# Expression analysis of amphioxus orthologues of genes expressed in vertebrate lateral plate or pharyngeal mesoderm

Anaël Soubigou, Lydvina Meister, Lucie Subirana, Lucille Cornand, Hector Escriva, Stéphanie Bertrand

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Anaël Soubigou, Lydvina Meister, Lucie Subirana, Lucille Cornand, Hector Escriva, et al.. Expression analysis of amphioxus orthologues of genes expressed in vertebrate lateral plate or pharyngeal mesoderm. Gene Expression Patterns, 2025, 56, pp.119397. 10.1016/j.gep.2025.119397. hal-05133546

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# Expression analysis of amphioxus orthologues of genes expressed in vertebrate lateral plate or pharyngeal mesoderm

Anaël Soubigou<sup>a</sup>, Lydvina Meister<sup>a</sup>, Lucie Subirana<sup>a</sup>, Lucille Cornand<sup>a</sup>, Hector Escriva<sup>a, *</sup>, Stéphanie Bertrand<sup>a,b,**</sup>

## ARTICLE INFO

Handling editor: M. Roberto

## ABSTRACT

The lateral plate mesoderm of vertebrates, which borders the other mesodermal territories, develops during embryogenesis into a variety of tissues and organs such as blood, heart, vasculature, kidney and smooth muscles. This mesoderm compartment, as well as the unsegmented pharyngeal mesoderm which gives rise to head muscles and part of the heart, have been proposed as vertebrate innovations. Indeed, in the two other chordate clades, the tunicates and the cephalochordates, no such mesoderm regions are formed during development. However, in ascidians, the most studied tunicate group, some cells in the larva which participate to siphon muscles and heart formation are thought to be homologous to the cardiopharyngeal field of vertebrates. Moreover, in the cephalochordate amphioxus, lateral plate and pharyngeal mesoderm marker genes were shown to be expressed in different regions of the fully segmented paraxial mesoderm. In this work, we decided to look at the embryonic expression in amphioxus of several of these mesoderm marker genes, that could give new insights into the putative homology between cephalochordate somite regions and vertebrates’ mesoderm compartments. Here, we describe the expression pattern of *Erg/Fli1a*, *Erg/Fli1b*, *Lmo2*, *Mesp*, *Npas4/4l*, *Osr1/2a*, *Osr1/2b*, *Tcf21/Msc* and *Tcf21/Mscb*. Our results highlight the presence of a putative hematopoietic field in the first somite pair as previously proposed, and suggest that some genes were probably specifically recruited during vertebrate evolution for the development of pharyngeal or lateral plate mesoderm derivatives.

## 1. Introduction

Bilaterian emergence was accompanied by the appearance of a new germ layer, the mesoderm, that forms a variety of tissues and organ, including bilaterian innovations. In vertebrates, the mesoderm, after gastrulation, is subdivided in various territories: (i) the axial notochord in the trunk, (ii) the prechordal plate anterior to the notochord, (iii) the pharyngeal mesoderm in the head, (iv) the paraxial mesoderm that forms the somites in the trunk, and (v) the lateral plate mesoderm also called ventral mesoderm. The latest develops during embryogenesis into a variety of tissues and organs among which the heart and the vascular system, the excretory system, blood cells, some smooth muscles and the limb buds in gnathostomes (Prummel et al., 2020).

Vertebrates belong, together with their sister group the tunicates, and with the cephalochordates (i.e. amphioxus), to the chordate clade

(Bourlat et al., 2006; Delsuc et al., 2006, 2008). The three groups share a common bodyplan characterized by several features among which a dorsal notochord deriving from the axial mesoderm (Annona et al., 2015). In ascidian tunicates, the notochord is located in the larval tail and is flanked by segmentally arranged muscles. However, unlike vertebrates, these muscles do not originate from segmented somites (Lemaire, 2011). Moreover, tunicates do not show any embryonic structure recognized as a putative lateral plate mesoderm homolog. However, in the head/trunk region, the ascidian tadpole possesses cells, the trunk ventral cells (TVC), that develop into the heart of the juvenile and that express orthologues of genes expressed in the lateral plate mesoderm region from which the heart forms in vertebrates (Diogo et al., 2015). In the third group of chordates, the cephalochordates, there are only two mesoderm compartments: the notochord that runs axially all along the body and the paraxial mesoderm which is fully segmented

