node Cells for Left-Right Symmetry Breaking. Dev Cell. 2017 Mar 13;40(5):439-452.e4.

614 30. MacGrogan D, Münch J, de la Pompa JL. Notch and interacting signalling pathways in cardiac development, disease, and regeneration. Nat Rev Cardiol. 2018 Nov;15(11):685-704.

617 31. Williams R, Lendahl U, Lardelli M. Complementary and combinatorial patterns of *Notch* gene family expression during early mouse development. Mech Dev. 1995 Nov 1;53(3):357-68.

620 32. Klaus A, Müller M, Schulz H, Saga Y, Martin JF, Birchmeier W. Wnt/β-catenin and Bmp signals control distinct sets of transcription factors in cardiac progenitor cells. Proc Natl Acad Sci. 2012 Jul 3;109(27):10921-6.

623 33. Kwon C, Qian L, Cheng P, Nigam V, Arnold J, Srivastava D. A Regulatory Pathway Involving Notch1/β-Catenin/Isl1 Determines Cardiac Progenitor Cell Fate. Nat Cell Biol. 2009 Aug;11(8):951-7.

626 34. Bardot ES, Jadhav B, Wickramasinghe N, Rezza A, Rendl M, Sharp AJ, et al. Notch Signaling Commits Mesoderm to the Cardiac Lineage [Internet]. bioRxiv; 2020 [cited 2024 Feb 21]. p. 2020.02.20.958348. Available from: https://www.biorxiv.org/content/10.1101/2020.02.20.958348v1

630 35. Del Monte-Nieto G, Ramialison M, Adam AAS, Wu B, Aharonov A, D'Uva G, et al. Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation. Nature. 2018 May;557(7705):439-45.

633 36. Timmerman LA, Grego-Bessa J, Raya A, Bertrán E, Pérez-Pomares JM, Díez J, et al. Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation. Genes Dev. 2004 Jan 1;18(1):99-115.

636 37. MacGrogan D, D'Amato G, Travisano S, Martinez-Poveda B, Luxán G, del Monte-Nieto G, et al. Sequential Ligand-Dependent Notch Signaling Activation Regulates Valve Primordium Formation and Morphogenesis. Circ Res. 2016 May 13;118(10):1480-97.

639 38. Krebs LT, Xue Y, Norton CR, Sundberg JP, Beatus P, Lendahl U, et al. Characterization of Notch3-deficient mice: normal embryonic development and absence of genetic interactions with a Notch1 mutation. Genesis. 2003 Nov;37(3):139-43.

642 39. Volz KS, Jacobs AH, Chen HI, Poduri A, McKay AS, Riordan DP, et al. Pericytes are progenitors for coronary artery smooth muscle. eLife. 2015 Oct 19;4:e10036.

644 40. Domenga V, Fardoux P, Lacombe P, Monet M, Maciazek J, Krebs LT, et al. Notch3 is required for arterial identity and maturation of vascular smooth muscle cells. Genes Dev. 2004 Nov 15;18(22):2730-5.

647 41. Wang Q, Zhao N, Kennard S, Lilly B. Notch2 and Notch3 function together to regulate vascular smooth muscle development. PloS One. 2012;7(5):e37365.
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653 43. Joutel A, Vahedi K, Corpechot C, Troesch A, Chabriat H, Vayssière C, et al. Strong clustering and stereotyped nature of Notch3 mutations in CADASIL patients. Lancet Lond 654 Engl. 1997 Nov 22;350(9090):1511-5. 655

656 44. Haffner C, Malik R, Dichgans M. Genetic factors in cerebral small vessel disease and their impact on stroke and dementia. J Cereb Blood Flow Metab Off J Int Soc Cereb Blood Flow 657 658 Metab. 2016 Jan;36(1):158-71.

