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. Author manuscript; available in PMC: 2026 Feb 25.
Published in final edited form as: Gene Expr Patterns. 2025 Jun 26;56:119398. doi: 10.1016/j.gep.2025.119398

Expression of Netrin-1 in the developing mouse heart

Adrianna Matos-Nieves 1,†, Sarah C Greskovich 1,†, Talita Z Choudhury 1, Sathiyanarayanan Manivannan 1, Yukie Ueyama 1, Anupama S Rao 1, Emily M Cameron 1, Vidu Garg 1,2
PMCID: PMC12930406  NIHMSID: NIHMS2148903  PMID: 40578544

Abstract

Axon guidance signaling pathways, including the Eph/ephrin, Semaphorin, and Slit/Robo pathways, have been found to play crucial roles in cardiac development. Netrin signaling is another well-studied signaling pathway important for axon guidance, but its role in the developing heart has not been investigated. Here, we describe the novel expression pattern of Netrin-1 in the developing murine heart. Transcriptomic analysis of embryonic mouse hearts shows dynamic Netrin-1 expression from E8.5 through E14.5, where Netrin-1 expression preferentially co-localizes with developing trabecular cardiomyocytes. We further demonstrate the spatiotemporal expression pattern of Netrin-1 using a combination of RNA in situ hybridization and Netrin-1Bgeo/+ reporter mice. Netrin-1 is expressed in the developing cardiomyocytes with the highest degree of expression within the left ventricular trabecular myocardium, which has not been previously recognized. Additionally, Netrin-1 expression is observed at lower levels in the cardiomyocytes of the right ventricle and atria. This expression pattern supports a role for Netrin signaling in the developing murine myocardium requiring further functional characterization.

Keywords: Netrin, axon guidance signaling, heart development, trabecular myocardium, RNA in situ hybridization

Introduction

Congenital heart disease (CHD) is the leading cause of infant mortality, affecting 1% of live births annually (Pierpont et al., 2018). Chromosomal aneuploidies, copy number variation and established CHD variants capture approximately 40–45% CHD cases (Yasuhara & Garg, 2021; Morton et al., 2021). However, approximately 55–60% of CHD cases remain of unknown etiology, and will require additional investigation to identify novel causal mechanisms. First described in the context of nervous system development, axon guidance signaling has been identified to be crucial for embryogenesis, including heart development (Zhao & Mommersteeg, 2018). Among the axon guidance signaling pathways, disruption of Slit/Robo, Semaphorin and EphrinB2/EphB4 signaling results in CHD (Grego-Bessa et al., 2007; Zhao & Mommersteeg, 2018; Sanchez-Castro et al., 2014; Jaouadi et al., 2023). Loss of function variants in ROBO1 have been identified in CHD patients with ventricular septal defects, tetralogy of Fallot and bicuspid aortic valve (Zhao & Mommersteeg, 2018; Jaouadi et al., 2022). Additionally, a patient with transposition of the great arteries, ventricular septal defect, and coarctation of the aorta was found to have disruption of the SEMA3D gene, a signaling ligand of the Semaphorin pathway (Sanchez-Castro et al., 2014). CHD phenotypes have also been recapitulated in mouse models; Sema3C null mice display interrupted aortic arch and improper septation of the cardiac outflow tract and disrupted Slit/Robo signaling in mice contributes to partially penetrant ventricular septal defects in Robo1−/− and Slit3−/− embryos, while Robo1−/−;Robo2−/− compound mutants display near complete penetrance of bicuspid aortic valve. (Feiner et al., 2001; Mommersteeg, et al., 2015). Additionally, EphrinB2/EphB4 signaling was found to be required for cardiomyocyte differentiation and trabeculation (Gerety et al., 1999; Grego-Bessa et al., 2007).

Netrin signaling is another major pathway involved in axon guidance. Netrins are extracellular, lamin-related proteins that function as chemotropic guidance cues. In mammals, there are four secreted Netrin ligands, Netrin-1, Netrin-3, Netrin-4 and Netrin-5, as well as two membrane-bound glycophosphate idylinositol (GPI)-linked Netrins, Netrin-G1 and Netrin-G2 (Yamagishi et al., 2015; Nakashiba et al., 2000; Serafini et al., 1996; Wang et al., 1999; Yin, Sanes, & Miner, 2000). Due to differences in homology among the Netrin family members, the proteins also exhibit differences in receptor binding (Bruikman et al., 2019). Receptors for secreted Netrins include Mcam, Dcc, Neo1, Unc5A-D, and Dscam (Tu et al., 2015; Andrews et al., 2008; Vries & Cooper, 2008; Geisbrecht et al., 2003; Kruger et al., 2004; Leonardo et al., 1997).

Netrin-1 is the most extensively studied member of this protein class; orthologous genes in mammals along with zebrafish, frog, fruit fly, and nematode have been found to perform a highly conserved role during neural development (Bradford et at., 2019; Bruikman et al., 2019). Netrin-1 functions as both a chemoattractant and repellant of growing axons, depending on which of its receptors are presented by the axonal growth cone (Boyer & Gupton, 2018). Netrin-1 is significant in the central nervous system not only for its role in axon guidance, but for its contribution to axon branching, synaptogenesis, cell migration and axon regeneration (Dent et al., 2004; Dun & Parkinson, 2017; Flores, 2011; Mehlen & Rama, 2007; Ylivinkka, Keski-Oja, & Hyytiäinen, 2016). Outside of the nervous system, Netrins facilitate mammary gland, pancreas and lung morphogenesis as well as angiogenesis (Yebra et al., 2003; Srinivasan, et al., 2003; Liu et al., 2004; Park et al., 2004). Similar to its bifunctional role in axon guidance, Netrin-1 has different effects on the vascular system depending on receptor binding: signaling via UNC5B receptor repulses vascularization while angiogenesis is promoted by DCC binding (Claro & Ferro 2020; Xia et al., 2022; Lu et al., 2004).