E-mail addresses: soubigou@obs-banyuls.fr (A. Soubigou), lydvina.meister@gmail.com (L. Meister), subirana@obs-banyuls.fr (L. Subirana), cornand@obs-banyuls.fr (L. Cornand), hescriva@obs-banyuls.fr (H. Escriva), stephanie.bertrand@obs-banyuls.fr (S. Bertrand).

https://doi.org/10.1016/j.gep.2025.119397

Received 9 May 2025; Received in revised form 10 June 2025; Accepted 11 June 2025

Available online 13 June 2025

1567-133X/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

<footnote><sup>a</sup> Sorbonne Université, CNRS, Biologie Intégrative des Organismes Marins, BIOM, F-66650, Banyuls-sur-Mer, France</footnote>

<footnote><sup>b</sup> Institut Universitaire de France, IUF, Paris, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author. Sorbonne Université, CNRS, Biologie Intégrative des Organismes Marins, BIOM, F-66650, Banyuls-sur-Mer, France.</footnote>
into somites (Bertrand and Escriva, 2011). There is hence no unseg-
mented embryonic structure bordering the somites similar to the lateral
plate mesoderm of vertebrates or to the pharyngeal head mesoderm.
However, the amphioxus somites, which are spherical at first, change
their shape during development and elongate towards the ventral region
of the embryo, with the ventralmost cells intercalating between the
epidermis and the endoderm (Kozmik et al., 2001; Mansfield et al.,
2015). It has also been demonstrated that these somites are compart-
mentalized at the molecular level, with a ventral region expressing
orthologues of vertebrate lateral plate mesoderm marker genes (Holland
et al., 2003; Kozmik et al., 2001; Onimaru et al., 2011; Pascual-Anaya
et al., 2013; Yong et al., 2021). Moreover, the vasculature, hemal cells
and the excretory organ of the larva called the Hatschek's nephridium,
were proposed to develop from subregions of the first pair of somites
(Holland, 2017, 2018; Kozmik et al., 1999; Langeland et al., 2006;
Pascual-Anaya et al., 2013). It has also been suggested that the
pharyngeal mesoderm of vertebrates would in part be homologous to the
amphioxus first somite pair (Grau-Bové et al., 2024; Meister et al.,
2022). Hypotheses about the homology between amphioxus somite
parts and vertebrate mesoderm regions primarily rely on gene expres-
sion patterns of orthologous vertebrate mesoderm markers. To bolster
these homology arguments, our study examines the expression of new
vertebrate mesoderm marker genes in amphioxus embryos. Specifically,
we describe the expression of amphioxus orthologues of genes expressed
in vertebrate lateral plate mesoderm or derivatives: *Erg/Fli1*, *Lmo2*,
*Mesp*, *Npas4/4l*, *Osr1/2* and *Tcf21/Msc*.

## 2. Results

2.1. Identification of orthologues

We searched for orthologues of vertebrate *Erg/Fli1*, *Lmo2*, *Mesp*, *Npas4/4l*, *Osr1/2*, *Tcf21/Msc* genes in the genome of *Branchiostoma*

*lanceolatum*. The sequences retrieved were first used to reconstruct phylogenetic trees in order to confirm their orthology relationships (Supplementary Figs. 1–6). We found two genes coding for orthologues of vertebrates *Erg* and *Fli1* (named *Erg/Fli1a* and *Erg/Fli1b*) and two genes coding for orthologues of *Osr1* and *Osr2* (named *Osr1/2a* and *Osr1/2b*), corresponding to specific duplications in the *Branchiostoma* lineage. For *Tcf21/Msc*, the three *Branchiostoma* species included in the study (*B. belcheri*, *B. floridae* and *B. lanceolatum*) have three paralogs. One was already described and is called below *Tcf21/Msc*. The two other genes were named *Tcf21/Msca* and *Tcf21/Mscb*. Concerning *Lmo2*, *Mesp* and *Npas4/4l*, a unique ohnologue was found in available cephalochordate genomic data.

