LRP2 is a member of the LDL receptor gene family, a class of multifunctional endocytic receptors that play key roles in embryonic development and that cause severe developmental malformations in humans and animal models when dysfunctional (1) . During neurulation, LRP2 is prominently expressed in the neuroepithelium that gives rise to the various parts of the developing central nervous system. Loss of receptor expression in this tissue in genetargeted mice results in fusion of the forebrain hemispheres (holoprosencephaly) (2, 3) and in overgrowth of the eye globe (buphthalmia) (4, 5) . Similar defects are seen in patients with Donnai-Barrow/ Facio-oculo-acoustico-renal (DB/FOAR) syndrome, an autosomal recessive disorder caused by inheritable LRP2 mutations (6) (7) (8) (9) .
Concerning its mode of action, LRP2 has been shown to act as an auxiliary receptor for sonic hedgehog (SHH) and to activate or inhibit this morphogen pathway dependent on the biological context. In the neuroepithelium, LRP2 acts as a co-receptor to Patched1 (PTCH1) to promote SHH signaling and to pattern the ventral midline of the forebrain (10) .
By contrast, in the developing eye, it operates as a clearance receptor for SHH to antagonize growth promoting signals by this morphogen in the retina (11) . Conceptually, these molecular functions of LRP2 in control of SHH signaling explain the forebrain and eye phenotypes observed in patients with DB/FOAR syndrome. However, LRP2 has also been shown to bind fibroblast growth factor (FGF) 8 (12) and bone morphogenetic protein (BMP) 4 (3) , and may thus have the potential to regulate multiple morphogen pathways during organogenesis.
Surprisingly, unbiased screens using exome sequencing now implicated mutations in LRP2 in congenital heart disease in humans, a receptor function not considered thus far (13) .
Additional evidence for a role in heart development came with ENU-induced mutagenesis studies that revealed Lrp2 mutations as a prominent cause of cardiac outflow tract (OFT) defects in mice (14) . The cardiac OFT is a transient structure at the arterial pole of the embryonic heart. During development, it separates into the ascending aorta and pulmonary trunk, outlets of the definitive left and right ventricle respectively. Defects in OFT formation produce conotruncal malformations characterized by incomplete or absent septation of the aorta and pulmonary trunk, resulting in low oxygen supply due to provision of mixed blood to the circulation. OFT malformations account for almost 30% of all congenital heart defects in humans (15) . Recently, conotruncal malformations were also documented in mice with targeted Lrp2 gene disruption, but the mechanism of receptor action remained unexplained (16) .
We now demonstrate that LRP2 is specifically expressed in SHH-responsive progenitor cells in the dorsal pericardial wall (DPW) that contribute to formation of the OFT.
Loss of receptor activity in Lrp2 mutant mice impairs SHH signaling in the DPW, resulting in decreased numbers and disturbed myocardial differentiation of progenitors that migrate into the OFT myocardium. Ultimately, these morphogenetic defects cause insufficient elongation of the OFT, the reason for the conotruncal malformations seen in mice, and possible patients lacking the morphogen receptor LRP2.
The OFT, positioned above the right ventricle, connects the embryonic ventricles with the aortic sac. During heart tube elongation and heart looping, the single OFT vessel is remodeled to undergo septation. Septation results in formation of the ascending aorta (Ao) from the left ventricle and the pulmonary trunk (Pa) arising from the right ventricle and ensures delivery of oxygenated blood to the body but deoxygenated blood to the lungs. To clarify the impact of LRP2 deficiency on OFT formation, we studied mice homozygous for a targeted Lrp2 gene disruption (Lrp2 -/-) generated by us previously (2) . LRP2 mutant embryos were compared to their respective wild-type or heterozygous littermates, as no defects were observed in Lrp2 +/animals. The latter two genotypes are jointly referred to as controls herein. Loss of LRP2 activity in Lrp2 -/mice lead to defects in OFT formation characterized by incomplete or absent septation of the Ao and Pa, a defect referred to as a common arterial trunk (CAT; persistent truncus arteriosus). CAT formation was evident at embryonic (E) day 15.5 and 18.5 when control embryos showed properly separated Ao and Pa (Fig. 1A) , while Lrp2 -/embryos exhibited a single vessel exiting the heart from the right ventricle (Fig. 1A, asterisks) Fig. S1 ), or showed normal OFT development with nicely separated Ao and Pa (16 out of 33; Fig. S1 ). This observation suggested genetic modifiers to impact LRP2 function in heart morphogenesis.
