Understanding the role of Shroom3 in the developing mouse myocardium

Jennifer L Carleton; Rami R Halabi; Jessica A Willson; Timothy F Plageman Jr; Darren Bridgewater; Qingping Feng; Thomas A Drysdale

doi:10.1371/journal.pone.0331583

. 2025 Sep 8;20(9):e0331583. doi: 10.1371/journal.pone.0331583

Understanding the role of Shroom3 in the developing mouse myocardium

Jennifer L Carleton ^1,^2,^*,^#, Rami R Halabi ^1,^2,^#, Jessica A Willson ^1,³, Timothy F Plageman Jr ⁴, Darren Bridgewater ⁵, Qingping Feng ^1,^2,⁶, Thomas A Drysdale ^1,^2,³

Editor: Federica Limana⁷

PMCID: PMC12416694 PMID: 40920782

Abstract

Loss of actin cytoskeleton control can hinder integral developmental and physiological processes and can be the basis for a subset of developmental defects. SHROOM3 is an actin binding protein, best characterized as being essential for neural tube closure in vertebrates. Shroom3 expression has also been identified in the developing heart, with some associated congenital heart defects. Here we show that the expression pattern of Shroom3 in the developing and adult mouse heart is specific to the myocardium. Using a gene trap line, we show that embryos with homozygous full-body Shroom3 loss die at birth due to exencephaly but also show congenital heart defects. This includes ventricular septal defects, semilunar valve abnormalities, and ventricle wall thinning. Adult mice heterozygous for Shroom3 loss also show ventricular thinning due to decreased cardiomyocyte size. To explore if SHROOM3 is operating in a cell autonomous manner in the cardiomyocytes, we utilized a floxed Shroom3 mouse line, allowing for spatial and temporal control of Shroom3 loss. Using an Nkx2–5-Cre recombinase, we targeted Shroom3 loss to the myocardium of the developing heart. Neonate pups with myocardial specific Shroom3 loss showed no significant impact on heart development, including no septal or valve defects, no ventricular thinning, and no change in viability into adulthood. Adult mice with myocardial specific Shroom3 loss showed no ventricular thinning and no change in cardiomyocyte size. These results show that the heart defects seen in full-body Shroom3 loss do not arise from myocardial specific loss. Rather, other cell types expressing Shroom3, such as the cardiac neural crest cells, may be directly contributing to cardiac development.

Introduction

Congenital heart defects (CHDs) are seen in approximately 1% of human births, making them one of the most prevalent congenital disorders in newborns [1–3]. With the progression of specialized surgical and diagnostic techniques, over 90% of children born with CHDs survive to adulthood [4,5]. The ability to reach adulthood presents a new challenge to researchers and clinicians: how does this impact our understanding of the potential heritability of CHDs? As a candidate gene for CHDs, SHROOM3 is necessary for proper heart development. Full-body loss of Shroom3 in the developing mouse shows a spectrum of CHDs including ventricular septal defects, double outlet right ventricle, and thinning of the myocardium in the left ventricle [6]. Shroom3 missense mutations have also been identified and are associated with heterotaxy and hemophagocytic lymphohistiocytosis in patient populations [7,8].

The SHROOM3 protein contains two major functional domains: the central Apx/Shrm-specific domain 1 (ASD1) allows for direct binding with f-actin, and the C-terminal ASD2 domain allows for direct binding with ROCK [9–16]. These functions allow for myosin light chain phosphorylation, inducing actomyosin constriction and thus SHROOM3-induced apical constriction [10,17–23]. There have also been indications that SHROOM3 is able to drive the re-distribution of γ-tubulin, allowing for apical-basal elongation, however the basis for this activity is less understood [20,24]. This cell shape change is described in polarized epithelial cells, where it plays a pivotal role in many developmental processes, most notably neural tube folding and closure, but also lens pit invagination, directional gut tube looping, and maintenance of glomerular structural integrity in the kidney [9–11,14,16,18,22,23,25–28]. In humans, Shroom3 loss-of-function variants have been linked with anencephaly, spina bifida, and cleft lip and palate [29,30]. While promoting rolling of the neural plate into the neural tube is the best-established function of SHROOM3, epithelial folding has not been documented during mammalian heart development. As such, the functional contributions of Shroom3 to cardiac morphogenesis remain unclear. We set out to understand where and when Shroom3 is expressed in the mouse heart, and what the consequences of Shroom3 loss are on cardiac development.

In this study we document Shroom3 expression specifically in the myocardium of the developing and adult mouse heart. We found that full-body loss of Shroom3 produces CHDs in embryos, similar to what has been described in Durbin et al. 2020. Adult mice with full-body Shroom3 loss show ventricular thinning due to decreased cardiomyocyte size. Here we also utilize a novel mouse line containing a floxed Shroom3 allele, allowing for spatial and temporal control of Shroom3 loss. In combination with a cardiac specific Nkx2–5 promoter driven Cre recombinase, we have selectively eliminated Shroom3 in the myocardium of the developing heart. Despite the loss, there were no notable CHDs or long-term effects on adult hearts seen in these mice. These results indicate that while SHROOM3 plays a role in proper heart development, myocardial SHROOM3 is not necessary for this role.

Methods

Mouse lines

All animal experiments were approved by Western University ACC (Protocol #015–2011 and #064–2019). All procedures were approved by the Council on Animal Care at The University of Western Ontario, in accordance with the guidelines of the Canadian Council on Animal Care.

Mouse line B6.129S4-Shroom3^{Gt(ROSA53)Sor/J} was purchased from The Jackson Laboratory on a C57BL⁄6 background. This line was generated using a gene trap assay to insert a SaβgalCrepA cassette under the control of the endogenous Shroom3 promoter [31]. This line will be referred to as Shroom3^Gt. A schematic for the gene trap vector insertion is depicted in S1A Fig.

Mouse line Tg(Nkx2–5-cre)9Eno (MGI:3514028) was donated to us by the laboratory of Dr. Qingping Feng and was maintained on a C57BL⁄6 background. This transgenic line shows the highest level of expression in the myocardium of the developing heart, beginning in the developing heart tube. Later during development this expression is localized to the left and right ventricular myocardium [32]. This transgenic line will be referred to as Nkx2–5-Cre.

Cyagen Biosciences was contracted to create a novel floxed Shroom3 allele mouse line. Using C57BL⁄6 embryonic stem cells, two loxP sites were inserted into the Shroom3 allele using a targeting vector containing a Neo cassette flanked by self-deletion anchor sites. These loxP sites were inserted to flank exon 5 of the Shroom3 gene on mouse chromosome 5. Upon Cre-mediated recombination of the floxed allele, exon 5 is excised, resulting in a constitutive knockout (KO) allele and a loss of function of the Shroom3 gene. This floxed Shroom3 mouse line has been named C57BL/6-Shroom3^tm1Shrc. A schematic for the targeting vector, targeted allele, and KO allele is depicted in S1B Fig. This line will be referred to as Shroom3^fl. A more in-depth description of the targeting vector and line generation can be found in Herstine et al., 2025.

Pregnant dams were assessed for a vaginal plug in the morning following mating. The morning that the vaginal plug was seen was designated as embryonic day 0.5 (E0.5). For embryonic studies, pregnant dams were euthanized using CO₂ gas inhalation and embryos were dissected at the required developmental time-point. For neonate studies, pups were taken on the day of birth and euthanized via decapitation. For adult studies, mice were euthanized using CO₂ gas inhalation at the desired age according to standard ACC guidelines.