Within the landscape of cardiovascular health and disease, Netrin-1 expression is associated with atherosclerosis as well as cardioprotective action against ischemia-reperfusion injury (Layne, Ferro, & Passacquale, 2015). Netrin-1 plays a complex role in atherosclerosis; endothelial Netrin-1 acts as an atheroprotective factor while macrophage-derived expression promotes disease progression (Xia et al., 2022; van Gils et al., 2012). Netrin signaling has also been implicated in CHD; a de novo microdeletion between the second and third exons of NETRIN-1 (NTN1) was identified in a patient with a ventricular septal defect, atrial septal defect, and patent ductus arteriosus (Opitz et al., 2015). In addition, rare damaging genetic variants have been identified in the cognate receptors for NETRIN-1 (UNC5B and NEO1) in patients with conotruncal heart malformations, suggesting a role for Netrin signaling in cardiac development (Jin et al., 2017). Knockdown of ntn1a (paralogous homolog of NTN1) in zebrafish embryos results in abnormal aortic arch artery formation and cardiac laterality defects (Opitz et al., 2015). However, the expression pattern and functional role of Netrin-1 in the developing mammalian heart has not been described and warrants further investigation. Here, we characterize the expression pattern of Netrin-1 during mouse cardiac development.

Results

Netrin-1 is expressed in the developing mouse heart

We sought to determine whether Netrin-1 is expressed in the developing heart by using publicly available single-cell RNA-sequencing data (scRNA-Seq) of wildtype embryonic mouse hearts (Figure 1) (DeLaughter et al., 2016; Li et al., 2016). We merged the datasets and performed unbiased clustering of embryonic day (E)8.5- E14.5 timepoints, then assigned cell types to the clusters based on known cell markers for each cell type, as described in Feng et al (Supplementary Figures 1A–1C). In this analysis, we conducted expression overlap analysis of Netrin-1 in the developing mouse heart from E8.5 to E14.5 with various cell type markers (Figure 1). Utilizing gene markers to label endothelial cells and cardiomyocytes (Pecam1+ and Tnnt2+ cells, respectively), we examined the proportion of cells that show Netrin-1 co-expression with these markers. We observed that Netrin-1 and Tnnt2+ showed a strong overlap in expression pattern. We also noted Netrin-1 predominantly co-localizes with Nppa+;Tnnt2+ cells, which label the developing trabecular cardiomyocytes (Tian et al., 2017). Comparably, there was less overlap of Netrin-1 with Hey2+;Tnnt2 indicating limited Netrin-1 expression in compact myocardium (Supplementary Figures 1D and 1E) (Tian et al., 2017). These results suggest that Netrin-1, the major signaling ligand of the Netrin signaling pathway, is largely expressed in the developing trabecular myocardium of the mouse heart.

Figure 1. scRNA-Seq data derived from publicly available datasets shows Netrin-1 expression during heart development.

Figure 1.

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Expression profile of Netrin-1 (Ntn1), Pecam1 and Tnnt2 overlayed on an UMAP projection of single-cell data from mouse embryonic stage E8.5 to E14.5. In each row, the first three panels show the expression pattern of the three markers (Ntn1, Pecam1, Tnnt2) as log transformed normalized gene expression at each developmental stage. The subsequent panel (Overlap) shows the UMAP projection of cells color coded to reflect the overlap in the expression of Ntn1-alone positive (red), Ntn1-Tnnt2 double positive (purple), Ntn1, Tnnt2, Pecam1 triple positive (orange), and Ntn1-Pecam1 double positive (yellow) cells. Cells that do not show any expression of Netrin1 are shown in Grey. The last panel in each row shows a pie chart quantifying percentage of marker overlap shown in the UMAP projection in Overlap panel as percent of Ntn1 positive cells.

Netrin-1 is highly expressed in the trabecular myocardium of the developing heart

To visualize the spatiotemporal expression pattern of Netrin-1 transcripts in the developing heart, we performed in situ hybridization on embryonic mouse hearts at E11.5 - E14.5 (Figure 2). From E11.5 - E14.5, Netrin-1 expression is observed most prominently in the trabecular myocardium of the left ventricle (Figure 2). As observed in Kathiriya, et al (Kathiriya et al., 2024), a gradient across the interventricular septum was most notable at E13.5 (Figure 2H), but this was not remarkable at earlier timepoints (i.e., E11.5, E12.5). To a lesser extent, Netrin-1 expression is also observed in the right ventricular trabecular myocardium (Figure 2A–B, D–E, G–H, J–K) as well as atrial cardiomyocytes (Figure 2A–B, D–E, G–H, J–K). Notably at E13.5 and E14.5, we also observed Netrin-1 transcript expression in the compact myocardium (Figure 2C, F, I, L) as suggested by our prior transcriptomic analysis (Supplementary Figure 1E). Additionally, Netrin-1 expression was present in the lungs within the epithelium and smooth muscle cells surrounding the airways, as previously described (Figure 2K) (Dalvin et al., 2003).

Figure 2. In situ hybridization of E11.5 – E14.5 embryos shows Netrin-1 transcript expression in the developing myocardium.

Figure 2.