In the following, we focused our studies on Lrp2 mutants on a pure C57BL/6N genetic background exhibiting CAT formation. Histological alterations indicative of an OFT defect manifested around E12.5 to E13.5 when OFT septation normally occurs. Separation of Ao and Pa through the formation of endocardial cushions was seen in control embryos (Fig 1B) .
By contrast, LRP2-deficient embryos failed to form distinct endocardial cushions but exhibited an unorganized cell cluster in the OFT (Fig. 1B, arrows) . In addition, blebbing of the epicardial layer as well as hemorrhages were visible in mutant hearts (Fig. 1B, arrowheads).
We further investigated defects in endocardial cushion formation in Lrp2 -/embryos by in situ hybridization (ISH) for Sox9 ( Fig. 2A) . Sox9 is expressed in the cardiac cushion mesenchyme and promotes cardiac precursor cell expansion during heart valve development.
At E10.5, two Sox9 positive endocardial cushions were visible in control embryos (arrows) while these distinct tissue swellings were not detectable in Lrp2 -/embryos, despite the presence of Sox9 positive cells. By E11.5, these spiraling clusters of Sox9 positive cells (arrows) were less compacted in Lrp2 -/compared to control embryos and failed to develop into the endocardial swellings of the aortic valve (E12.5, stippled circles in controls). Rather, they formed an unorganized Sox9 positive cell cluster (E12.5, stippled circle in Lrp2 -/-).
Failure of the development of the endocardial cushions lead to a malformed outlet septum ( Fig. 2B , asterisk) that subsequently resulted in the formation of a CAT in LRP2-deficient embryos.
The formation, elongation, and septation of the cardiac OFT depends on the interaction of two distinct cell populations, namely cardiac neural crest cells (CNCC) and second heart field (SHF) cells. CNCC are a subpopulation of neural crest cells that originate from the dorsal neural tube and migrate into pharyngeal arches 3, 4, and 6. Starting from E9.5, CNCC populate the cardiac outflow tract and migrate into the outflow tract cushions (17) . There, they give rise to the condensed mesenchyme in the endocardial cushions forming the aorticopulmonary septation complex that divides the distal OFT into Ao and Pa (18) . SHF cells are cardiac progenitor cells located in the pharyngeal mesoderm. During heart development they are added to the arterial pole of the OFT driving OFT elongation.
To interrogate the role of LRP2 during OFT formation, we investigated the expression pattern of the receptor during OFT separation using immunohistology. At E10.5, when 
While LRP2 expression in neural crest cells had been reported before (19, 20) , we failed to detect the receptor in the CNCC population by immunohistology (Fig. 4A) . Still, the endocardial cushion defect observed in Lrp2 -/embryos ( Fig. 2 ) was consistent with a potential defect in the CNCC population. To query an indirect effect of receptor deficiency on this cell population, we crossed the Lrp2 deficient mouse strain with the transgenic Wnt1-Cre_LacZ reporter line to specifically mark CNCCs (21) . Staining for lacZ activity at E10.5 revealed a comparable pattern of CNCCs in the OFT in Lrp2 -/and control embryos (Fig. 4B ).
Subtle differences were seen in more proximal regions of the spiraling OFT cushions, where CNCCs appeared less compacted in Lrp2 -/embryos compared with controls ( Fig. 4B , stippled circle). We concluded that LRP2 deficiency did not affect the migration of CNCCs into the OFT and their integration in the endocardial cushions. Rather subtle alterations in 8 CNCC organization seen in the proximal OFT of mutant mice were considered secondary to defects in cushion formation described in Fig. 2 .
Since Lrp2 was not expressed in CNCC and LRP2 deficiency did not affect the migration of CNCC into the heart, we turned our attention to SHF cells that express LRP2 and are necessary for OFT septation. Among other markers, these cells are characterized by expression of insulin gene enhancer protein (Islet1) (22) . Detection of Islet1 protein as well as (24) . Since LRP2 is known to act in multiple morphogen pathways, we investigated these pathways in Lrp2 -/embryos during OFT development.