Genotyping

Adult, neonate, and embryonic mouse genotypes were determined using tail tip DNA. Tissue specific genotyping was carried out using small pieces of the tissues of interest. DNA was isolated with phenol/chloroform extraction and gradient ethanol washed. Shroom3^Gt LacZ cassette insertion primer pairs: GT-F: 5’-GGTGACTGAGGAGTAGAGTCC-3’ and GT-R: 5’-GAGTTTGTGCTCAACCGCGAGC-3’. Nkx2–5-Cre transgene primer pairs: NCre-F: 5’-ACTGATTTCGACCAGGTTCGTT-3’ and NCre-R: 5’-CCCAGGCTAAGTGCCTTCTCTA-3’. Shroom3^fl loxP insertion primer pairs: loxP-F: 5’-CCAGGAAGGTTGCCAGAGTCTAGCT-3’ and loxP-R: 5’-CTGTCCGTTGTGGATGCTCGTG-3’. All PCR products were run on a 1% agarose gel with 1 Kb Plus DNA Ladder (Invitrogen, 10787018).

mRNA isolation, cDNA synthesis, RT-PCR

mRNA isolation from tissues of interest was performed with Trizol/chloroform extraction. 1ug of mRNA was used for cDNA synthesis with the qScript cDNA synthesis kit (QuantaBio, 95047). TBP was used as the housekeeping gene for RT-PCR. Shroom3^fl loxP recombination primer pairs, to be used with cDNA: ExonRec-F: 5’-TATCTCAGGGCACAATGGGC-3’, ExonRec-R: 5’-GGAGAAAGGAGATGGCAGGG-3’, and ExonRec-Ko: 5’-TTCCTGCTGAGAGTGGCCTA-3’. TBP housekeeping gene primer pairs, to be used with cDNA: TBP-F: 5’-ACAGGAGCCAAGAGTGAAGA-3’ and TBP-R: 5’-CTACTGAACTGCTGGTGGGT-3’.

Wholemount X-gal staining and clearing

Whole embryos from E9.5 to E13.5, or excised hearts of E14.5 embryos and older were used for wholemount X-gal staining. Heterozygous and wild type control samples were stained in parallel. n = 10 for embryonic and adult stages. A mixed cohort was used for all X-gal staining.

Tissues were fixed for 20 minutes to 2 hours depending on specimen thickness in 4% PFA, and washed overnight at 4°C in PBST. Samples were stained with X-gal staining solution (0.5 mg/ml X-Gal in DMF, 400µM potassium ferricyanide, 400µM potassium ferrocyanide, 2mM MgCl2) in PBST, at 37°C overnight or until the desired level of staining was developed. Tissues were post fixed in 4% PFA for 2 hours. Samples were then cleared as described in [33] with the following modifications: tissues were cleared in scintillation vials for one day each in 20% and 50% glycerol (v/v) with 1% KOH (w/v) in PBS at room temperature, followed by 2–3 days in 80% glycerol (v/v) with 1% KOH (w/v) in PBS at 37°C and finally at 100% glycerol (v/v) with 1% KOH (w/v) in PBS at room temperature until desired translucencies were achieved.

Hematoxylin and eosin staining

Embryonic, neonate, and adult hearts were fixed in 4% PFA and embedded in paraffin. Tissues were sectioned at 5μm and stained with CAT hematoxylin (BioCare Medical), followed with Tasha’s Bluing Solution (BioCare Medical), and then Eosin Y (0.25% in ethanol, Fisher Scientific). A mixed cohort was used for all H&E staining.

Congenital and postnatal heart morphology assessment

Adult, neonate, and embryonic hearts were collected, processed, and H&E stained as described above. Shroom3^Gt hearts were serially sectioned in the transverse plane. Nkx2–5-Cre;Shroom3^fl hearts were serially sectioned in the frontal plane. A mixed cohort was used for all morphological analysis.

Ventricular septal defects (VSDs) were counted when a break in the ventricular septum was found in the histological section. Membranous VSDs were classified as those found within the top third of the cardiac septum. Muscular VSDs were counted when the defect was localized to the thicker, muscular portion of the ventricular septum. n = 24 for Shroom3^+/+ and Shroom^+/Gt E18.5 embryos and 33 for Shroom3^Gt/Gt E18.5 embryos. n = 23 for Nkx2–5-Cre;Shroom3^fl/fl and Shroom3^fl/fl neonate mice and 8 for Nkx2–5-Cre;Shroom3^+/fl neonate mice.

Semilunar valve defects were based on visual observation. Thick pulmonary valves and malformed aortic valves were comparable to the abnormalities seen in the ADAM17 knockout mouse which exhibits semilunar valve defects [34]. This was used as a guide for valve defects, in addition to valve comparison in wild type littermate controls. n = 18 for Shroom3^+/+ and Shroom^+/Gt E18.5 embryos and 27 for Shroom3^Gt/Gt E18.5 embryos. n = 23 for Nkx2–5-Cre;Shroom3^fl/fl and Shroom3^fl/fl neonate mice and 8 for Nkx2–5-Cre;Shroom3^+/fl neonate mice.

To measure ventricular thickness in transverse sections, 10 measurements were taken per section on 3 sequential sections per sample, each 5μm apart. Compact layer was defined as the region between the outside of the epicardium and beginning of the trabecular zone. In example images the compact myocardial layer is delineated from the trabeculae with a dotted line. Only sections where all four chambers were visible in transverse sectioned were used for embryonic compact layer measurements. n = 6 for Shroom3^Gt/Gt, Shroom3^+/Gt, and Shroom3^+/+ E18.5 embryos, and n = 3 for Shroom3^+/Gt and Shroom3^+/+ adult hearts.

To measure ventricular thickness in frontal sections, measurements were taken from 4 sequential sections, each 10μm apart, providing a 30μm area of average ventricle thickness. This was repeated and averaged from 4 locations within the middle of the ventricle. In example images the compact myocardial layer is delineated from the trabeculae with a dotted line. Only heart sections in which the interior of the entire ventricle was visible were used. n = 6 for Nkx2–5-Cre;Shroom3^fl/fl, Nkx2–5-Cre;Shroom3^+/fl and Shroom3^fl/fl neonate hearts and adult hearts.

Immunofluorescence

Deparaffinized tissue sections underwent antigen retrieval in a 95°C bath of sodium citrate buffer (10mM sodium citrate, 0.1% Triton-X, pH 6.0) for 25 minutes. Sections were then permeabilized with 0.2% TBST (Tween-20) for 10 minutes. Tissue sections were blocked in 10% Goat serum in 1% BSA in TBST for 1 hour. Wheat germ agglutinin Alexa Fluor 594 conjugate (5 µg/ml; Invitrogen) was used to visualize myocardial cell borders. Slides were counterstained with DAPI (1:1000, Invitrogen), cover slipped with PermaFluor Aqueous Mounting Medium (Fisher Scientific) and stored in the dark at −20°C.

Cardiomyocyte area measurements

Adult mouse hearts were stained with Wheat germ agglutinin Alexa Fluor 594 conjugate at P8m. Cardiomyocyte cell area was measured only if cells had completely labeled membranes and a centrally located nucleus. To standardize where cardiomyocyte measurements were taken, only sections where the papillary muscle was visible were used. n = 3 for Shroom3^+/Gt and Shroom3^+/+ hearts and n = 3 for Nkx2–5-Cre;Shroom3^fl/fl, Nkx2–5-Cre;Shroom3^+/fl and Shroom3^fl/fl hearts.

Statistical analysis

One way ANOVA was used to assess the statistical significance of compact layer thickness differences in Shroom3^Gt embryos, with a post-hoc Tukey’s test. P < 0.05 was considered significant. An unpaired two-tailed t-test was used to determine statistical significance for compact layer and cross-sectional area measurements in adult Shroom3^Gt mice. P < 0.05 was considered significant. One way ANOVA was used to assess the statistical significance of the heart weight, body weight, and heart weight to body weight ratios in the Shroom3^fl adult mice, as well as the cross-sectional measurements of adult Shroom3^fl cardiomyocytes, and of the compact layer thickness differences in Shroom3^fl neonates. All statistical analysis performed using GraphPad Prism 8.0 software.