2.2. Expression of *Erg/Fli1a* and *Erg/Fli1b*

The expression of *Erg/Fli1a* is first detected in the anterior somites at the N2 stage (Fig. 1a and b), with a stronger labelling in the two first right somites and the second left somite. At N4, the pattern is similar, with a very faint labelling in the first left somite and a stronger labelling in the right first somite (Fig. 1c and d). Expression is also detected at this stage in the ventral region of the tailbud territory (Fig. 1c and d). In N5 stage embryos, the somitic expression is restricted to subregions of the first left and right somites (Fig. 1e–g). This pattern is very similar to what has been described for *Pdvegfr* and *Scl* at the same stage, both genes being expressed in the Hatschek’s nephridium anlage on the left side and in a region proposed as the presumptive glomus, a richly vascularized and potentially hematopoietic region, on the right side (Pascual-Anaya et al., 2013). At this stage, the expression of *Erg/Fli1a* is still observed posteriorly in the tailbud (Fig. 1e–g). As neurulation proceeds, the expression in the mesoderm stays very similar to what has been described for *Pdvegfr* and *Scl* (Pascual-Anaya et al., 2013), with a posterior expansion of the expression on the right side at T0 (Fig. 1h–j). The tailbud labelling is still observed and expression is detected in a few cells

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

**Fig. 1.** Expression of *Erg/Fli1a* during amphioxus embryogenesis.** Dorsal view with anterior to the left (a) and left side view with dorsal to the top (b) of a N2 stage embryo. Dorsal view with anterior to the left (c) and right side view with dorsal to the top and anterior to the right (d) of a N4 stage embryo. The arrow marks the first right somite in both panels and the double arrow indicates labelling in the ventral region of the tailbud. Dorsal view with anterior to the left (e) and left side views with dorsal to the top (f, g) of a N5 stage embryo. A black arrow indicates the right somite labelled region while a black arrowhead indicates the left labelling in the Hatschek's nephridium anlage. Dorsal view of a T0 stage embryo with anterior to the left (h), and left side view of the anterior region of the same specimen with dorsal to the top (i, j). A black arrow indicates the right mesodermal cells while a black arrowhead indicates the Hatschek's nephridium anlage. The white arrowhead signals the cerebral vesicle. Left side views with dorsal to the top of the anterior (k) and posterior (l) regions of a larva. The double black and white arrowheads indicate labelling in cells of the dorsal aorta and of the subintestinal vessel, respectively. Scale bar: 50 µm.
of the cerebral vesicle (Fig. 1h–j). In the larva, *Erg/Fli1a* is expressed in a few cells in the presumptive subintestinal vessel and dorsal aorta, as previously reported for *Pdvegfr* (Pascual-Anaya et al., 2013), and in the tailbud (Fig. 1k and l).

The expression of *Erg/Fli1b* is very similar to what we observed for *Erg/Fli1a* (Fig. 2). The main difference is that its expression is first detected later, at the N4 stage, and that at this stage the labelling is already strong in both the right and left first somites contrary to *Erg/Fli1a* (Fig. 2a and b). Finally, at the larva stage, the expression in the cerebral vesicle that begins at T0 is still observed only for *Erg/Fli1b* (Fig. 2j).