659 45. Kitamoto T, Hanaoka K. Notch3 Null Mutation in Mice Causes Muscle Hyperplasia by Repetitive Muscle Regeneration. Stem Cells. 2010 Dec 1;28(12):2205-16. 660

661 46. Lafkas D, Rodilla V, Huyghe M, Mourao L, Kiaris H, Fre S. Notch3 marks clonogenic mammary luminal progenitor cells in vivo. J Cell Biol. 2013 Oct 7;203(1):47-56. 662

663 47. Kawai H, Kawaguchi D, Kuebrich BD, Kitamoto T, Yamaguchi M, Gotoh Y, et al. Area-Specific Regulation of Quiescent Neural Stem Cells by Notch3 in the Adult Mouse Subependymal Zone. J Neurosci. 2017 Dec 6;37(49):11867-80. 664 665

666 48. Than-Trong E, Ortica-Gatti S, Mella S, Nepal C, Alunni A, Bally-Cuif L. Neural stem cell quiescence and stemness are molecularly distinct outputs of the Notch3 signalling cascade in the vertebrate adult brain. Development. 2018 May 15;145(10):dev161034. 667 668

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671 50. Tyser RCV, Ibarra-Soria X, Pedroza M, Miranda AMA, Brand TAH van den, Scialdone A, et al. Molecular characterization of Left-Right symmetry breaking in the mouse embryo [Internet]. bioRxiv; 2022 [cited 2024 Feb 21]. p. 2022.12.26.521947. Available from: https://www.biorxiv.org/content/10.1101/2022.12.26.521947v1 672 673 674

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679 52. Vincent SD, Norris DP, Ann Le Good J, Constam DB, Robertson EJ. Asymmetric *Nodal* expression in the mouse is governed by the combinatorial activities of two distinct regulatory elements. Mech Dev. 2004 Nov 1;121(11):1403-15. 680 681

682 53. Piedra ME, Icardo JM, Albajar M, Rodriguez-Rey JC, Ros MA. Pitx2 Participates in the Late Phase of the Pathway Controlling Left-Right Asymmetry. Cell. 1998 Aug 7;94(3):319-24. 683

684 54. Saijoh Y, Adachi H, Sakuma R, Yeo CY, Yashiro K, Watanabe M, et al. Left-Right Asymmetric Expression of lefty2 and nodal Is Induced by a Signaling Pathway that Includes the Transcription Factor FAST2. Mol Cell. 2000 Jan 1;5(1):35-47. 685 686
649 42. Sanketi BD, Mantri M, Huang L, Tavallaei MA, Hu S, Wang MFZ, et al. Origin and adult renewal of the gut lacteal musculature from villus myofibroblasts [Internet]. bioRxiv; 650 2024 [cited 2024 Feb 21]. p. 2023.01.19.523242. Available from: 651 https://www.biorxiv.org/content/10.1101/2023.01.19.523242v2 652

653 43. Joutel A, Vahedi K, Corpechot C, Troesch A, Chabriat H, Vayssière C, et al. Strong clustering and stereotyped nature of Notch3 mutations in CADASIL patients. Lancet Lond 654 Engl. 1997 Nov 22;350(9090):1511-5. 655

656 44. Haffner C, Malik R, Dichgans M. Genetic factors in cerebral small vessel disease and their impact on stroke and dementia. J Cereb Blood Flow Metab Off J Int Soc Cereb Blood Flow 657 658 Metab. 2016 Jan;36(1):158-71.

659 45. Kitamoto T, Hanaoka K. Notch3 Null Mutation in Mice Causes Muscle Hyperplasia by Repetitive Muscle Regeneration. Stem Cells. 2010 Dec 1;28(12):2205-16. 660

661 46. Lafkas D, Rodilla V, Huyghe M, Mourao L, Kiaris H, Fre S. Notch3 marks clonogenic mammary luminal progenitor cells in vivo. J Cell Biol. 2013 Oct 7;203(1):47-56. 662

663 47. Kawai H, Kawaguchi D, Kuebrich BD, Kitamoto T, Yamaguchi M, Gotoh Y, et al. Area-Specific Regulation of Quiescent Neural Stem Cells by Notch3 in the Adult Mouse Subependymal Zone. J Neurosci. 2017 Dec 6;37(49):11867-80. 664 665