Results

Shroom3 is expressed throughout the embryonic and adult myocardium

The expression range of Shroom3 was assessed using the reporter lacZ gene found within the Shroom3^Gt cassette. Whole embryos were used from E9.5 – E13.5 and excised hearts were used from E14.5 into adulthood. Wild type littermate controls were stained in parallel.

Using the reporter gene, Shroom3 expression was detected in wholemount embryonic hearts beginning at E10.5 (Fig 1A, right) and was observed at all subsequent developmental stages including E18.5 (Fig 1I, right). Representative images for each embryonic day between these time-points are pictured in Fig 1. This expression was specific to the myocardium of the atria and ventricles of the heart and was not seen in the outflow tracts or great arteries. X-gal staining intensity increased as development progressed. When assessing sectioned embryonic hearts, Shroom3 expression was seen earlier in development at E9.5 within the compact myocardium (Fig 2A). At E14.5 staining was seen in the trabeculae and septum, and the myocardium surrounding the base of the outflow tracts (Fig 2B–D). Shroom3 expression was not observed in the epicardium, endocardial cushions or aortic valves at any timepoint.

Fig 2 — X-gal staining in paraffin sections of *Shroom3*^+/Gt embryonic heart. A) The primitive ventricle of an E9.5 mouse embryo showed presence of lacZ in the myocardium (arrow) but not the endocardium. B-D) Hearts sections of an E14.5 embryo showed widespread staining in the compact layer myocardium, trabecular myocardium, ventricular septum and the base of the aorta. No staining was detected in the endocardium, epicardium (arrows), aortic valve, or endocardial cushions. Ao: aorta; e: epicardium; en: endocardium; m: myocardium.

In wholemount postnatal hearts, Shroom3 expression was also seen in the myocardium of the ventricles and atria, and the myocardium surrounding the base of the outflow tracts. The widespread, high intensity staining which is seen in newborn hearts (Fig 3A) is similar to staining seen in late-stage embryonic hearts. However, this staining intensity dramatically decreases by three months of age (Fig 3B) and eight months of age (Fig 3C). Notably, the staining intensity of the left atrium remained strong in comparison to the right atrium in adulthood (Fig 3C). Assessing sectioned adult hearts, Shroom3 expression was again specific to the compact and trabecular myocardium and absent from the epicardium and endocardium, with increased staining in the compact layer (Fig 4A, B). This is a similar pattern to that seen in embryonic heart sections. Reflecting the findings in wholemount hearts, increased staining in the left atrium compared to the right atrium was also observed in section (Fig 4C, D).

Fig 3 — X-gal staining in postnatal mouse hearts detecting for presence of lacZ in *Shroom3*^+/Gt (right), or littermate controls (left). Wholemount detection began on the day of birth (A), where an expression pattern similar to embryonic hearts was observed. Expression of lacZ in 3-month-old (B) and 8-month-old (C) mice, respectively, showed increased staining in the left atrium compared to the right atrium. Widespread expression throughout the ventricles appeared to be consistent with embryonic expression, however this was difficult to observe in wholemount. Expression was observed in the myocardium surrounding the base of the outflow tracts. Wild type control hearts stained in parallel showed no background staining. LA: left atrium; LV: left ventricle; OFT: outflow tracts; RA: right atrium; RV: right ventricle.

Fig 4 — X-gal staining in paraffin sections of *Shroom3*^+/Gt 8-month-old adult heart. Transverse sections through the left (A) and right (B) ventricle showed intense staining in the compact layer myocardium and to a lesser extent the trabeculae. No staining was seen in the epicardium. Sagittal sections through the right (C) and left (D) atrium showed increased staining intensity in the left atrium. No staining was found within the endocardium or epicardium. ca: coronary artery; e: epicardium; en: endocardium; m: myocardium; tr: trabeculae.

Full-body loss of Shroom3 during development shows congenital heart defects in embryos and ventricular thinning and decreased cardiomyocyte size in adults

As Shroom3 was seen in much of the developing heart and was present for the majority of cardiogenesis, embryos were assessed at the end of development for CHDs after full-body developmental Shroom3 loss. In E18.5 embryos three types of CHDs were found. Firstly, 55% of Shroom3^Gt/Gt embryos contained VSDs (18 of 33 embryos). Of these VSDs, 72% were membranous, occurring in the first third of the ventricles (Fig 5B, C), and 28% were muscular, occurring lower in the muscular septum (Fig 5E, F). Secondly, thinning of the left ventricle wall was also found in these embryonic hearts, where a significant, stepwise reduction in compact myocardium thickness was seen between WT, Shroom3^+/Gt, Shroom3^Gt/Gt hearts (Fig 6C). The compact myocardium was defined as the layer between the epicardium and the trabecular myocardium boundary (Fig 6B, dotted line). No significant difference in compact myocardium thickness was seen between genotypes in the right ventricle. The morphology of the trabeculae networks was also assessed at this time, however there was no difference seen in network branching between genotypes. Finally, abnormal aortic valves were seen in 15% of Shroom3^Gt/Gt embryos (4 of 27 embryos), and abnormal pulmonary valves were seen in 26% of Shroom3^Gt/Gt embryos (7 of 27 embryos). This appeared as aortic valves with no clear delineation of the leaflet cusps (Fig 7C and D), and pulmonary valves where leaflet cusps were clustered within the valve lumen (Fig 7G and H). In all embryonic hearts, there was no observed incidence of cardia bifida, failure to loop or form chambers, failure to trabeculate, nor hypertrophy. As seen in the representative images in Fig 2, normal gross morphology was observed for all hearts at all developmental time points, where no obvious alterations in overall size and shape were seen.

Fig 5 — H&E staining in E18.5 hearts. A&D) Transverse sections of wild type hearts show an intact ventricular septum between the two ventricles, at two different heights of the heart. B) Transverse section of a *Shroom3*^Gt/Gt heart with a membranous VSD (black arrow). C) Magnification shows an otherwise intact tissue morphology. E) Transverse section of a *Shroom3*^Gt/Gt heart with a muscular VSD (black arrow). F) Magnification shows an otherwise intact tissue morphology. Incidence: *Shroom3*^+/+ = 0/24; *Shroom3*^+/Gt = 1/24; *Shroom3*^Gt/Gt = 18/33. LA: left atrium; LV: left ventricle; RA: right atrium; RV: right ventricle.

Fig 6 — Measurements of the compact layer thickness in the wall of the right and left ventricle of E18.5 embryos. The compact layer was defined as the space between the outer epicardium and where the trabeculae begin. A&B) Transverse section demonstrating where the compact layer measurements were taken for each heart section. Lines in B indicate the border of the compact layer. C) Quantification of the compact layer width showed no significant differences in the right ventricle across all genotypes. In the left ventricle, compact layer thickness was significantly reduced in *Shroom3*^+⁄Gt (P < 0.01) and *Shroom3*^Gt/Gt (P < 0.001) hearts compared to littermate controls. Additionally, *Shroom3*^Gt/Gt left ventricle compact layer thickness was significantly thinner than *Shroom3*^+⁄Gt (P < 0.01). ***P < 0.001, **P < 0.01. ± SD. RV: right ventricle; LV: left ventricle. n = 6 per genotype.