## 2.3. Expression of *Npas4/4l*, *Lmo2* and *Mesp*

*Npas4/4l* expression was only detected at N4 and N5 stages (Fig. 3a–d), similarly to what has been described previously in *B. floridae* (Li et al., 2014), while *Lmo2* expression could only be observed in a restricted region in N4 embryos (Fig. 3e and f). Interestingly, both genes are expressed in neurula within the subregions of the first left and right somites, which also exhibit expression of *Erg/Fli1a*, *Erg/Fli1b* as well as of *Pdvegfr* and *Scl* (Pascual-Anaya et al., 2013). However, the expression in these specific domains is here only transient and we could not detect expression at earlier or later stages by *in situ* hybridization. Concerning *Mesp*, we only observed expression in the tailbud from T0 stage onwards, in a smaller domain than *Erg/Flia* and *Erg/Flib* (Fig. 3g and h).

## 2.4. Expression of *Osr1/2a* and *Osr1/2b*

Restricted embryonic expression was only detected for *Osr1/2a* (Fig. 4), with no labelling detectable at any of the stages studied for *Osr1/2b*. Among the genes studied in this work, *Osr1/2a* is the first to be expressed and labelling is observed at the N1 stage in the somites that have just formed (Fig. 4a and b). At N2, N3 and N4, *Osr1/2a* is still expressed in the somites, but a new expression domain in the endoderm posterior to the presumptive pharyngeal region is detected (Fig. 4c–h). This pattern persists until the T0 stage, characterized by a sharply defined anterior expression boundary just posterior to the developing pharynx in the endoderm and a progressively weakening signal in the somite domain from which muscles develop (Fig. 4i and j). Finally, no specific expression could be detected at the larva stage.

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Expression of *Npas4/4l* (a–d), *Lmo2* (e, f) and *Mesp* (g, h). Dorsal view with anterior to the left (a) and left side view with dorsal to the top (b) of a N4 stage embryo showing the expression of *Npas4/4l* in the first left (arrowhead) and right (arrow) somites. Equivalent views of a N5 stage embryo showing the expression of *Npas4/4l* in the same domains (c, d). Same views of a N4 stage embryo showing the expression of *Lmo2* in the left (arrowhead) and right (arrow) first somites (e, f). Left side views with dorsal to the top of a T0 stage embryo (g) and of a larva (h) showing the expression of *Mesp* in the tailbud. Scale bar: 50 µm.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Expression of *Erg/Fli1b* during amphioxus embryogenesis. Dorsal view with anterior to the left (a) and left side view with dorsal to the top (b) of a N2 stage embryo. Dorsal view with anterior to the left (c) and left side views with dorsal to the top (d, e) of a N4 stage embryo. The arrow and the arrowhead mark the first right and first left somites in the three panels, respectively. Dorsal views with anterior to the left (f, g) and left side views of the anterior region with dorsal to the top (h, i) of a N5 stage embryo. A black arrow indicates the right somite labelled region while a black arrowhead indicates the left labelling in the Hatschek's nephridium anlage. The cerebral vesicle is indicated by a white arrowhead. Left side views with dorsal to the top of the anterior (j) and posterior (k) regions of a larva. The double black and white arrowheads indicate labelling in cells of the dorsal aorta and of the subintestinal vessel, respectively. Scale bar: 50 µm.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Expression of *Osr1/2a*. Dorsal views with anterior to the left (a, c, e, g and i) and left side views with dorsal to the top (b, d, f, h and j) of embryos at the N1 (a, b), N2 (c, d) and N3 (e, f), N5 (g, h) and T0 (i, j) stages. A black arrow indicates the anterior sharp limit of expression in the endoderm. Scale bar: 50 µm.</figcaption></figure>

## 2.5. Expression of *Tcf21/Msc*

We found, in the three *Branchiostoma* species with a publicly available genome sequence, three genes orthologous to both *Tcf21* and *Msc* of vertebrates. Specific expression could only be detected for the *Tcf21/Msc* gene for which a partial expression pattern was previously described (Grau-Bové et al., 2024) and for *Tcf21/Mscb*. We found that *Tcf21/Msc* is first expressed at the N2 stage in the first somite pair (Fig. 5a and b). This restricted expression is maintained until the T0 stage in the ventral posterior region of these two first somites (Fig. 5c–h). At the larva stage, the expression is detected in the forming Hatschek’s nephridium as well as in some individual cells at the level of the pharynx (Fig. 5i). *Tcf21/Mscb* expression was first detected at the T0 stage in the left first somite (Fig. 5j). At the larva stage, labelling was observed in the Hatschek’s nephridium as well as at a low level in the notochord (Fig. 5l).