666 48. Than-Trong E, Ortica-Gatti S, Mella S, Nepal C, Alunni A, Bally-Cuif L. Neural stem cell quiescence and stemness are molecularly distinct outputs of the Notch3 signalling cascade in the vertebrate adult brain. Development. 2018 May 15;145(10):dev161034. 667 668

669 49. Krebs LT, Xue Y, Norton CR, Shutter JR, Maguire M, Sundberg JP, et al. Notch signaling is essential for vascular morphogenesis in mice. Genes Dev. 2000 Jun 1;14(11):1343-52. 670

671 50. Tyser RCV, Ibarra-Soria X, Pedroza M, Miranda AMA, Brand TAH van den, Scialdone A, et al. Molecular characterization of Left-Right symmetry breaking in the mouse embryo [Internet]. bioRxiv; 2022 [cited 2024 Feb 21]. p. 2022.12.26.521947. Available from: https://www.biorxiv.org/content/10.1101/2022.12.26.521947v1 672 673 674

675 51. Yagi H, Cui C, Saydmohammed M, Gabriel G, Baker C, Devine W, et al. Spatial transcriptome profiling uncovers metabolic regulation of left-right patterning [Internet]. 676 bioRxiv; 2023 [cited 2024 Feb 21]. p. 2023.04.21.537827. Available from: 677 https://www.biorxiv.org/content/10.1101/2023.04.21.537827v1 678

679 52. Vincent SD, Norris DP, Ann Le Good J, Constam DB, Robertson EJ. Asymmetric *Nodal* expression in the mouse is governed by the combinatorial activities of two distinct regulatory elements. Mech Dev. 2004 Nov 1;121(11):1403-15. 680 681

682 53. Piedra ME, Icardo JM, Albajar M, Rodriguez-Rey JC, Ros MA. Pitx2 Participates in the Late Phase of the Pathway Controlling Left-Right Asymmetry. Cell. 1998 Aug 7;94(3):319-24. 683

684 54. Saijoh Y, Adachi H, Sakuma R, Yeo CY, Yashiro K, Watanabe M, et al. Left-Right Asymmetric Expression of lefty2 and nodal Is Induced by a Signaling Pathway that Includes the Transcription Factor FAST2. Mol Cell. 2000 Jan 1;5(1):35-47. 685 686
687 55. Liu C, Liu W, Lu MF, Brown NA, Martin JF. Regulation of left-right asymmetry by thresholds of Pitx2c activity. Development. 2001 Jun 1;128(11):2039-48.

689 56. Ammirabile G, Tessari A, Pignataro V, Szumska D, Sutera Sardo F, Benes J Jr, et al. Pitx2 confers left morphological, molecular, and functional identity to the sinus venosus myocardium. Cardiovasc Res. 2012 Feb 1;93(2):291-301.

692 57. Del Gaudio F, Liu D, Andaloussi Mäe M, Braune EB, Hansson EM, Wang QD, et al. Left ventricular hypertrophy and metabolic resetting in the Notch3-deficient adult mouse heart. Sci Rep. 2023 Sep 12;13(1):15022.

695 58. Watanabe K, Nagaoka T, Lee JM, Bianco C, Gonzales M, Castro NP, et al. Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1. J Cell Biol. 2009 Nov 2;187(3):343-53.

698 59. Wells JR, Padua MB, Ware SM. The genetic landscape of cardiovascular left-right patterning defects. Curr Opin Genet Dev. 2022 Aug 1;75:101937.

700 60. Lu CC, Robertson EJ. Multiple roles for *Nodal* in the epiblast of the mouse embryo in the establishment of anterior-posterior patterning. Dev Biol. 2004 Sep 1;273(1):149-59.

702 61. Bernheim S, Borgel A, Garrec JFL, Perthame E, Desgrange A, Michel C, et al. Identification of Greb1l as a genetic determinant of crisscross heart in mice showing torsion of the heart tube by shortage of progenitor cells. Dev Cell. 2023 Nov 6;58(21):2217-2234.e8.