Fig 7 — H&E staining in E18.5 hearts. Assessment of phenotype was based on comparison to the littermate controls. A&E) Transverse sections of wild type hearts with normal development of the aortic (A) and pulmonary (E) valve. Magnifications to the right (B&F). C) Transverse section of *Shroom3*^Gt/Gt heart at the site of the aortic valve with undefined cusp formation. D) Magnification of C. Compared to the control valve, leaflets of the aortic valve in *Shroom3*^Gt/Gt hearts are not clearly defined (black arrow) and do not extend into the luminal space of the valve. Incidence: *Shroom3*^+/+ = 0/18; *Shroom3*^+/Gt = 0/18; *Shroom3*^Gt/Gt = 4/27. G) Transverse section of *Shroom3*^Gt/Gt heart at the site of the pulmonary valve with thick pulmonary cusps. (H) Magnification of G. From visual comparison, pulmonary valves in *Shroom3*^Gt/Gt hearts were thickened compared to control valves. Incidence: *Shroom3*^+/+ = 0/18; *Shroom3*^+/Gt = 1/18; *Shroom3*^Gt/Gt = 7/27. Ao: aorta; AV: aortic valve; LA: left atrium; LV: left ventricle; PV: pulmonary valve; RA: right atrium; RV: right ventricle.

Due to lethal extra-cardiac developmental defects, Shroom3^Gt/Gt embryos are not able to survive into adulthood. However, Shroom3^+/Gt mice can survive past weaning and into adulthood. Therefore, mice heterozygous for full-body Shroom3 loss were assessed for long-term consequences of left ventricular thinning during development. At three months postnatal, Shroom3^Gt/+ hearts showed significantly reduced left ventricular wall thickness compared to WT controls (Fig 8A–C). This was also seen at eight months postnatal (Fig 8D–F). As ventricular wall thinning may result in stress on the ventricles and thus cause compensatory cardiomyocyte hypertrophy, we also assessed cardiomyocytes at eight months postnatal. Using wheat germ agglutinin to outline cardiomyocyte cell walls, surface areas of cardiomyocytes from the left ventricle compact myocardium were measured. Cardiomyocytes from Shroom3^Gt/+ left ventricles were found to be significantly smaller than the WT controls (Fig 9).

Fig 8 — Transverse sections of adult mouse hearts stained with hematoxylin and eosin. Only the compact layer of the left ventricle was compared between genotypes for thinning. The compact layer was defined as the space between the outside of the epicardium and where the trabeculae begin. A&B) Wild type and *Shroom3*^+/Gt 3-month-old hearts. Arrows and lines indicate where measurements in the left ventricle were taken. C) Quantification of the thickness of the compact layers of 3-month-old hearts showed a significant decrease in left ventricle wall thickness in the heterozygous mice (P < 0.0001) when compared to the wild-type. D&E) Transverse sections of wild type and *Shroom3*^+/Gt 8-month-old hearts. Arrows and lines indicate where measurements in the left ventricle were taken. F) Quantification of the thickness of the compact layers of 8-month-old hearts showed a significant decrease in left ventricle wall thickness in the heterozygous mice (P < 0.0001) when compared to the wild type. ****P < 0.0001. ± SD. RV: right ventricle; LV: left ventricle. n = 3 per genotype at both ages. Mixed cohorts used at 3 months and 8 months.

Fig 9 — Hearts from 8-month-old mice were stained with wheat germ agglutinin conjugated to Alexa Fluor 594 (red) to mark the cell membranes and DAPI (blue). Only cells with fully defined and intact cell membranes and a centralized nucleus were used for measurements. A) Wild type heart section showing normal cardiomyocytes in cross section. B) *Shroom3*^+/Gt heart cardiomyocytes in cross section. C) Hearts of heterozygous mice showed a significantly smaller cross-sectional area (P < 0.0001) when compared to the wild type. ****P < 0.0001. ± SD. n = 3 per genotype. Mixed cohort used.

Floxed allele recombination and verification

Following this data, we aimed to investigate if these developmental defects were due to Shroom3 loss in the myocardium during development. Our lab and two collaborating labs funded the creation of a novel floxed Shroom3 allele (Shroom3^fl). Placement of the loxP sites was designed to splice around exon 5, creating a null Shroom3 allele which does not express Shroom3 mRNA. This line has recently been published by our collaborators to study optic cup morphogenesis [28].

The specificity of the loxP targeting was assessed by making a compound allele. The Shroom3^fl line was crossed with the Shroom3^Gt line, resulting in embryos containing the compound Shroom3^Gt/fl alleles. This would create a null allele from the gene trap insertion and a null allele from recombination of the floxed Shroom3 allele, driven by the Cre recombinase inside the Shroom3^Gt cassette. Compound allele embryos phenocopied Shroom3^Gt/Gt embryos at E10.5, where similar neural tube closure defects were seen (Fig 10A and B). The same phenocopying was seen at E18.5, where the same exencephaly, gut tube looping, and open eye phenotypes were seen (Fig 10C and D). Using similar methods, our collaborators have obtained similar results at E12.5 [28].

Fig 10 — A) Full-body *Shroom3* loss using the gene trap line, visualized at E10.5. Homozygous *Shroom3* loss on the right, and littermate control on the left. On the right, neural tube closure defects can be seen. B) Embryos from a *Shroom3*^Gt/+ X *Shroom3*^fl/fl cross at E10.5. 8 embryos were produced. 4 showed the WT phenotype (left) and 4 showed the mutant phenotype (right). This mutant phenotype seen in A, right. Mating cross and data collection by T.J Plageman. C) Full-body *Shroom3* loss using the gene trap line, visualized at E18.5. Homozygous *Shroom3* loss on the right, and littermate control on the left. On the right, neural tube defects, eye defects, and gut tube looping defects can be seen. D) E18.5 embryos from *Shroom3*^Gt/+ X *Shroom3*^fl/fl. 6 embryos produced, 3 with WT phenotype (left), 3 with neural tube defects, open eye phenotype, gut tube looping defects (right).

To target Shroom3 loss to the developing myocardium, Shroom3^fl mice were crossed to an Nkx2–5-Cre recombinase line. This transgenic Cre recombinase begins expression at E7.5 in cardiac precursor cells, and expression remains on for the duration of development and into adult life [35]. Primers to amplify exon 5 of the conditional KO Shroom3^fl allele, and the recombined Shroom3^fl allele were used with cDNA. Anticipated band sizes for the conditional KO allele (181 bp) and the recombined allele (306 bp) were seen in agarose gel in both neonate and adult heart samples (S2A Fig). We also verified that this loss was specific to the heart by using kidney samples from the same mice. cDNA samples from neonate control hearts and neonate knockout hearts (n = 4) were then amplified for the conditional KO allele or the recombined allele (S2B Fig). Band densitometry was used to quantify recombination efficacy in the Nkx2–5-Cre;Shroom3^fl hearts. The average difference in band density between Shroom3^fl littermate controls and Nkx2–5-Cre;Shroom3^fl hearts was 74.5% (S2C Fig). This indicates at least a 74% difference in Shroom3 expression between Nkx2–5-Cre;Shroom3^fl mice and littermate controls, or a 74% knockout of the Shroom3^fl allele from our myocardial specific Cre driver.

Myocardial Shroom3 loss during development does not produce congenital heart defects in embryos and does not impact adult cardiac or cardiomyocyte morphology

To assess for perinatal lethality in our myocardial specific knockout, four Nkx2–5-Cre;Shroom3^+/fl X Shroom3^fl/fl breeding pairs were set up, two with paternally inherited Cre, two with maternally inherited Cre. Neonates born from these crosses were monitored for the first 12 hours of life. All observed neonates were born alive, however any deaths within the first 12 hours of life were recorded and bodies were collected. From a final n-value of 44, 21 neonates were born with the Cre-recombinase, and 23 were born without. Within the first 12 hours of life, 5 neonates from both groups died. This indicates a mortality rate of 23% across Nkx2–5-Cre;Shroom3^+/fl and Nkx2–5-Cre;Shroom3^fl/fl neonates, and 21% across Shroom3^+/fl and Shroom3^+/fl neonates. Overall, no trend in mortality was seen across genotypes in neonates. Additionally, observed genotypes across all neonates did not differ from expected mendelian ratios (X² = 0.72, df = 3, p = 0.05), indicating no significant in-utero mortality.