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RNAscope in situ hybridization assay was performed for Netrin-1 on E11.5-E14.5 embryos utilizing one litter (n=3 embryos) per time point. (A-L) Representative sections were chosen for each timepoint. (A-B, D-E, G-H, J-K) Sections were imaged at 10x and (C, F, I, L) 40x magnification. *, airway smooth muscle, Scale bar = 100 μm.

To further support our observation of the cardiac expression pattern of Netrin-1 in vivo, we utilized the previously described Netrin-1Bgeo/+ reporter mice, in which the N-terminal signal sequence of endogenous Netrin-1 is used to generate an active B-galactosidase fusion protein (Skarnes et al., 1995). We expanded our analysis to earlier timepoints and found Netrin-1 expression in the neural tube from E8.5 to E10.5 (Figures 3A–D). A posterior view of the E8.5 Netrin-1Bgeo/+ embryo shows expression in the otic placode, neural groove and neural fold (Figure 3B; Supplementary Figure 2A). In addition to the neural tube, Netrin-1 was observed in the developing mesencephalon, metencephalon and myelencephalon at E9.5 as well as the diencephalon and telencephalon at E10.5 (Supplementary Figures 2B – 2D). Examination of Netrin-1 expression during early cardiac development demonstrated it to be localized to the left ventricle at timepoints of E9.5 and E10.5 (Figure 3C and 3D). In agreement with our in situ hybridization experiments, Netrin-1 expression predominates in the left ventricle at E11.5, but also has expanded into the right ventricular trabecular myocardium (Figure 3E and F). Atrial expression of Netrin-1 is noted in the right and left atria starting at ~E12.5 and is equally expressed in both atria by E14.5 (Figure 3I, M, Q). Expression of PECAM1 by immunohistochemistry, which labels endothelial and endocardial cells, demonstrated that Netrin-1 is not expressed in these cells during mouse heart development (Figure 3H, L, P, T). Expression was not visualized in the compact myocardium using the Netrin-1Bgeo/+ reporter potentially due to the sensitivity of this methodology or heterozygosity of the Netrin-1Bgeo allele (which results in loss of function) as Netrin-1 RNA transcripts were noted at low levels in the compact myocardium by scRNA-seq analysis and in situ hybridization (Figure 3G, K, O, S). Outside of the heart, expression was observed in the developing somites, kidneys, lungs, renal/ iliac vessels, esophagus and stomach from E12.5-E14.5 in these Netrin-1Bgeo/+ reporter mice (Supplementary Figure 2E–J).

Figure 3. Pattern of cardiac expression of Netrin-1 in E8.5 -E14.5 Netrin-1Bgeo/+ mouse embryos using X-gal staining.

Figure 3.

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(A-D) Staining of E8.5 - E10.5 whole Netrin-1Bgeo/+ embryos was performed (n of at least 3 embryos examined per each timepoint). Positive X-gal staining is noted by dark blue precipitate. A representative image of an excised E10.5 Netrin-1Bgeo/+ stained heart is shown to the right of (D). (E, I, M, Q) Embryonic hearts were dissected from E11.5 - E14.5 Netrin-1Bgeo/+ embryos and stained. (F-G, J-K, N-O, R-S) Histological tissue sections derived from E11.5 – E14.5 stained hearts were counterstained using Nuclear Fast Red and imaged at 10x and 40x magnification. (H,L,P,T) Immunohistochemistry shows that PECAM does not colocalize with positive X-gal stains at the timepoints examined. Scale bar: A, B, E, I, M, Q = 200 μm, C, D = 500 μm, F, J, N, R = 100 μm, G, H, K, L, O, S, T = 50 μm.

Discussion

Through this study, we have characterized the expression pattern of Netrin-1 in the developing murine myocardium. By utilizing publicly available scRNA-Seq datasets, we were able to identify Netrin-1 as a novel gene expressed in the developing mouse heart. Through performing in situ hybridization on wildtype mice and utilizing the Netrin-1Bgeo/+ reporter mouse line, we were able to spatiotemporally characterize this expression pattern in vivo and found Netrin-1 has the highest expression in the left ventricular trabecular myocardium throughout multiple stages of murine cardiac development expanding upon the expression pattern found by others (Kathiriya et al., 2024).

This embryonic cardiac expression pattern suggested a potential functional role for Netrin-1 for normal heart development. As previously reported, global knockout of Netrin-1 results in embryonic and/or early neonatal lethality (Bin et al. 2015; Yung, Nishitani, & Goodrich, 2015). We uncovered embryonic lethality in Netrin-1 null mice and failed to recover null embryos past E11.5 in a C57Bl/6J background strain (Supplemental Table 1). Therefore, we also investigated if cardiomyocyte-specific deletion of Netrin-1 expression leads to a cardiac phenotype. Surprisingly, mice with conditional deletion of Netrin-1 (Myh6Cre+;Netrin-1flox/flox) in the myocardium survived postnatally with normal cardiac function (Supplemental Table 2). Echocardiography of 8-week-old Myh6Cre+;Netrin-1flox/flox mice demonstrated no differences in ventricular size and function between mutants and controls except for mutants having an increased but clinical insignificant interventricular septal thickness during systole only (Supplementary Figure 3C–K) and similarly, histologic analysis of 12 week old Myh6Cre+;Netrin-1flox/flox murine hearts showed no obvious cardiac phenotype (Supplementary Figure 3A–B). The finding that conditional deletion of Netrin-1 in developing cardiomyocytes does not result in an overt cardiac phenotype is similar to previous functional analysis of Netrin-1 and Netrin-4 in lung morphogenesis, in which no significant morphological difference was observed in murine lungs lacking genes encoding either Netrin ligand (Liu et al., 2004). Additionally, they found no obvious phenotype in mutants lacking Netrin receptor Dcc. Our findings may be in part due to functional redundancy of the axon guidance molecules; analysis of scRNA-Seq datasets predicts intermittent expression of Netrin-3 and Netrin-4 during mouse embryonic heart development (Supplementary Figures 4A - 4B). Comparison of Netrin family expression at different developmental stages shows Netrin-1 expression is primarily restricted to myocardial cells and expression is noted through all studied stages (Supplementary Figures 5A and 5B). We hypothesize that deletion of multiple genes within the Netrin family may be required to observe a cardiac phenotype. As the function of Netrin-1 in other tissues and pathologies is highly specific to the presence of its receptors, further investigation into the receptor expression could determine the significance of Netrin signaling in heart development. Future work should focus on characterizing ligand-receptor interaction in the developing heart and identify mechanisms that might compensate for Netrin-1 function in developing cardiomyocytes.