705 62. Susaki EA, Tainaka K, Perrin D, Yukinaga H, Kuno A, Ueda HR. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc. 2015 Nov;10(11):1709-27.

708 63. de Chaumont F, Dallongeville S, Chenouard N, Hervé N, Pop S, Provoost T, et al. Icy: an open bioimage informatics platform for extended reproducible research. Nat Methods. 2012 Jun 28;9(7):690-6.

711 64. Lun ATL, McCarthy DJ, Marioni JC. A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconductor. F1000Research. 2016;5:2122.

713 65. Stuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM, et al. Comprehensive Integration of Single-Cell Data. Cell. 2019 Jun 13;177(7):1888-1902.e21.

715 66. Feng W, Bais A, He H, Rios C, Jiang S, Xu J, et al. Single-cell transcriptomic analysis identifies murine heart molecular features at embryonic and neonatal stages. Nat Commun. 2022 Dec 27;13(1):7960.

718 67. Van Praagh R. The segmental approach to diagnosis in congenital heart disease. Birth Defects Orig Artic Ser. 1972;8:4-23.

720

721
687 55. Liu C, Liu W, Lu MF, Brown NA, Martin JF. Regulation of left-right asymmetry by thresholds of Pitx2c activity. Development. 2001 Jun 1;128(11):2039-48.

689 56. Ammirabile G, Tessari A, Pignataro V, Szumska D, Sutera Sardo F, Benes J Jr, et al. Pitx2 confers left morphological, molecular, and functional identity to the sinus venosus myocardium. Cardiovasc Res. 2012 Feb 1;93(2):291-301.

692 57. Del Gaudio F, Liu D, Andaloussi Mäe M, Braune EB, Hansson EM, Wang QD, et al. Left ventricular hypertrophy and metabolic resetting in the Notch3-deficient adult mouse heart. Sci Rep. 2023 Sep 12;13(1):15022.

695 58. Watanabe K, Nagaoka T, Lee JM, Bianco C, Gonzales M, Castro NP, et al. Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1. J Cell Biol. 2009 Nov 2;187(3):343-53.

698 59. Wells JR, Padua MB, Ware SM. The genetic landscape of cardiovascular left-right patterning defects. Curr Opin Genet Dev. 2022 Aug 1;75:101937.

700 60. Lu CC, Robertson EJ. Multiple roles for *Nodal* in the epiblast of the mouse embryo in the establishment of anterior-posterior patterning. Dev Biol. 2004 Sep 1;273(1):149-59.

702 61. Bernheim S, Borgel A, Garrec JFL, Perthame E, Desgrange A, Michel C, et al. Identification of Greb1l as a genetic determinant of crisscross heart in mice showing torsion of the heart tube by shortage of progenitor cells. Dev Cell. 2023 Nov 6;58(21):2217-2234.e8.

705 62. Susaki EA, Tainaka K, Perrin D, Yukinaga H, Kuno A, Ueda HR. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc. 2015 Nov;10(11):1709-27.

708 63. de Chaumont F, Dallongeville S, Chenouard N, Hervé N, Pop S, Provoost T, et al. Icy: an open bioimage informatics platform for extended reproducible research. Nat Methods. 2012 Jun 28;9(7):690-6.

711 64. Lun ATL, McCarthy DJ, Marioni JC. A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconductor. F1000Research. 2016;5:2122.

713 65. Stuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM, et al. Comprehensive Integration of Single-Cell Data. Cell. 2019 Jun 13;177(7):1888-1902.e21.

715 66. Feng W, Bais A, He H, Rios C, Jiang S, Xu J, et al. Single-cell transcriptomic analysis identifies murine heart molecular features at embryonic and neonatal stages. Nat Commun. 2022 Dec 27;13(1):7960.

718 67. Van Praagh R. The segmental approach to diagnosis in congenital heart disease. Birth Defects Orig Artic Ser. 1972;8:4-23.