Canonical Wnt signaling has been shown to act upstream of other signaling pathways in control of Islet1 positive progenitor cell proliferation (25) . Non-canonical Wnt signaling is implicated in OFT development (26, 27) and important for planar cell polarity during myocardialization of the OFT cushions. It regulates polarity and intracellular cytoskeletal rearrangements, important to prevent premature differentiation of SHF progenitor cells into cardiomyocytes (28) (29) (30) . To explore potential defects in canonical Wnt signaling during OFT formation, we crossed the LRP2 mutant strain with the Tcf/Lef_LacZ reporter line. Detecting lacZ activity as a read-out of Wnt signaling, we failed to observe any changes in Wnt activity in the SHF of embryos lacking LRP2 (Fig. S3A) . Also, the non-canonical Wnt pathway was unchanged as deduced from investigating the expression pattern of Wnt11 in the OFT using ISH ( Fig S3B) . BMP signaling downregulates proliferation when SHF cells enter the OFT and it drives myocardial differentiation (31). Analyzing the BMP pathway by studying Bmp4 expression in the distal OFT failed to detect obvious changes in LRP2-deficient as compared with control embryos (Fig. S3B ).
Another essential morphogenetic signal in SHF patterning is provided by SHH secreted by the pharyngeal endoderm. SHH regulates both OFT development and CNCC survival. In addition, in the pharyngeal endoderm, SHH signaling coordinates a secondary, so far unknown pathway to control SHF survival and development (32). Using the Gli1-LacZ During OFT formation, SHF progenitor cells located in the DPW move along a path of differentiation to the transition zone (Tz), where they initiate a myocardial gene expression program. They finally reach the OFT myocardium to fully differentiate into cardiomyocytes.
By this movement, cardiac SHF progenitor cells contribute to the elongation of the OFT, necessary for correct alignment. Disturbances in this differentiation program result in a shortened OFT and can give rise to a CAT (33). A significant decrease in OFT length was seen in the hearts of Lrp2 -/embryos compared with controls ( Fig. 7D and E) , arguing for a defect in progenitor movement in Lrp2 -/embryos.
To investigate the consequences of reduced SHH signaling in DPW progenitors for their differentiation potential, we studied sagittal sections of Gli1-CreER T2 control and LRP2deficient embryos at E10.5. In controls, SHH-responsive cells (as deduced by YFP expression) concentrated in the DPW and distal Tz, a region overlapping with the LRP2 expression domain in the DPW (Fig. 8A ). Almost no SHH-responsive cells were detected in the OFT myocardium of control embryos as evidenced by an absence of co-staining of YFP with the cardiomyocyte marker MF20 (Fig. 8A ). By contrast, in Lrp2 -/embryos, an increased number of SHH-responsive cells were co-stained by YFP and MF20 in the OFT myocardium 
To further substantiate a role for LRP2 in control of SHF progenitor cell differentiation along a path from the DPW to the Tz and finally to the OFT myocardium, we performed immunodetection of markers representative for these three tissues in control and receptor Vanishing E-cadherin protein levels were detected in the posterior region of the DPW closer to the venous pole. By contrast, in Lrp2 -/embryos, the localization of E-cadherin-positive cells in the DPW was shifted to more posterior regions in the DPW (Fig. 8C , compare localization of the stippled lines). This shifted cell identity was also confirmed by a shift in localization of cardiac troponin I, a marker for mature cardiomyocytes (Fig. 8C) . Cardiac troponin I was exclusively expressed in cells of the OFT myocardium in controls. In Lrp2 -/-OFT tissue, also cells in the TZ stained positive for cardiac troponin I. Furthermore, laminin, a marker for the basal lamina showed an altered localization in Lrp2 -/embryos. While in the control DPW and Tz, laminin showed a punctuated pattern in the apical cell region, this punctuated pattern was lost in the Lrp2 -/-DPW and Tz (Fig. 8C, arrowheads) . In addition, in control embryos, the basal lamina formed a continuous pattern in the OFT region but not in the Tz, whereas in Lrp2 -/hearts, this continuous pattern of the basal lamina reached inside the Tz (Fig. 8C) . Collectively, these findings argue for a reduction in numbers and for premature differentiation of Lrp2 -/-SHF progenitor cells as they transit from the DWP via the Tz into OFT myocardium. These defects confound the contribution of SHF progenitor cells to OFT elongation and result in shortening of the OFT, the cause of CAT formation in Lrp2 -/embryos.