Neonates from this cross were assessed for CHDs using the same methods as in Shroom3^Gt embryos. In both Nkx2–5-Cre;Shroom3^+/fl and Nkx2–5-Cre;Shroom3^fl/fl neonate hearts, no VSDs were seen (Fig 11). This includes neither membranous nor muscular VSDs, both of which had been seen in Shroom3^Gt/Gt embryos. Additionally, in comparing to littermate controls, aortic and pulmonary valves were morphologically normal regardless of phenotype (Fig 12). Additional images of these valves are displayed in S3 Fig. Finally, no significant differences were seen in the ventricular compact layer thickness between Nkx2–5-Cre;Shroom3^+/fl, Nkx2–5-Cre;Shroom3^fl/fl neonates and littermate controls (Fig 13). This was consistent in both the right (p = 0.1522) and left ventricle (p = 0.8859) (Fig 13C).

Fig 11 — Neonates from the *Nkx2-5-Cre;Shroom3*^+/fl X *Shroom3*^fl/fl cross who died within 24 hours of birth display no VSDs. This was consistent between littermate neonates which did not contain Cre (A), littermates heterozygous for *Shroom3* recombination (B), and littermates homozygous for *Shroom3* recombination (C). n = 23 for *Nkx2-5-Cre;Shroom3*^fl/fl and *Shroom3*^fl/fl. n = 8 for *Nkx2-5-Cre;Shroom3*^+/fl. Sectioned in the frontal plane.

Fig 12 — Neonates from the *Nkx2-5-Cre;Shroom3*^+/fl X *Shroom3*^fl/fl cross show normal pulmonary and aortic valve leaflets compared to littermate controls. This was consistent between littermate neonates which did not contain Cre (A), littermates heterozygous for *Shroom3* recombination (B), and littermates homozygous for *Shroom3* recombination (C). n = 23 for *Nkx2-5-Cre;Shroom3*^fl/fl and *Shroom3*^fl/fl. n = 8 for *Nkx2-5-Cre;Shroom3*^+/fl. Sectioned in the frontal plane.

Fig 13 — Measurements of compact myocardium were taken from *Nkx2-5-Cre;Shroom3*^+/fl X *Shroom3*^fl/fl neonate litters. B) Measurements were taken between the two lines, for both the left and right ventricle. C) No significant differences were found in wall thickness between the different genotypes for both the right ventricle (p = 0.1522) and left ventricle (p = 0.8859). Control label indicates *Shroom3*^+/fl and *Shroom3*^fl/fl samples. One way ANOVA ±SD. n = 6 per genotype.

Mice born from this cross were also monitored for a year with no notable issues in overall health or breeding. At one year of age no alterations to gross morphology in adult hearts were observed. This includes no minor VSDs which persisted into adulthood and no alterations to ventricular wall thickness (Fig 14A, first and second column). Additionally, there were no morphological alterations in the semilunar valves of these hearts (Fig 14A, third and fourth column). No alterations in body weight (Fig 14B, left) or heart weight (Fig 14B, middle) were seen in these mice, reflecting no changes in heart weight to body weight ratios (Fig 14B, right). Thus, there is no indication of compensatory cardiac hypertrophy after one year of myocardial Shroom3 loss.

Fig 14 — A) Representative images of 1y adult hearts from myocardial *Shroom3* loss during development. *Shroom3*^fl, *Nkx2-5-Cre;Shroom3*^+/fl, and *Nkx2-5-Cre;Shroom3*^fl/fl mice were observed to show no changes in heart shape or size externally and internally, with no changes in ventricular thickness, and no alterations to the semilunar valves. All hearts were equal in appearance. B) Body weight (left), heart weight (middle), and heart weight to body weight ratios (right) from *Nkx2-5-Cre;Shroom3*^+/fl X *Shroom3*^fl/fl offspring at 1y, by genotype. No statistical differences seen (left, p = 0.3490; middle, p = 0.9156; right, p = 0.4829). Control label indicates *Shroom3*^+/fl and *Shroom3*^fl/fl samples. One-way ANOVA ±SD. n = 19 for *Shroom3*^fl, n = 19 for *Nkx2-5-Cre;Shroom3*^+/fl, n = 12 for *Nkx2-5-Cre;Shroom3*^fl/fl. No significance seen in sex segregated analysis.

Adult hearts were also assessed at 8 months of age for any changes in cardiomyocyte morphology using the same wheat germ agglutinin staining as in Shroom3^Gt adults. The average cardiomyocyte area between littermate controls, Nkx2–5-Cre;Shroom3^+/fl, and Nkx2–5-Cre;Shroom3^fl/fl mice were not significantly different (p = 0.1956) (Fig 15).

Fig 15 — Hearts from 8-month-old mice were stained with wheat germ agglutinin conjugated to Alexa Fluor 594 (red) to mark the cell membranes and DAPI (blue). Only cells with well-defined and intact cell membranes and a centralized nucleus were used for measurements. A) Littermate control heart section showing normal cardiomyocytes. B) *Nkx2-5-Cre;Shroom3*^+/fl heart cardiomyocytes in cross section. C) *Nkx2-5-Cre;Shroom3*^fl/fl heart cardiomyocytes in cross section. D) No significant differences in cardiomyocyte cross sectional area were seen in between all three genotypes. Control label indicates *Shroom3*^+/fl and *Shroom3*^fl/fl samples. (p = 0.1956) ±SD. n = 3 per genotype.

Discussion

Shroom3 expression begins early in heart development and continues in the adult heart

We aimed to assess when expression of Shroom3 began during heart development, if it was sustained during and after development, and where this expression was localized. Through X-gal staining, we have demonstrated that Shroom3 is expressed in the developing heart as early as E9.5, continues for the entirety of development, and into adult life. This expression was localized to the myocardium and trabeculae of the atria and the ventricles, and myocardium surrounding the base of the outflow tracts. Shroom3 expression was not found in the outflow tracts, epicardium, endocardium, or the valves of the developing and adult heart. Due to the increase of X-gal staining intensity over the course of development, it is likely that Shroom3 expression is increasing in the embryonic heart as gestation progresses. However, staining intensity decreased in the adult heart. This shift in expression between embryonic and adult hearts may be due to the functionality of the heart at different stages of life. In utero, trabeculae contribute to the force of cardiac output, and act as an important biomechanical support structure for proper downstream heart development [36–38], whereas adult hearts rely on the muscular compacted myocardium in the ventricular walls.

A recent study has established spatially distinct transcriptomes in cardiomyocytes of adult mice which radiate out from an area of infarct [39]. In this study, Shroom3 was expressed in a transitional cell population found between non-ischemic tissue and cardiomyocytes within the infarct zone, but not highly expressed within either of these areas. It is known that as the heart tries to recover from ischemia, developmental programming within the cells is re-established in a compensatory, regenerative manner, for example the change from adult α-myosin heavy chain to embryonic β-myosin heavy chain [40,41]. This presents the possibility that under conditions of heart failure or ischemic damage, Shroom3 may be upregulated in the adult heart in a compensatory manner. This supports our findings from X-gal staining that Shroom3 expression is decreased from developmental levels in normal adult mouse hearts.