720

721
722 **Supporting Information**

723 **Figure S1 related to Fig. 1. Transcriptomic approach. (A)** Brightfield images of wild-type
724 embryos used for RNA sequencing at E8.5f. In the left panel, an outline of the dissected areas
725 is shown. The identification number of embryos is given. **(B)** Normalised read counts of genes
726 used to validate the threshold of expression in the transcriptomic analysis. The osteocyte gene
727 *Dmp1*, inner ear marker *Oc90*, neuronal marker *Neurod1* are used as negative controls and
728 *Mmp9* as a positive control, lowly expressed in left heart progenitors. Whisker plots show the
729 median, 25th- and 75th quartiles (boxes), and the extreme data points (whiskers). **(C)**
730 Normalised read counts of genes used as markers to control sample micro-dissection. *En2*,
731 *Wnt8b* are anterior markers, *Hoxa5*, *Hoxb6* posterior markers, *Rfx4*, *Tfap2b* back markers, *Isl1*,
732 *Six2*, *Fgf8* second heart field markers, *Tbx5*, *Mab21l2* juxta-cardiac field (JCF) markers and
733 *Myh7b*, *Myoz1*, *Tnnt1* cardiomyocyte markers. The dotted line indicates the threshold of
734 background expression. **(D)** Normalised read counts of genes used as markers to validate the
735 left-right dissection of samples. NODAL targets *Lefty2* and *Pitx2*, as well as *Six2* label the left
736 side. *Nodal* is turned off at E8.5f. *p*-value between the left and right sides < 0.05, ***
737 Benjamini-Hochberg corrected *p*-value < 0.00001 (LimmaVoom, n=4). **(E)** Violin plot of *Dtx4*
738 expression in single cells at E8.5 from (18), clustered as annotated (n= 89 Ec2, 59 Me2, 713
739 Me3, 221 Me4, 355 Me5, 65 Me6, 514 Me7). Dots are normalised reads per cell. **(F)** Violin plot
740 of *Notch3* expression in single *Nodal*-negative (n=1559) and *Nodal*-positive (n=309) cells of
741 cardiac clusters (Me3-7) of (18) at E8.5 (stage 1 to Late Heart Tube, wild-type embryos). 75%
742 (234/309) of *Nodal*-positive cells also express *Notch3*.

743 **Figure S2 related to Fig. 2. Normal Nodal expression in Notch3 mutants. (A-B)** Whole
744 mount RNAscope ISH of *Nodal* in *Notch3*⁻/⁻ mutant embryos at E8.5c in anterior (A) and
745 posterior (B) frontal views. The midline of the embryo is indicated by a yellow dotted line. A
722 **Supporting Information**

723 **Figure S1 related to Fig. 1. Transcriptomic approach. (A)** Brightfield images of wild-type
724 embryos used for RNA sequencing at E8.5f. In the left panel, an outline of the dissected areas
725 is shown. The identification number of embryos is given. **(B)** Normalised read counts of genes
726 used to validate the threshold of expression in the transcriptomic analysis. The osteocyte gene
727 *Dmp1*, inner ear marker *Oc90*, neuronal marker *Neurod1* are used as negative controls and
728 *Mmp9* as a positive control, lowly expressed in left heart progenitors. Whisker plots show the
729 median, 25th- and 75th quartiles (boxes), and the extreme data points (whiskers). **(C)**
730 Normalised read counts of genes used as markers to control sample micro-dissection. *En2*,
731 *Wnt8b* are anterior markers, *Hoxa5*, *Hoxb6* posterior markers, *Rfx4*, *Tfap2b* back markers, *Isl1*,
732 *Six2*, *Fgf8* second heart field markers, *Tbx5*, *Mab21l2* juxta-cardiac field (JCF) markers and
733 *Myh7b*, *Myoz1*, *Tnnt1* cardiomyocyte markers. The dotted line indicates the threshold of
734 background expression. **(D)** Normalised read counts of genes used as markers to validate the
735 left-right dissection of samples. NODAL targets *Lefty2* and *Pitx2*, as well as *Six2* label the left
736 side. *Nodal* is turned off at E8.5f. *p*-value between the left and right sides < 0.05, ***
737 Benjamini-Hochberg corrected *p*-value < 0.00001 (LimmaVoom, n=4). **(E)** Violin plot of *Dtx4*
738 expression in single cells at E8.5 from (18), clustered as annotated (n= 89 Ec2, 59 Me2, 713
739 Me3, 221 Me4, 355 Me5, 65 Me6, 514 Me7). Dots are normalised reads per cell. **(F)** Violin plot
740 of *Notch3* expression in single *Nodal*-negative (n=1559) and *Nodal*-positive (n=309) cells of
741 cardiac clusters (Me3-7) of (18) at E8.5 (stage 1 to Late Heart Tube, wild-type embryos). 75%
742 (234/309) of *Nodal*-positive cells also express *Notch3*.