# 3. Discussion

## 3.1. Genes coexpressed in the first somite pair

Among the genes studied in this work, five (*Erg/Fli1a*, *Erg/Fli1b*, *Lmo2*, *Npas4/4l*, *Tcf21/Msc*) are expressed at least transiently during neurulation in the first left and right somites, in regions that were proposed to correspond to the Hatschek’s nephridium anlage, the excretory organ of the larva, and to a putative hematopoietic region.

*Erg* and *Fli1* are two vertebrate paralogous genes of the Ets family of transcription factor that are involved in endothelial and hematopoietic development. In zebrafish, *erg* and *fli1* are both expressed in the lateral plate mesoderm and then in the vasculature (Brown et al., 2000; Liu and Patient, 2008). In addition, *fli1* is expressed in hematopoietic regions and in the pharyngeal arches and finbuds (Brown et al., 2000). In mouse, *Erg* and *Fli1* genes exhibit similar expression profiles, also localized in

endothelial tissues, indicating the involvement of these factors in vasculogenesis. Both genes are also expressed in the mesodermal region of the pharyngeal arches, the urogenital tract and the renal mesenchyme (Vlaeminck-Guillem et al., 2000). In *Xenopus*, *Erg* and *Fli1* are expressed in endothelial tissues and hematopoietic cells (Baltzinger et al., 1999; Walmsley et al., 2002). Concerning *Lmo2*, it is expressed in zebrafish, *Xenopus*, chick and mouse in blood island and vasculature, with a pattern similar to *Scl* (Manaia et al., 2000; Mead et al., 2001; Minko et al., 2003; Thompson et al., 1998). *Npas4l* is a bHLH transcription factor gene, paralogous to *Npas4*, which is absent in mammals and was recently isolated as the gene mutated in the zebrafish *cloche* mutant characterized by a loss of endothelial and blood cells (Reischauer et al., 2016). *Npas4l* is expressed early in the zebrafish lateral plate mesoderm, in precursors of blood and vasculature, with a pattern similar to *Scl*, although its expression is detected earlier. In chick, *Npas4l* is also expressed in the lateral mesoderm transiently before *Scl* or *Lmo2* (Wei et al., 2020). On the other hand, *Npas4* was shown to be expressed in the nervous system of zebrafish and mouse (Klarić et al., 2014; Klaric et al., 2014).

*Kdr* (although known as *Vegfr2* or *Flk-1*), orthologous of amphioxus *Pdvegfr*, is a key gene for vascular and blood cells development in vertebrates (*Shalaby* et al., 1995). *Lmo2* interacts with *Scl* (also known as Tal-1) and *Gata* family transcription factors that, together with *Ets* family members such as *Erg*, regulate differentiation, proliferation and survival of hematopoietic cells in vertebrates (*Vagapova* et al., 2018). The co-expression of their amphioxus orthologues in the first somite pair, as described previously (*Pascual-Anaya* et al., 2013) and in this study, reinforces the hypothesis that this embryonic region corresponds to a hematopoietic field. Interestingly, these genes are expressed in the first right somite and in the first left somite, in the region that later forms the Hatschek’s nephridium. This suggests that at least a partial common regulatory network is controlling the development of both excretory