Methods

Single-cell RNA-Sequencing data visualization

Publicly available single-cell data from wildtype, embryonic mouse heart were collected from gene expression omnibus (GEO). (Feng et al, 2022; Xiao et al, 2018; Li et al, 2016). Raw counts were processed using Seurat (v5) (Hao et al, 2024) and integrated using harmony (Korsunsky et al, 2019). Data were log normalized and clustered using the first 30 harmony-generated principal components. Dimensionality reduction was also performed on the first 30 harmony-generated principal components to create uniform manifold approximation and projection (UMAP) coordinates for each cell. Cells were clustered using 30 harmony-generated principal components using Louvain clustering algorithm with a resolution of 0.5. Cluster makers were determined using Wilcoxon rank sum test using Seurat (v5). These markers were used to identify cell types as described in Feng et al., 2022. For expression overlap analysis, a cell was considered as positive for a gene if normalized expression of a gene was greater than 0. For the quantification of overlap, the percentage of cells that were single, double or triple positive for the three markers were evaluated. Cells that did not express the first marker genes (Ntn1 or Ntn3 or Ntn4) in their corresponding analysis were excluded from this overlap quantifications shown in the pie chart.

Mouse embryo collection

All mice were maintained on a 12-hour-light/dark cycle and fed a standard western diet. For timed breeding, noon of the day of observation of the vaginal plug was defined as E0.5. Pregnant dams were anesthetized using inhalation of 3% isoflurane in accordance with IACUC protocols; cervical dislocation and organ removal used as a secondary method of euthanasia. Netrin-1+/− mice (Jackson Laboratories # 028070) were bred in a heterozygous cross (Netrin-1+/− × Netrin-1+/−) and embryos collected at E8.5, E9.25, E9.5, E10.5, E11.5, and E12.5 to establish embryonic lethality of Netrin-1−/− in a C57BL6/J background. Wildtype C57BL6/J males were bred to wildtype C57BL6/J females (Jackson Laboratories # 000664) and embryos were collected at E11.5 – E14.5 for RNAscope assay. Previously described Netrin-1Bgeo/+ animals were provided by Dr. Alex Jaworski’s laboratory at Brown University (Skarnes et al., 1995). Netrin-1Bgeo/+ males were time bred to wildtype CD1 and C57BL6/J females (Charles River # 022, Jackson Laboratories # 000664). Embryos were collected at E8.5 – E14.5 for β-galactosidase staining and genotyped for the Netrin-1-Bgeo allele utilizing forward primer 5’-TCGTCTGCTCATCCATGACC and reverse primer 5’-GTCTCGTTGCTGCATAAACC. Mice harboring the Netrin-1 floxed (Netflox/flox) allele (Jackson Laboratories #028038), and Myh6-Cre allele (Jackson Laboratories #011038) were generated as previously described and genotyped according to Jackson Laboratory genotyping protocol (Bin et al., 2015; Agah et al., 1997). Heterozygous males were generated for the Myh6-Cre line and bred continuously to Netrin-1flox/flox females to establish Mendelian inheritance of Netrin-1.

Histological analysis of embryos

Whole embryos or adult hearts were harvested and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) overnight, washed with 1X PBS and processed using the Leica ASP2065 tissue processor and standard protocol. All tissue collected was paraffin embedded and sectioned at 6μm. A subset of β-galactosidase-stained embryos were cleared using an ethanol series prior to 4-hour processing.

RNA in situ hybridization

The RNAscope assay was performed on 6μm paraffin embedded tissue sections using the 2.5 HD-Red kit (Advanced Cell Diagnostics; ACD) (Cat#322350) according to manufacturer’s instructions. An n of 3 embryos per each timepoint were examined in this experiment. Tissue pretreatment was performed in accordance with manufacturer recommendation for mouse embryo tissue: tissue was boiled in Target Retrieval Reagents for 15 minutes and treated with Protease Plus for 30 minutes. The Netrin-1 probe used was Mm-Ntn1 (Cat#407621), designed and synthesized by the manufacturer. Universal negative control probe targeting the dapB gene (Cat# 310043) and a positive control probe targeting murine PPIB (Cat#313911), both generated by ACD, were used as reference. Images were taken using the Keyence BZ-X810 Fluorescence Microscope.