743 **Figure S2 related to Fig. 2. Normal Nodal expression in Notch3 mutants. (A-B)** Whole
744 mount RNAscope ISH of *Nodal* in *Notch3*⁻/⁻ mutant embryos at E8.5c in anterior (A) and
745 posterior (B) frontal views. The midline of the embryo is indicated by a yellow dotted line. A
746 transverse section at the level of the node (see b1) is shown in b2. Filled and empty
747 arrowheads point to high and absent expression, respectively. (C-D) Transverse section of the
748 left lateral plate mesoderm, labelled by double wholemount RNAscope ISH of *Nodal* (red) and
749 *Notch3* (white). The region of *Notch3* and *Nodal* co-expression is outlined in yellow (n=3). L,
750 left; LPM, lateral plate mesoderm; No, node; NT, neural tube; R, right.

751 **Figure S3 related to Fig. 3. Notch3 inactivation in mutants. (A) Schema of *Notch3* alleles in**
752 wild-types and *Notch3*/- mutants, indicating the localisation of exons and primers used for RT-
753 qPCR. (B) Relative expression of *Notch3* in micro-dissected heart fields of littermate wild-types
754 (n=5 E8.5e, 3 E8.5g), *Notch3*+/- (n=6 E8.5e, 6 E8.5g) and *Notch3*/- (n=6 E8.5e, 4 E8.5g) embryos,
755 quantified by RT-qPCR using the indicated primer pairs and normalised to wild-types. **p-
756 value<0.01, ***p-value<0.001 (Pairwise Mann-Whitney Wilcoxon tests with Benjamini-
757 Hochberg correction).

758 **Figure S4 related to Fig. 4. Notch3 expression after heart looping.** Violin plot of *Notch3*
759 expression in single cardiac cell transcriptomic between E11.5 and P9 (from (66)), clustered as
760 annotated (n= 5422 atrial cardiomyocytes, 10493 ventricular cardiomyocytes, 2361
761 endocardium, 1176 vascular endocardium, 1012 epicardium, 3309 fibroblast like cells, 182
762 smooth muscle cells, 349 pericytes, 1032 macrophages, 33 B cells, 42 T cells, 17 dendritic cells,
763 23 natural killer cells, 22 neutrophils, 244 blood cells). Dots are normalised reads per cell.

764 **Video S1 related to Fig. 1. Co-expression of Notch3 and Nodal.** Notch3 (white) and *Nodal*
765 (magenta) double whole-mount RNAscope ISH in a wild-type E8.5d embryo imaged in 3D by
766 lightsheet microscopy.

767 **Table S1 related to Fig. 1.** List of 597 differentially expressed genes used for Ingenuity
768 Pathway analysis, based on a fold change ≥1.2 and p-val ≤0.05 with either the LimmaVoom,
746 transverse section at the level of the node (see b1) is shown in b2. Filled and empty
747 arrowheads point to high and absent expression, respectively. (C-D) Transverse section of the
748 left lateral plate mesoderm, labelled by double wholemount RNAscope ISH of *Nodal* (red) and
749 *Notch3* (white). The region of *Notch3* and *Nodal* co-expression is outlined in yellow (n=3). L,
750 left; LPM, lateral plate mesoderm; No, node; NT, neural tube; R, right.