Previous studies implicated LRP2 in OFT formation (16) . Still, the exact mechanism whereby this multifunctional endocytic receptor may control formation of the cardiac OFT and why receptor dysfunction results in conotruncal malformations in patients and in mouse models remained unexplained. Here, we uncovered a crucial role for LRP2 in SHH signaling in progenitor cells of the DPW, signals required to ensure their timely differentiation into mature cardiomyocytes as they migrate into the OFT tissue. Loss of LRP2 results in a decrease in SHH-responsive progenitor cells in the DPW and in a concomitant appearance of cardiomyocytes in the Tz, arguing for a depletion of this progenitor niche due to premature differentiation of cardiac progenitors along their path to an OFT myocardial fate.
Formation and subsequent septation of the OFT is dependent on two cell populations, the SHF and the CNCCs. We traced the primary defect in LRP2 deficiency to the progenitor cells of the SHF that express this receptor (Fig. 5C ). While the CNCC population normally migrated into the OFT and reached the distal OFT cushions (Fig. 4) , the number of Islet1 positive progenitor cells in the SHF was significantly reduced in receptor mutant hearts (Fig.   5 ). Cardiac progenitors in the DPW contribute to growth and elongation of the OFT, a process essential for proper septation into Ao and Pa (33). Lrp2 -/mice exhibit a significantly shortened OFT (Fig 7E) 
To facilitate OFT elongation, SHF progenitor cells exhibit an increased proliferative capacity and a delayed propensity to differentiate into myocardium (39) . Several signaling pathways have been implicated in balancing proliferative versus differentiation fate decisions in the SHF (24, 40) -when defective, they cause CAT. These pathways include canonical and noncanonical Wnt signaling, as well as signals downstream of FGF, BMP, and SHH (27, (41) (42) (43) (44) (45) (46) (47) .
Our studies failed to provide evidence for defects in Wnt and BMP signaling as primary cause of CAT formation in the LRP2-deficient heart. Rather, our findings from the Gli1_LacZ (Fig.   6 ) and the Gli1_CreER T2 (Fig. 7A-C, 8A , B) reporter strains strongly argue for a defect in SHH signaling in the progenitor niche of the DPW as the underlying pathological mechanism.
Our model is consistent with the established role of LRP2 in promoting SHH signal reception in other embryonic tissues, such as the neuroepithelium (10).
The manifold cardiac phenotypes observed upon interception with the SHH pathway uncovered multiple roles for this morphogen pathway in heart morphogenesis (48) . SHH is necessary for arterial pole formation as loss of SHH signaling in Shh -/mice or in chick embryos treated with the SHH antagonist cyclopamine results in CAT formation (49, 50) . In addition to CAT formation, Lrp2 -/embryos show ventricular septal as well as aortic arch defects as reported by others. Such malformations also occur in Shh -/mice and in animals defective in the ciliary signaling compartment (14, 50, 55) . While it is conceivable that ventricular septal defects in Lrp2 mutants documented before (16) may also be caused by SHH signaling defects, our study focused on the consequences of impaired SHH signaling in the anterior SHF region for CAT formation. Using the Gli1-CreER T2 reporter line, we show an overall reduction in the number of SHH-responsive Islet1 positive progenitor cells in the Lrp2 -/distal OFT tissue (Fig. 7A, C) . Also, while these cells were mainly restricted to the DPW and the Tz in controls (Fig. 8A) , they showed a more dispersed localization in the OFT tissue in mutants, overlapping with MF20 positive cardiac cells in Tz and OFT myocardium.
In line with an impaired demarcation of DPW, Tz, and OFT myocardium in mutants, the expression domain for cardiac troponin I reached into the Tz, while E-cadherin expression, restricted to the Tz in controls, aberrantly extended more posteriorly into the mutant DPW (Fig. 8C) . Finally, the pattern of laminin, indicative of a more mature development of the basal lamina was restricted to proximal regions of the Tz and the OFT myocardium in controls but extended inside the distal Tz and anterior DPW in Lrp2 -/heart tissue.