β-galactosidase staining

A modified β- galactosidase staining protocols based upon prior publications was used (Dodou, Xu, & Black, 2003; Yamagishi, Olson, & Srivastava, 2000). Embryos were harvested in sterile, 4°C 1X PBS. The tissue was fixed using a solution made of 2% formaldehyde, 0.02% glutaraldehyde, 1X sterile PBS over ice for 30 minutes – 1 hour depending on the size of the embryo. Embryos (n of at least 3 embryos per each timepoint) were permeabilized using a solution, referred henceforth as rinse buffer, composed of 1X sterile PBS, 2mM magnesium chloride (Sigma-Aldrich), 0.01% sodium deoxycholate (Acros Organics), 0.02% NP-40 (Abcam) for thirty minutes – 1 hour depending on the size of the embryo. Embryos were incubated at room temperature overnight on a shaker in stain solution made up of 0.1% X-gal (Fisher), 5mM Potassium hexacyanoferrate(II) trihydrate (Sigma-Aldrich), 5mM Potassium ferricyanide (Acros Organics), 2mM MgCl2 (Sigma Aldrich) in rinse buffer (see previous), protected from the light. The following day, embryos were rinsed in 1X PBS for 30 minutes, fixed overnight in 4% formaldehyde and washed with 1X PBS. Embryos were dehydrated in glass scintillation vials using 50%, 70% and 100% ethanol in 30-minute intervals and stored in 100% ethanol overnight. To clear, embryos were incubated in 100% ethanol for 30-minutes and later cleared in a 1:1 benzyl benzoate and benzyl alcohol mixture. Tissue was imaged using Olympus BX51.

Echocardiography

Transthoracic echocardiography was performed using a Vevo 2100 Imaging System equipped with 18- to 38-MHz linear-array transducer with a digital ultrasound system (VisualSonics Inc., Canada), as previously described (Majumdar et al., 2021). Mice examined by echocardiogram included Myh6Cre+;Ntn1fl/+(n=4), Myh6Cre+;Ntn1fl/fl (n=6), with Myh6Cre-;Ntn1fl/fl (n=11) and Myh6Cre-;Ntn1fl/+ (n=8) included as controls. The mice were anesthetized with isoflurane (2% in 100% oxygen at a flow rate of 1.0 liters/min) and body temperature was monitored throughout the procedure using a rectal probe thermometer. Pulse-wave Doppler analysis of three consecutive cardiac cycles across the aortic valve was performed and averaged along the parasternal long axis to obtain maximal transvalvular velocity along with additional measurements of ventricular function. All echocardiographic experiments and analyses were performed by an experienced investigator in a genotype blinded fashion.

Statistics

Statistical analysis was performed utilizing GraphPad Prism. The Shapiro-Wilk test was applied to quantitative data to test for normality. Echocardiogram measurements were analyzed using Student’s t-test for normally distributed data. Additionally, we performed the Mann-Whitney U test for nonparametric comparisons on the echocardiographic data to strengthen our statistical analysis. Chi-squared test was performed on categorical data. P-value ≤ 0.05 was considered significant.

Supplementary Material

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Acknowledgments

The authors would like to acknowledge Dr. Tessier-Lavigne and Dr. Jaworski for sharing the Netrin-1Bgeo/+ reporter animals. In addition, we would like to thank Dr. Fang Bu for assistance in anatomical analysis of mouse embryos.

Funding:

Research reported in this publication was supported by the National Heart, Lung, and Blood Institute, National Institutes of Health Award Number T32HL134616 (AMN), Award Number T32HL098039 (SM), Award Number T32HL166149 (ASR), and Award Number R01-HL121797 (VG).