751 **Figure S3 related to Fig. 3. Notch3 inactivation in mutants. (A) Schema of *Notch3* alleles in**
752 wild-types and *Notch3*/- mutants, indicating the localisation of exons and primers used for RT-
753 qPCR. (B) Relative expression of *Notch3* in micro-dissected heart fields of littermate wild-types
754 (n=5 E8.5e, 3 E8.5g), *Notch3*+/- (n=6 E8.5e, 6 E8.5g) and *Notch3*/- (n=6 E8.5e, 4 E8.5g) embryos,
755 quantified by RT-qPCR using the indicated primer pairs and normalised to wild-types. **p-
756 value<0.01, ***p-value<0.001 (Pairwise Mann-Whitney Wilcoxon tests with Benjamini-
757 Hochberg correction).

758 **Figure S4 related to Fig. 4. Notch3 expression after heart looping.** Violin plot of *Notch3*
759 expression in single cardiac cell transcriptomic between E11.5 and P9 (from (66)), clustered as
760 annotated (n= 5422 atrial cardiomyocytes, 10493 ventricular cardiomyocytes, 2361
761 endocardium, 1176 vascular endocardium, 1012 epicardium, 3309 fibroblast like cells, 182
762 smooth muscle cells, 349 pericytes, 1032 macrophages, 33 B cells, 42 T cells, 17 dendritic cells,
763 23 natural killer cells, 22 neutrophils, 244 blood cells). Dots are normalised reads per cell.

764 **Video S1 related to Fig. 1. Co-expression of Notch3 and Nodal.** Notch3 (white) and *Nodal*
765 (magenta) double whole-mount RNAscope ISH in a wild-type E8.5d embryo imaged in 3D by
766 lightsheet microscopy.

767 **Table S1 related to Fig. 1.** List of 597 differentially expressed genes used for Ingenuity
768 Pathway analysis, based on a fold change ≥1.2 and p-val ≤0.05 with either the LimmaVoom,
769

DESeq2 or edgeR methods. Flags indicate the number of samples in which normalised read

770

counts are ≥ 20 (n=4 embryos). Lf, left heart field at E8.5f; Rf, right heart field at E8.5f.

771

**Table S2. List of primers** used for genotyping and RT-qPCR.

772

**Table S3. Data.** Numerical data used to generate figure graphs along with statistical tests.

773

774
769 DESeq2 or edgeR methods. Flags indicate the number of samples in which normalised read

770 counts are ≥ 20 (n=4 embryos). Lf, left heart field at E8.5f; Rf, right heart field at E8.5f.

771 **Table S2. List of primers** used for genotyping and RT-qPCR.

772 **Table S3. Data.** Numerical data used to generate figure graphs along with statistical tests.

773

774
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_7.png" /></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2024.03.25.586543; this version posted March 28, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

<caption>Figure 3</caption>
A B C

<figure><img src="image_8.png" /></figure>

Coronal section

Transverse section

Initial section

Notch3<sup>+/+</sup>
No phenotype

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

Notch3<sup>-/-</sup>
Congenital heart defects

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<footnote>bioRxiv preprint doi: https://doi.org/10.1101/2024.03.25.586543; this version posted on January 28, 2024. The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint provided that the preprint is made available under a CC-BY 4.0 international license.</footnote>

<footnote>This version posted under a license that permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</footnote>
A B C

<figure><img src="image_8.png" /></figure>

Coronal section

Transverse section

Initial section

Notch3<sup>+/+</sup>
No phenotype

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

Notch3<sup>-/-</sup>
Congenital heart defects

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<footnote>bioRxiv preprint doi: https://doi.org/10.1101/2024.03.25.586543; this version posted on January 28, 2024. The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint provided that the preprint is made available under a CC-BY 4.0 international license.</footnote>

<footnote>This version posted under a license that permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</footnote>
<figure><img src="image_16.png" /><figcaption>Figure 5</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>Figure 5</figcaption></figure>