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Expression of *Tcf21/Msc* (a–i) and *Tcf21/Mscb* (j–l). Dorsal views with anterior to the left (a, c, e, g and i) and left side views with dorsal to the top (b, d, f, h, i, k and l) of embryos at the N2 (a, b), N3 (c, d), N4 (e, f), T0 (g, h, j, k) and larva (i, l) stages. At N2, N3, N4 and T0 stages, *Tcf21/Msc* is expressed in the left (arrowhead) and right (arrow) first somites (a–h). At T0, *Tcf21/Mscb* is only expressed in the left first somite (arrowhead) (j–k). At the larva stage, both *Tcf21/Msc* and *Tcf21/Mscb* are expressed in the Hatschek’s nephridium region (arrowhead, i, l) while *Tcf21/Mscb* is also expressed in the notochord (white arrowhead, l). Scale bar: 50 µm.</figcaption></figure>
system and hematopoietic fields in amphioxus. Remarkably, this also seems to be the case in vertebrates, as highlighted recently by the study of the function of Npas4l (Mattonet et al., 2022). Indeed, in zebrafish, Npas4l has been shown to be a key regulator controlling the acquisition of pronephros or hematoendothelial fate by lateral plate mesoderm precursors (Mattonet et al., 2022). The amphioxus transient Npas4/4l expression is therefore consistent with such a function pre-existing in the chordate ancestor.

Finally, *Tcf21/Msc* is orthologous to both *Tcf21*, also known as *Capsulin*, and to *Musculin* (Msc) also known as *MyoR*. Both genes are expressed in zebrafish, *Xenopus*, chick and mouse in the myogenic region of the head mesoderm (Lee et al., 2011; Nogueira et al., 2015; von Scheven et al., 2006; White et al., 2010). In *Xenopus*, chick and mouse, *Tcf21* is also expressed in the intermediate mesoderm and/or kidney, and in other lateral plate mesoderm derivatives (von Scheven et al., 2006; White et al., 2010). Thus, the expression of amphioxus *Tcf21/Msc* aligns with the idea that the vertebrate pharyngeal mesoderm and lateral plate mesoderm partially share a developmental program with both the ventral region of amphioxus somites and the first somite pair. This also suggests that *Tcf21* and *Msc* were co-opted for myogenesis in head muscles that do not originate from the somites. (Aldea et al., 2019; Meister et al., 2022).

### 3.2. *Mesp* gene expression

In vertebrates, *Mesp1* and *Mesp2* are key regulators of heart formation and somitogenesis, respectively. KO mouse for *Mesp1* show cardiac abnormal development while mutants for *Mesp2* show strong segmentation defects of the paraxial mesoderm (Liang et al., 2015). Interestingly, it was also shown that *Mesp* in the ascidian *Ciona* is a key determinant for the development of the trunk ventral cells which develop into cells precursor of the juvenile heart (Satou et al., 2004). The late expression of amphioxus *Mesp* in the tailbud region argue for either a loss of *Mesp* function in somitogenesis and heart field development in amphioxus, or a specific acquisition of *Mesp* genes function in the ancestor of tunicates and vertebrates. In both cases, the absence of *Mesp* expression in the ventral region of amphioxus somite expressing cardiac marker genes such as *Nkx2.5* (Holland et al., 2003) could explain the absence of a true heart in cephalochordates compared to the two other chordate clades.