References

  1. Agah R, Frenkel PA, French BA, Michael LH, Overbeek PA, & Schneider MD (1997). Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo. Journal of Clinical Investigation, 100(1), 169–179. 10.1172/jci119509 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andrews GL, Tanglao S, Farmer WT, Morin S, Brotman S, Berberoglu MA, Price H, Fernandez GC, Mastick GS, Charron F, & Kidd T (2008). Dscam guides embryonic axons by Netrin-dependent and -independent functions. Development, 135(23), 3839–3848. 10.1242/dev.023739 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bin Jenea M., Han D, Lai Wing Sun K, Croteau L-P, Dumontier E, Cloutier J-F, Kania A, & Kennedy Timothy E. (2015). Complete Loss of Netrin-1 Results in Embryonic Lethality and Severe Axon Guidance Defects without Increased Neural Cell Death. Cell Reports, 12(7), 1099–1106. 10.1016/j.celrep.2015.07.028 [DOI] [PubMed] [Google Scholar]
  4. Boyer NP, & Gupton SL (2018). Revisiting Netrin-1: One Who Guides (Axons). Frontiers in cellular neuroscience, 12, 221. 10.3389/fncel.2018.00221 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradford D, Cole SJ, & Cooper HM (2009). Netrin-1: Diversity in development. The International Journal of Biochemistry & Cell Biology, 41(3), 487–493. 10.1016/j.biocel.2008.03.014 [DOI] [PubMed] [Google Scholar]
  6. Bruikman CS, Zhang H, Kemper AM, & van Gils JM (2019). Netrin Family: Role for Protein Isoforms in Cancer. Journal of Nucleic Acids, 2019(1), 1–9. 10.1155/2019/3947123 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Claro V, & Ferro A (2020). Netrin-1: Focus on its role in cardiovascular physiology and atherosclerosis. JRSM Cardiovascular Disease. 10.1177/2048004020959574 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dalvin S, Anselmo MA, Prodhan P, Komatsuzaki K, Schnitzer JJ, & Kinane T. Bernard. (2003). Expression of Netrin-1 and its two receptors DCC and UNC5H2 in the developing mouse lung. Gene Expression Patterns, 3(3), 279–283. 10.1016/s1567-133x(03)00047-4 [DOI] [PubMed] [Google Scholar]
  9. DeLaughter DM, Bick AG, Wakimoto H, McKean D, Gorham JM, Kathiriya IS, Hinson JT, Homsy J, Gray J, Pu W, Bruneau BG, Seidman JG, & Seidman CE (2016). Single-Cell Resolution of Temporal Gene Expression during Heart Development. Developmental Cell, 39(4), 480–490. 10.1016/j.devcel.2016.10.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dent EW, Barnes AM, Tang F, & Kalil K (2004). Netrin-1 and Semaphorin 3A promote or inhibit cortical axon branching, respectively, by reorganization of the cytoskeleton. Journal of Neuroscience, 24(12), 3002–3012. 10.1523/JNEUROSCI.4963-03.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dodou E, Xu S-M, & Black BL (2003). mef2c is activated directly by myogenic basic helix-loop-helix proteins during skeletal muscle development in vivo. Mechanisms of Development, 120(9), 1021–1032. 10.1016/s0925-4773(03)00178-3 [DOI] [PubMed] [Google Scholar]
  12. Dun X-P, & Parkinson DB (2017). Role of Netrin-1 Signaling in Nerve Regeneration. International Journal of Molecular Sciences, 18(3). 10.3390/ijms18030491 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feiner L, Webber AL, Brown CB, Lu MM, Jia L, Feinstein P, Mombaerts P, Epstein JA, & Raper JA (2001). Targeted disruption of semaphorin 3C leads to persistent truncus arteriosus and aortic arch interruption. Development, 128(16), 3061–3070. 10.1242/dev.128.16.3061 [DOI] [PubMed] [Google Scholar]
  14. Feng W, Bais A, He H, Rios C, Jiang S, Xu J, Chang C, Kostka D, & Li G (2022). Single-cell transcriptomic analysis identifies murine heart molecular features at embryonic and neonatal stages. Nature communications, 13(1), 7960. 10.1038/s41467-022-35691-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Flores C (2011). Role of netrin-1 in the organization and function of the mesocorticolimbic dopamine system. Journal of Psychiatry & Neuroscience, 36(5), 296–310. 10.1503/jpn.100171 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Geisbrecht BV, Dowd KA, Barfield RW, Longo PA, & Leahy DJ (2003). Netrin Binds Discrete Subdomains of DCC and UNC5 and Mediates Interactions between DCC and Heparin. Journal of Biological Chemistry, 278(35), 32561–32568. 10.1074/jbc.m302943200 [DOI] [PubMed] [Google Scholar]
  17. Gerety SS, Wang HU, Chen Z-F, & Anderson DJ (1999). Symmetrical Mutant Phenotypes of the Receptor EphB4 and Its Specific Transmembrane Ligand ephrin-B2 in Cardiovascular Development. Molecular Cell, 4(3), 403–414. 10.1016/s1097-2765(00)80342-1 [DOI] [PubMed] [Google Scholar]
  18. Grego-Bessa J, Luna-Zurita L, del Monte G, Bolós V, Melgar P, Arandilla A, Garratt AN, Zang H, Mukouyama Y, Chen H, Shou W, Ballestar E, Esteller M, Rojas A, Pérez-Pomares JM, & de la Pompa JL (2007). Notch Signaling Is Essential for Ventricular Chamber Development. Developmental Cell, 12(3), 415–429. 10.1016/j.devcel.2006.12.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hao Y, Stuart T, Kowalski MH, Choudhary S, Hoffman P, Hartman A, Srivastava A, Molla G, Madad S, Fernandez-Granda C, & Satija R (2024). Dictionary learning for integrative, multimodal and scalable single-cell analysis. Nature biotechnology, 42(2), 293–304. 10.1038/s41587-023-01767-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jaouadi H, Gérard H, Théron A, Collod-Béroud G, Collart F, Avierinos JF, & Zaffran S (2022). Identification of non-synonymous variations in ROBO1 and GATA5 genes in a family with bicuspid aortic valve disease. Journal of human genetics, 67(9), 515–518. 10.1038/s10038-022-01036-x [DOI] [PubMed] [Google Scholar]