Collectively, these findings suggest a role for LRP2 in establishing a SHH-dependent niche for cardiac progenitor cells in the DPW and in controlling their differentiation along their path to the OFT.
In the LRP2 mutant OFT, both the number of Islet1 positive cells receptive to SHH signals (double positive for Islet1 and YFP) and the overall expression domain of Islet1 positive progenitor cells are reduced (Fig. 5) . Thus, we cannot distinguish with certainty whether (a) LRP2 deficiency directly impacts SHH signaling in progenitor cells, thereby decreasing their numbers, or whether (b) LRP2 activity is required for other aspects of progenitor cell maintenance and loss of SHH activity in this niche as a secondary consequence of progenitor cell loss. However, based on evidence provided in this study and in published work, an instructive role for LRP2 in facilitation SHH signaling in SHF progenitors seems most likely.
The SHH signaling pathway is well characterized at the molecular level. In addition to the primary SHH receptor PTCH1, several auxiliary SHH binding proteins have been identified that are necessary to activate or inhibit the pathway in a context dependent manner.
This modulatory activity of SHH binding proteins has mainly been documented in the embryonic neuroepithelium (56, 57) , with LRP2 being one of these essential SHH coreceptors. Dependent on the biological context, LRP2 acts as pathway activator (by facilitating cell surface binding of the morphogen) (10) or as pathway inhibitor (by directing SHH to lysosomal degradation) (11) . Data presented herein argue that LRP2 acts as agonist to promote responsiveness of SHF progenitor cells to SHH signals during OFT formation. This hypothesis is supported by a recent study documenting a crucial role for SHH in activating progenitor gene expression and in inhibiting premature differentiation, thereby maintaining the progenitor cell population in the SHF (58) . We also show that LRP2 ensures the correct migration pattern of SHH-responsive SHF progenitors during OFT elongation. These results are in line with findings that SHH-responsive progenitors from the anterior SHF migrate into the pulmonary vessel (51) . Finally, a recent study on Gata4-regulated SHH signaling in cardiac progenitor cells documented the importance of SHH signals for SHF cell migration in mice (59) . In this study, the migratory defect of Gata4 haploinsufficient SHF progenitor cells was rescued by over-activation of the Hedgehog pathway using the constitutively activated In conclusion, we document a crucial role for LRP2 in maintenance of a pool of SHHdependent progenitors in the SHF and in ensuring their differentiative fate as they migrate into the OFT tissue. Our findings have identified a novel component of the SHH signaling machinery essential for heart development and uncovered the molecular cause of conotruncal malformations in humans and mouse models lacking this receptor.
Generation of mice with targeted Lrp2 gene disruption (2) or ENU-induced gene disruption (60) has been described before. The Lrp2 gene defect was analyzed in receptor-deficient and somite-matched littermates either wild-type (Lrp2 +/+ ) or heterozygous for the mutant Lrp2 
For hemotoxilin and eosin (H+E) staining, embryos were fixed overnight in 4% paraformaldehyde in PBS at 4°C, followed by routine paraffin embedding and sectioning at 10 μm thickness. For lacZ or immunofluorescence stainings, embryos were fixed for 1 -4 hrs in 4% paraformaldehyde in PBS at 4°C. Fixed embryos were infiltrated with 30% sucrose in PBS overnight at 4°C, followed by 2 hrs incubation in 50% cryoprotectant Tissue-Tek® OCT in 30% sucrose, followed by 2 hrs incubation in 75% Tissue-Tek® OCT in 30% sucrose. 
In situ hybridization on paraffin sections was described earlier (61) . Plasmids for digoxigenin 
Tamoxifen was prepared at 20 mg/ml in peanut butter oil. It was injected at a final dose of 2 mg into pregnant females at E7.5. Embryos were collected at E10.5 and fixed for 4 hrs, 
The length of the OFT was measured on 5 sagittal sections per embryo for a total of 6 control and 7 Lrp2 -/-E10.5 embryos using ImageJ.
Statistical significance was tested using the GraphPad Prism 7 software. Results comparing two groups were analyzed by Student's t test (Figure 7 