### 3.3. *Osr1/2* genes expression

Concerning *Osr1* and *Osr2*, both are expressed in vertebrate intermediate mesoderm, the region of the lateral mesoderm developing into excretory organs, and in renal progenitors at different developmental stages (Lan et al., 2001; So and Danielian, 1999; Tena et al., 2007). In zebrafish, *Osr2* is also expressed in the pectoral fin buds while in mouse *Osr1* and *Osr2* are expressed in branchial arches and limb buds (Lan et al., 2001; So and Danielian, 1999). The expression of vertebrate *Osr1* and/or *Osr2* in the intermediate mesoderm precedes the expression of the pronephros markers *Pax8* or *Pax2* and *Lim1* (Tena et al., 2007). In amphioxus, *Osr1/2a* is expressed in the first three-somite pairs just after their formation. However, no further specific expression in the Hatschek's nephridium anlage is observed although *Osr1/2a* is expressed in the dorsal part of the somites until the T0 stage. Interestingly, the three *Osr1/2* orthologues in *Drosophila*, *odd*, *drm* and *sob*, are mainly expressed in the gut (Tena et al., 2007), as observed for amphioxus *Osr1/2a* from the N2 stage. These comparisons of expression patterns suggest that the role in renal development of *Osr1* and *Osr2* might have been acquired specifically in vertebrates, except maybe for a precocious role before the expression of the Pax and Lim genes in the embryonic field from which the excretory organs develop.

## 4. Experimental procedure

### 4.1. Phylogenetic tree reconstruction

Amino acid sequences from several species were retrieved from various databases (see Supplementary Table 1) for each gene of interest. Sequences were aligned using ClustalX (Larkin et al., 2007) and informative sites were selected using GBLOCKS with the less stringent options (Castresana, 2000). Maximum Likelihood phylogenetic trees were built using IQ-TREE (Minh et al., 2020) with default parameters (fast bootstrapping and automatic best model search) and customized using FigTree v1.4.3.

### 4.2. Probe synthesis

Partial sequences of each gene were either cloned in pBluescript II KS+ after RT-PCR amplification of *Branchiostoma lanceolatum* total RNA, or purchased as DNA fragments flanked by the T3 and T7 promoter sequences at IDT™ (Integrated DNA Technology). The sequences used for the probe are given in Supplementary Table 2. For the fragments inserted in pBluescript II KS+, the plasmid was linearized with the appropriate restriction enzyme. DNA fragments were amplified by PCR using the universal T3 and T7 primers. In both cases, probes were synthesized using the DIG labelling system (Roche) with the T3 or T7 RNA polymerases.

### 4.3. Whole mount in situ hybridization

Adult amphioxus of the *Branchiostoma lanceolatum* species were collected at the Racou beach near Argelès-sur-Mer, France, (latitude 42° 32' 53" N and longitude 3° 03' 27" E) with a specific permission delivered by the Prefect of Region Provence Alpes Côte d'Azur. Spawning was induced using heat stimulation (Fuentes et al., 2004, 2007). Embryos were obtained by *in vitro* fertilization in Petri dishes filled with filtered seawater and fixed at the desired stage (Bertrand et al., 2021; Carvalho et al., 2021) in 4 % PFA (paraformaldehyde) in MOPS-EGTA buffer (0,1 M MOPS pH7,5, 2 mM MgSO4, 1 mM EGTA, 0,5 M NaCl) overnight at 4 °C. They were then transferred to Ethanol 70 % and kept at -20 °C. Colorimetric *in situ* hybridizations were performed as previously described in (Somorjai et al., 2008) on Blastula, G1, G4, N0, N1, N2, N3, N4, N5, T0 and larva stage samples for all the genes studied.

### CRediT authorship contribution statement

Anaël Soubigou: Writing – original draft, Investigation. Lydvina Meister: Writing – review & editing, Supervision, Investigation. Lucie Subirana: Writing – review & editing, Investigation. Lucille Cornand: Investigation. Hector Escriva: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Stéphanie Bertrand: Writing – original draft, Supervision, Funding acquisition, Conceptualization.

### Funding

The laboratory of H.E. and S.B. was supported by the CNRS, and by the "Agence Nationale de la Recherche" under the grants ANR-19-CE13-0011, ANR-21-CE13-0034 and ANR-24-CE13-2279. S.B. is supported by the Institut Universitaire de France (IUF).

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
## Acknowledgements

This work benefited from access to the Observatoire Océanologique de Banyuls-sur-Mer, an EMBRC-France and EMBRC-ERIC site.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.gep.2025.119397.

## Data availability

Data will be available on request.

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