  21. Jaouadi H, Jopling C, Bajolle F, Théron A, Faucherre A, Gerard H, Al Dybiat S, Ovaert C, Bonnet D, Avierinos JF, & Zaffran S (2023). Expanding the phenome and variome of the ROBO-SLIT pathway in congenital heart defects: toward improving the genetic testing yield of CHD. Journal of translational medicine, 21(1), 160. 10.1186/s12967-023-03994-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jin SC, Homsy J, Zaidi S, Lu Q, Morton S, DePalma SR, Zeng X, Qi H, Chang W, Sierant MC, Hung W-C, Haider S, Zhang J, Knight J, Bjornson RD, Castaldi C, Tikhonoa IR, Bilguvar K, Mane SM, & Sanders SJ (2017). Contribution of rare inherited and de novo variants in 2,871 congenital heart disease probands. Nature Genetics, 49(11), 1593–1601. 10.1038/ng.3970 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kathiriya IS, Dominguez MH, Rao KS, Muncie-Vasic JM, Devine WP, Hu KM, Hota SK, Garay BI, Quintero D, Goyal P and Matthews MN, (2024). A disrupted compartment boundary underlies abnormal cardiac patterning and congenital heart defects. bioRxiv. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Korsunsky I, Millard N, Fan J, Slowikowski K, Zhang F, Wei K, Baglaenko Y, Brenner M, Loh PR, & Raychaudhuri S (2019). Fast, sensitive and accurate integration of single-cell data with Harmony. Nature methods, 16(12), 1289–1296. 10.1038/s41592-019-0619-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kruger RP (2004). Mapping Netrin Receptor Binding Reveals Domains of Unc5 Regulating Its Tyrosine Phosphorylation. Journal of Neuroscience, 24(48), 10826–10834. 10.1523/jneurosci.3715-04.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Layne K, Ferro A, & Passacquale G (2015). Netrin-1 as a novel therapeutic target in cardiovascular disease: to activate or inhibit? Cardiovascular Research, 107(4), 410–419. 10.1093/cvr/cvv201 [DOI] [PubMed] [Google Scholar]
  27. Leonardo ED, Hinck L, Masu M, Keino-Masu K, Ackerman SL, & Tessier-Lavigne M (1997). Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors. Nature, 386(6627), 833–838. 10.1038/386833a0 [DOI] [PubMed] [Google Scholar]
  28. Li G, Xu AF, Sim S, Priest JR, Tian X, Khan T, Quertermous T, Zhou B, Tsao PS, Quake SR, & Wu S (2016). Transcriptomic Profiling Maps Anatomically Patterned Subpopulations among Single Embryonic Cardiac Cells. Developmental Cell, 39(4), 491–507. 10.1016/j.devcel.2016.10.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liu Y, Stein E, Oliver T, Li Y, Brunken WJ, Koch M, Tessier-Lavigne M, & Hogan BLM (2004). Novel Role for Netrins in Regulating Epithelial Behavior during Lung Branching Morphogenesis. Current Biology, 14(10), 897–905. 10.1016/j.cub.2004.05.020 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lu X, Le Noble F, Yuan L, Jiang Q, De Lafarge B, Sugiyama D, Bréant C, Claes F, De Smet F, Thomas JL, Autiero M, Carmeliet P, Tessier-Lavigne M, & Eichmann A (2004). The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system. Nature, 432(7014), 179–186. 10.1038/nature03080 [DOI] [PubMed] [Google Scholar]
  31. Majumdar U, Manivannan S, Basu M, Ueyama Y, Blaser MC, Cameron E, McDermott MR, Lincoln J, Cole SE, Wood S, Aikawa E, Lilly B, & Garg V (2021). Nitric oxide prevents aortic valve calcification by S-nitrosylation of USP9X to activate NOTCH signaling. Science Advances, 7(6). 10.1126/sciadv.abe3706 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Matos-Nieves A, Manivannan S, Majumdar U, McBride KL, White P, & Garg V (2021). A Multi-Omics Approach Using a Mouse Model of Cardiac Malformations for Prioritization of Human Congenital Heart Disease Contributing Genes. Frontiers in Cardiovascular Medicine, 8. 10.3389/fcvm.2021.683074 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mehlen P, & Rama N (2007). Nétrine-1 et guidage axonal. M S-Medecine Sciences, 23(3), 311–316. 10.1051/medsci/2007233311 [DOI] [PubMed] [Google Scholar]
  34. Mommersteeg MTM, Yeh ML, Parnavelas JG, & Andrews WD (2015). Disrupted Slit-Robo signalling results in membranous ventricular septum defects and bicuspid aortic valves. Cardiovascular Research, 106(1), 55–66. 10.1093/cvr/cvv040 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Morton SU, Quiat D, Seidman JG, & Seidman CE (2021). Genomic frontiers in congenital heart disease. Nature Reviews Cardiology, 19(1). 10.1038/s41569-021-00587-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nakashiba T, Ikeda T, Nishimura S, Tashiro K, Honjo T, Culotti JG, & Itohara S (2000). Netrin-G1: a Novel Glycosyl Phosphatidylinositol-Linked Mammalian Netrin That Is Functionally Divergent from Classical Netrins. The Journal of Neuroscience, 20(17), 6540–6550. 10.1523/jneurosci.20-17-06540.2000 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Opitz R, Hitz M-P, Vandernoot I, Trubiroha Achim, Abu-Khudir Rasha, Samuels M, Désilets Valérie, Costagliola S, Andelfinger Gregor, & Deladoëy J. (2015). Functional Zebrafish Studies Based on Human Genotyping Point to Netrin-1 as a Link Between Aberrant Cardiovascular Development and Thyroid Dysgenesis. Endocrinology, 156(1), 377–388. 10.1210/en.2014-1628 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Park KW, Crouse D, Lee M, Karnik SK, Sorensen LK, Murphy KJ, Kuo CJ, & Li DY (2004). The axonal attractant Netrin-1 is an angiogenic factor. Proceedings of the National Academy of Sciences, 101(46), 16210–16215. 10.1073/pnas.0405984101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pierpont ME, Brueckner M, Chung WK, Garg V, Lacro RV, McGuire AL, Mital S, Priest JR, Pu WT, Roberts A, Ware SM, Gelb BD, & Russell MW (2018). Genetic Basis for Congenital Heart Disease: Revisited: A Scientific Statement From the American Heart Association. Circulation, 138(21). 10.1161/cir.0000000000000606 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sanchez-Castro M, Pichon O, Briand A, Poulain D, Gournay Véronique, David A, & Le Caignec Cédric. (2014). Disruption of theSEMA3DGene in a Patient with Congenital Heart Defects. Human Mutation, 36(1), 30–33. 10.1002/humu.22702 [DOI] [PubMed] [Google Scholar]