In adult hearts, there was also a notable difference in X-gal staining intensity between the left and right atria, with higher Shroom3 expression in the left atrium compared to the right. This staining pattern is similar to that of Pitx2, which also shows greater expression in the left mouse atrium than the right [42]. While Shroom3 is activated by Pitx2 in the developing gut, this interaction has not been studied in the heart at any time-point [17,27]. Additionally, as cell shape changes like those seen during gut tube looping are not present in adult atria, the purpose for this interaction is unknown. Pitx2c is known to be involved in left-right asymmetry during embryonic development [43]. While a Shroom3 missense mutation has been identified in a human patient with heterotaxy [7], it seems unlikely that Shroom3 is implicated in left-right asymmetry in the heart, as no looping defects were seen in this study. Using the same Shroom3^Gt line Durbin et al., (2020) had also investigated Shroom3 expression patterns in the mouse heart, however they did not document differential staining of the adult atria.

SHROOM3, but not myocardial SHROOM3, is required for normal cardiac morphogenesis

As Shroom3 is so widespread in the developing heart, we aimed to investigate the implications of full-body Shroom3 loss on cardiogenesis using the Shroom3^Gt mouse line. In E18.5 embryos, this loss resulted in thinning of the compact myocardium isolated to the left ventricle. Both membranous and muscular ventricular septal defects were also observed in these embryos, as well as malformation of the semilunar valve leaflets. These phenotypes were not completely penetrant amongst all embryos and did not always appear together in the same combinations. These results can be compared to Durbin et al., (2020) who also showed ventricular septal defects and thinning of the left ventricle in E14.5 Shroom3^Gt/Gt embryos. However, this group did not demonstrate remodelling complications of the semilunar valves. This may be due to assessment of embryos at different developmental time-points. At E14.5, the ventricles of the mouse heart have not completed proliferation and compaction, which occurs from E15.5 until birth [44]. Downstream of this, cardiac valves, but particularly the semilunar valves, are known to be morphologically influenced by cardiac hemodynamics and pressure during development [45–47]. It is possible that valve defects from Shroom3 loss were only seen for the first time in our study due to the influence of the hemodynamic changes from the hearts undergoing ventricular compaction at later developmental stages.

Following this, we investigated if it was SHROOM3 arising from the myocardium which, when lost, was causing these defects. However, when Shroom3 was eliminated using an Nkx2–5 promoter driven Cre recombinase, no abnormal phenotypes were seen in neonate or adult hearts. This included no VSDs, valve deformities, and no ventricular thinning.

With this data, we have demonstrated that Shroom3 loss does cause CHDs, but these defects are not due to loss of Shroom3 in the myocardium. Thus, we hypothesize that the defects seen in the Shroom3^Gt knockout must be due to loss of Shroom3 in extracardiac cell populations which also contribute to cardiac development. The best candidate for this is the cardiac neural crest cell (cNCCs). cNCCs are a migratory cell population which arise dorsal to the neural tube. They have been well established in the formation of the great arteries, including the patterning and smooth muscle of the aorta, and septal formation between the aorta and pulmonary artery, as well as the patterning of semilunar valve leaflets and the cardiac cushions, the structure which gives rise to the semilunar valves [48,49]. Recently cNCCs have been demonstrated to contribute to the cardiac musculature, where fluorescently labelled cNCCs were stably integrated into the ventricular myocardium and began expressing Troponin and Myosin Heavy Chain in chick and mouse embryos [50]. Additionally, these labelled cNCCs were seen in the cardiac cushion. This is important, as the semilunar valve remodelling complications presented in our work were not ascribable to myocardial Shroom3 loss, as the cardiac cushion is endocardial in origin [45,51,52]. Supporting this hypothesis, Durbin et al., (2020) documented Shroom3 expression in the cardiac neural crest cells at E9.5. cNCC loss contributes to many CHDs including outflow tract septation defects, double outlet right ventricle, VSDs, and valve malformations [49,53–59]. Thus, it is possible that cNCCs expressing Shroom3 may integrate into the myocardium and the cardiac cushions during embryonic development. Loss of Shroom3 in this cell population, rather than Shroom3 arising from cardiac mesodermal cells, may be sufficient to produce the CHDs described here. Supporting this, Nkx2–5 is not expressed in the cNCCs of the mouse embryo, thus our conditional knockout model of Shroom3 would not have targeted this specific subset of cells. This evidence allows for the conclusion that Shroom3 in the heart does not act in a cell autonomous manner, and that external cell types which also express Shroom3, particularly the cNCCs, likely contribute to the proper development of the heart. It should be noted that while we were able to show high levels of recombination, we cannot formally rule out that a small number of myocardial cells may still be expressing Shroom3 and could contribute to cardiac morphogenesis.

Shroom3 loss causes left ventricular thinning and decreased cardiomyocyte size

Finally, we assessed the impact of this developmental loss on the adult heart. After full body Shroom3 loss, three-month and eight-month-old mice showed significant thinning in the left ventricle, a phenotype which continued from development. This was found to be due to decreased cardiomyocyte cross-sectional area compared to littermate controls. However, in adult hearts with myocardial specific loss of Shroom3 during development, neither left ventricular thinning, nor smaller cardiomyocytes, were seen. Again, this was similar to developmental phenotypes which also found no ventricular thinning. The best-established function of SHROOM3, altering apical-basal cell shape in epithelial cells, is not seen in the myocardium. Rather, myocardial cells are polarized via the planar cell polarity pathway (PCP) [60–62]. Direct functional interactions between SHROOM3 and the PCP pathway during neural tube closure have been identified, as SHROOM3 is directly associated with Lrp2 and Dishevelled2 [14,63]. This interaction between Dishevelled2 has already been validated in the heart [6]. It is then possible that this interaction between SHROOM3 and the PCP pathway may support a function for SHROOM3 in actin organization within the cell, rather than causing cell shape changes in an apical-basal polarity system. This is supported by findings of SHROOM3 function during axon development, where SHROOM3 has been shown to regulate the projection of the axons through the recruitment and organization of F-actin [64]. Thus, actin organization controlled by SHROOM3 may allow for proper heart muscle development and maintenance of shape through the polarization and cell structure pathways.

Paramount to following this work, a Cre recombinase line should be used to selectively knock out Shroom3 in the cNCCs during development. Markers for this cell type, particularly Wnt1, are readily available. It is currently unknown if SHROOM3 is implicated in the induction or migration of the cNCCs. Further investigation is needed to determine the role of SHROOM in the cNCCs, and how this cell population impacts heart development. Additionally future analysis should assess the three-dimensional structure of the heart as initial morphogenesis of the organ can contribute to later maturation and development of the structure [36]. Cardiac functionality, including internal pressure changes, affect later cellular proliferation [46,65]. Thus, to understand the true geometry and arrangements of the cells and their actin networks, and to better understand the consequences of Shroom3 loss, 3D reconstruction is needed.

Conclusion

From this data, we conclude that while SHROOM3 is an important contributor to mammalian heart development and postnatal heart structure, SHROOM3 derived from the myocardium of the developing heart is not the sole contributor. While loss of Shroom3 in the entire developing mouse body resulted in CHDs, including ventricular and septal defects, semilunar valve defects, and smaller cardiomyocytes, myocardial specific loss of Shroom3 during development has no impact on heart morphology developmentally or postnatally. In this report, we have also presented the verification that our novel Shroom3^fl line produces a null allele upon recombination, and that this allele can be activated in a temporally and spatially specific manner.

Supporting information

S1 Fig. Schematic of Shroom3 gene trap allele and novel floxed Shroom3 allele.

A) Schematic of Shroom3 gene trap line B6.129S4-Shroom3^{Gt(ROSA53)Sor/J}. The inserted cassette includes a Splice Acceptor site (SA), an E.coli lacZ, Cre recombinase (Cre), and a polyadenylation sequence (pA). This inserted cassette is under control of the endogenous Shroom3 promoter. Insertion between exon 3 and exon 4 prevents proper mRNA formation, preventing functional protein from being made. B) Schematic of the novel floxed Shroom3 allele, created by Cyagen. LoxP sites were inserted to flank exon 5 of the endogenous Shroom3 gene. Upon recombination, the constitutive knockout allele was designed to produce a null allele.

(TIF)

pone.0331583.s001.tif^{(504.4KB, tif)}

S2 Fig. Semi-quantitative PCR for Shroom3^fl recombination and band quantification with densitometry.

A) Primers were designed to detect recombination of the floxed Shroom3 allele recombination. Forward and reverse primers create 181 bp band (lower band, right gel). Forward and knockout primers create 306 bp band (upper band, right gel). Samples from neonate and 10m adult mouse heart, with kidney for tissue specificity B) cDNA samples from E18.5 hearts. Genotypes for samples are indicated. Samples were run with F-R primer sets and F-Ko primer sets. C) Average band density from F-R primer set, compared to band density from F-Ko. n = 4. F = Forward, R = Reverse, Ko = Knockout, H = Heart, K = Kidney. Raw gel images and lane annotations can be found at Open Science Framework (https://doi.org/10.17605/OSF.IO/NUMF2).

(TIF)

pone.0331583.s002.tif^{(3.1MB, tif)}

S3 Fig. Additional images of Nkx2–5-Cre;Shroom3^fl/fl neonate semilunar valve morphologies.

Images of semilunar valves taken from neonate mice from the Nkx2–5-Cre;Shroom3^+/fl X Shroom3^fl/fl cross. Aortic valves are displayed on the left and pulmonary valves are displayed on the right. Images present differing angles and depths of sectioning. Sectioned in the frontal plane.

(TIF)

pone.0331583.s003.tif^{(25.4MB, tif)}

Acknowledgments

We thank the lab of Qingping Feng for their donation of the Nkx2–5 Cre recombinase line.

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

Natural Sciences and Engineering Research Council of Canada Award Number: RGPIN-2016-06536 Canadian Institutes of Health Research Award Number: MOP133593 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0331583.r001

Decision Letter 0

Federica Limana

16 Apr 2025

PONE-D-25-16398Loss of Shroom3 in the developing mouse myocardium does not impact heart developmentPLOS ONE

Dear Dr. Carleton,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Specifically, the reviewers have significant concerns about the small dataset used and suggest to refocus the manuscript on the novel findings obtained with a robust dataset using an established model. Further, they also ask to add updated citations on human heart defects in SHROOM3.

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: SHROOM3 expression has been shown in the developing heart where it has been shown to play an important role in heart development, although the cell type responsible for this effect is not known and is the focus of the study.

The authors generated a floxed Shroom3 mouse line to test loss of Shroom3 specifically in myocardial cells. The overall conclusion is that loss of Shroom3 expression in these cells is not responsible for defects in cardiac development associated with full body loss of Shroom3.

Specific findings:

• Shroom expression starting at E10.5 in developing mice.

• Expression can be detected even earlier within myocardial cells but not in endocardial cells.

• Expression of Shroom fades after birth in the heart overall but remains within the myocardium and within the left atrium.

• The authors show a significant decrease in cardiac cross-sectional area in full body Shroom3 loss but this loss is not recapitulated within the mice where Shroom3 is only lost in the myocardial cells. Similar effects are seen when examining Shroom3 associated heart defects and ventricular thinning.

Overall, the manuscript demonstrates the reported findings. I did have a few minor points that should be taken within the context of trying to make the data more accessible to readers with less background in physiology or tissue development.

Minor points:

In the quantification of compact layer thickness in Figure8C and 8F, the effect is clear between wt and heterozygous Shroom3 GT hearts but this reviewer would appreciate seeing the data points used for these graphs so that I can appreciate how representative the data in the rest of the figure is.

Additionally, is there another measurement that is not expected to change as a result of Shroom3 expression that could be quantified as a negative control?

In a related note, for measurements of heart wall thickness, is there a way to normalize these values to overall heart size or dimension so that we can be sure that the effect is not due to overall differences in heart size.

For Fig9C, as you are measuring the cross-sectional area for a large number of cells, a violin-plot would allow us to see the distribution of the data. Once again, I have no doubt as to the effect being shown, but I’d love to see if the effect was uniform for all cells or just a subset of the population (presumably a large subset of the population). This is more of a concern in Figure15 however.

For figure13, I appreciate that the point is to measure the thickness of both left and right ventricular walls in either wt of myocardial shroom loss mice but the placement of the lines used for measurements does not appear to reflect the actual thickness of the wall in 13B. The difference is dramatic and would change the conclusions for the figures. Since this is not my specific area of expertise, this reviewer would appreciate more explanation for how the boundaries are chosen (especially for the inside boundary). Once again, data points on the bar graphs would also help a reader appreciate the data with more nuance.

Reviewer #2: The manuscript by Carleton et al describe an important role for a novel candidate in human disease called SHROOM3. SHROOM3 has been implicated in human disease including primarily kidney defects, and more recently, cardiac defects. A manuscript from 2020 describes heart defects due to SHROOM3 loss of function in embryos. Carleton et al. have expanded these findings, describing a more detailed expression pattern, additional cardiac defects, including valve defects, and pathology in adult cardiomyocytes, with smaller cardiomyocytes persisting to adulthood. These findings are important to our understanding of cardiac development and relevant to human disease. In terms of placing this novel data in the literature, the manuscript describes a role for SHROOM3 in patients with heterotaxy, however a brief literature review reveals two more recent studies, that have shown a role for SHROOM3 in human cardiac disease, including patients with CHD (PMID: 36011280, PMID: 39202774) and upregulation of SHROOM3 after myocardial infarction (PMID: 39086770). The findings of Carleton et al. may have direct relevance to these studies, given their findings of left ventricle thinning and valve defects. Further their findings in adult mice, in terms of the expression pattern and smaller cardiomyocyte area, are relevant to upregulation of SHROOM3 post myocardial infarction. The study needs to highlight these more recent findings and describe their data in the full context of available literature surrounding SHROOM3. Carleton et al. also describe, for the first time, a SHROOM3 conditional loss of function mouse-line. However, this dataset is much smaller. Carleton et al describe that loss of function in the myocardium does not lead to heart defects. These findings may be important for mechanism, and the authors highlight that other cell lines like neural crest are likely contributors to cardiac defects. This finding is important data in narrowing SHROOM3‘s lineage-specific role. However, the conclusions are based on the evaluation of a small number of embryos. This small number is likely not sufficient given the partial penetrance of the described heart defects, and especially to draw primary conclusions for the manuscript. Carleton et al need to better highlight what has been previously reported in SHROOM3 loss of function mice, and the novel findings added here (which are important.) The manuscript would be stronger with enlarging this small dataset or refocusing onto their important findings in a more robust dataset using an established model. Below are comments which, if addressed, would make the manuscript suitable for publication.

1. SHROOM3 loss of function has been shown to result in heart defects in mouse embryos, however the current study adds novel findings. The study needs to describe what has been previously reported and what are the novel findings. The novel findings need to be highlighted. This includes: 1) Evaluation of embryos at e18.5, (versus 14.5 in the Durbin et all study, where E18.5 represent a more robust timepoint for evaluation of cardiac defects than Durbin et al.) 2) A more detailed expression pattern is described 3) A spectrum of valve defects are reported – for the first time. 4) Ventricle thinning is isolated to left ventricle 5) ventricle thinning persists to adulthood. 6) They measure cardiomyocyte area and the smaller size persists into adulthood

2. The manuscript needs to cite more recent literature PMID: 36011280, PMID: 39202774, PMID: 39086770), and place their data in context (for example, their findings related to SHROOM3 valve defects and ventricle thinning may be relevant in hypoplastic left heart syndrome patients, and SHROOM3 expression pattern post-myocardial infarction.