  41. Serafini T, Colamarino SA, Leonardo E. David., Wang H, Beddington R, Skarnes WC, & Tessier-Lavigne M. (1996). Netrin-1 Is Required for Commissural Axon Guidance in the Developing Vertebrate Nervous System. Cell, 87(6), 1001–1014. 10.1016/s0092-8674(00)81795-x [DOI] [PubMed] [Google Scholar]
  42. Skarnes WC, Moss JE, Hurtley SM, & Beddington RS (1995). Capturing genes encoding membrane and secreted proteins important for mouse development. Proceedings of the National Academy of Sciences, 92(14), 6592–6596. 10.1073/pnas.92.14.6592 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Srinivasan K, Strickland P, Valdes A, Shin GC, & Hinck L (2003). Netrin-1/Neogenin Interaction Stabilizes Multipotent Progenitor Cap Cells during Mammary Gland Morphogenesis. Developmental Cell, 4(3), 371–382. 10.1016/s1534-5807(03)00054-6 [DOI] [PubMed] [Google Scholar]
  44. Tian X, Li Y, He L, Zhang H, Huang X, Liu Q, Pu W, Zhang L, Li Y, Zhao H, Wang Z, Zhu J, Nie Y, Hu S, Sedmera D, Zhong TP, Yu Y, Zhang L, Yan Y, Qiao Z, … Zhou B (2017). Identification of a hybrid myocardial zone in the mammalian heart after birth. Nature communications, 8(1), 87. 10.1038/s41467-017-00118-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tu T, Zhang J, Yan H, Luo Y, Kong R, Wen P, Ye Z, Chen J, Feng J, Liu F, Wu JY, & Yan X (2015). CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development. Cell Research, 25(3), 275–287. 10.1038/cr.2015.15 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. van Gils JM, Derby MC, Fernandes LR, Ramkhelawon B, Ray TD, Rayner KJ, Parathath S, Distel E, Feig JL, Alvarez-Leite JI, Rayner AJ, McDonald TO, O’Brien KD, Stuart LM, Fisher EA, Lacy-Hulbert A, & Moore KJ (2012). The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques. Nature immunology, 13(2), 136–143. 10.1038/ni.2205 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vries MD, & Cooper HM (2008). Emerging roles for neogenin and its ligands in CNS development. Journal of Neurochemistry, 106(4), 1483–1492. 10.1111/j.1471-4159.2008.05485.x [DOI] [PubMed] [Google Scholar]
  48. Wang H, Copeland NG, Gilbert DJ, Jenkins NA, & Tessier-Lavigne M (1999). Netrin-3, a Mouse Homolog of Human NTN2L, Is Highly Expressed in Sensory Ganglia and Shows Differential Binding to Netrin Receptors. Journal of Neuroscience, 19(12), 4938–4947. 10.1523/jneurosci.19-12-04938.1999 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Xia X, Hu Z, Wang S, & Yin K (2022). Netrin-1: An emerging player in inflammatory diseases. Cytokine & growth factor reviews, 64, 46–56. 10.1016/j.cytogfr.2022.01.003 [DOI] [PubMed] [Google Scholar]
  50. Xiao Y, Hill MR, Zhang M, Martin TR, Morikawa Y, Wang S, Moise AR, Wythe JD, & Martin JF (2018). Hippo Signaling Plays an Essential Role in Cell State Transitions during Cardiac Fibroblast Development. Developmental Cell, 45(2), 153–169.e6. 10.1016/j.devcel.2018.03.019 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yamagishi H, Olson EN, & Srivastava D (2000). The basic helix-loop-helix transcription factor, dHAND, is required for vascular development. Journal of Clinical Investigation, 105(3), 261–270. 10.1172/jci8856 [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yamagishi S, Yamada K, Sawada M, Nakano S, Mori N, Sawamoto K, & Sato K (2015). Netrin-5 is highly expressed in neurogenic regions of the adult brain. Frontiers in Cellular Neuroscience, 9(1). 10.3389/fncel.2015.00146 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yasuhara J, & Garg V (2021). Genetics of congenital heart disease: a narrative review of recent advances and clinical implications. Translational pediatrics, 10(9), 2366–2386. 10.21037/tp-21-297 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yebra M, Montgomery AM, Diaferia GR, Kaido T, Silletti S, Perez B, Just ML, Hildbrand S, Hurford R, Florkiewicz E, Tessier-Lavigne M, & Cirulli V (2003). Recognition of the neural chemoattractant Netrin-1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration. Developmental cell, 5(5), 695–707. 10.1016/s1534-5807(03)00330-7 [DOI] [PubMed] [Google Scholar]
  55. Yin Y, Sanes JR, & Miner JH (2000). Identification and expression of mouse netrin-4. Mechanisms of Development, 96(1), 115–119. 10.1016/s0925-4773(00)00369-5 [DOI] [PubMed] [Google Scholar]
  56. Ylivinkka I, Keski-Oja J, & Hyytiäinen M (2016). Netrin-1: A regulator of cancer cell motility? European Journal of Cell Biology, 95(11), 513–520. 10.1016/j.ejcb.2016.10.002 [DOI] [PubMed] [Google Scholar]
  57. Yung AR, Nishitani AM, & Goodrich LV (2015). Phenotypic analysis of mice completely lacking netrin 1. Development (Cambridge, England), 142(21), 3686–3691. 10.1242/dev.128942 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zhao J, & Mommersteeg MTM (2018). Slit–Robo signaling in heart development. Cardiovascular Research, 114(6), 794–804. 10.1093/cvr/cvy061 [DOI] [PMC free article] [PubMed] [Google Scholar]

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