3. The manuscript presents a robust dataset from an established gene trap model. They then present a smaller dataset using a conditional loss of function approach. The later findings are important, given they are the first lineage-specific deletion of SHROOM3 reported. However, the dataset is much smaller than the evaluation of nearly 100 gene trap embryos. From my reading, the conclusions are based on n=8 flox/flox embryos for cardiac defects and n=3 for wall thickness. The results section needs to make more transparent how many embryos were analyzed in each group. Furthermore, 8 embryos does not seam sufficient to make a conclusion, given the partial penetrance of the cardiac defects. These numbers need to be increased, or, at least, the data needs to be described objectively without such strong conclusion.

4. The authors state their findings indicate other cell populations, like the neural crest cells, may be important for heart defects. Are neural crest cells important to cardiomyocyte size and ventricle thickness? Perhaps myocardial SHROOM3 expression is important in conjunction with other cell populations like the cardiac neural crest? If the authors have data about conditional loss of function using a Wnt1-Cre resulting in the neural crest deletion of SHROOM3, they should report it here.

5. To the best of their ability, the authors should refocus the manuscript on the novel findings obtained with a robust dataset using an established model, versus findings from a smaller number of embryos using a new model.

6. The title of the article needs to be modified to highlight the novel findings in the manuscript, obtained from a robust dataset, versus the conclusions drawn from a small cohort of embryos. Perhaps: (as stated in the first line of the conclusion), “SHROOM3 is an important contributor to mammalian heart development and postnatal heart structure.” or “The role of SHROOM3 in cardiac defects.”

7. The abstract also needs to be modified to highlight the novel findings obtained from a robust dataset versus the negative conclusions drawn from a much smaller number of embryos.

The manuscript presents important and novel findings. However, a better description of the numbers utilized in the dataset is needed. Furthermore, if possible, the numbers for the conditional loss of function embryos should be increased, and a second lineage-specific deletion should be performed. At the very least, they should describe their novel findings in the context of what was previously demonstrated in SHROOM3 loss of function embryo heart defects. The authors should also add updated citations on human heart defects in SHROOM3, properly placing the data in the literature. Overall, the authors should refocus the manuscript on the novel findings obtained with a robust dataset using an established model. These findings may have clinical relevance in heart disease given recent findings in patients.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2025 Sep 8;20(9):e0331583. doi: 10.1371/journal.pone.0331583.r002

Author response to Decision Letter 1

14 Jul 2025

Dear Dr. Chenette:

We would like to thank the reviewers for their constructive comments and suggestions. Their feedback is much appreciated. We have responded to the comments from Reviewer #1 and amended certain figures in our manuscript. The discussion and citations have been updated as requested by Reviewer #2, and n-values have been increased. Specific responses and updates are as follows.

Reviewer #1

1. In the quantification of compact layer thickness in Figure8C and 8F, the effect is clear between wt and heterozygous Shroom3 GT hearts but this reviewer would appreciate seeing the data points used for these graphs so that I can appreciate how representative the data in the rest of the figure is.

We agree that inclusion of the data points on all graphs in the manuscript is beneficial for supporting our conclusions and better appreciating the data. Unfortunately, this data was collected several years ago, and we have been unable to locate our original individual measurement values from the initial Shroom3Gt analysis. The student who did these measurements has graduated from the lab, and our computer for original data storage was corrupted several years ago. We have not been able to find those values on other lab computers. While this is clearly an issue, we can provide further corroborating data in the original thesis on this topic (https://ir.lib.uwo.ca/etd/1394/). Included in the thesis are additional ventricular wall measurements at E16.5, E17.5, and wall thickness measurements using in vitro ultrasound which all show significant thinning in the left ventricle in Shroom3Gt/Gt mice. In addition, analysis of the same mice in Durbin et al., 2020 also observe left ventricular wall thinning at E14.5.

In this manuscript, the most important point is that the myocardial specific knockout does not produce ventricular thinning. While we can provide the data points for that observation, we have not done so to maintain consistency with the Shroom3Gt/Gt figures. If reviewers feel otherwise, we would be willing to provide the scatter plot for that data.

2. Additionally, is there another measurement that is not expected to change as a result of Shroom3 expression that could be quantified as a negative control? In a related note, for measurements of heart wall thickness, is there a way to normalize these values to overall heart size or dimension so that we can be sure that the effect is not due to overall differences in heart size.

While this would be interesting, normalization of heart measurements is not normally done in the field of heart development and in our experience, it is not straightforward. When our embryo and neonate hearts are collected, we keep the torso (everything inside the ribcage) intact. While this has allowed us to assess any looping or symmetry defects, it precludes us from taking heart weight measurements. When these torsos are embedded in paraffin wax, although we do our best to embed in the same orientation, small changes to the angle of embedding do change the final viewing of the organ, even when sectioned in the same plane. One parameter which could be used for normalization is septal wall thickness. However, this measurement is subject to change depending on the angle of embedding, making it unreliable and comparisons difficult to interpret.

We have done measurements of total heart weight in adult mice and have not observed any effect based on genotype (Fig. 14).

3. For Fig9C, as you are measuring the cross-sectional area for a large number of cells, a violin-plot would allow us to see the distribution of the data. Once again, I have no doubt as to the effect being shown, but I’d love to see if the effect was uniform for all cells or just a subset of the population (presumably a large subset of the population). This is more of a concern in Figure15 however.

We do agree that a violin plot would better represent the data for this type of measurement. However, similarly to the response in comment 1, we have been unable to recover the original individual data points for the Shroom3Gt experiments. Making a violin plot would be possible for data from the Shroom3fl experiments, but again, for the sake of consistency we have chosen to present the Shroom3fl data in the same style as the Shroom3Gt data.

4. For figure13, I appreciate that the point is to measure the thickness of both left and right ventricular walls in either wt of myocardial shroom loss mice but the placement of the lines used for measurements does not appear to reflect the actual thickness of the wall in 13B. The difference is dramatic and would change the conclusions for the figures. Since this is not my specific area of expertise, this reviewer would appreciate more explanation for how the boundaries are chosen (especially for the inside boundary). Once again, data points on the bar graphs would also help a reader appreciate the data with more nuance.

We appreciate the reviewer’s comments as this is a common issue in the field and defining the exact boundary is often difficult. In this study, we have measured the thickness of the ventricular wall at the midpoint along the length of the ventricle. This is consistent with other studies in the field. Ventricular thickness can vary at different parts of the heart, particularly near the apex, and so the midpoint is often used for consistency. The compact myocardium is the contractile musculature where individual cells are aligned with the length of the heart. This area is densely packed, where the trabecular layer consists of finger-like, muscular projections that extend into the chamber of the heart. They are usually aligned more perpendicular to the length of the heart with gaps between the projections.

The example image used in Fig. 13 also had portions of the papillary muscle visible. These are muscles inside the ventricles which anchor the leaflets of the valves. We have used a new, hopefully clearer, example image in our revision. The image has been updated, and the delineation of the compact myocardium from the trabeculae has been indicated with a dotted line. For consistency, example images in Fig. 6 and Fig. 8 have been updated in the same way.

Reviewer #2

We thank the reviewer for these robust suggestions. A more in-depth comparison between our findings and those presented in Durbin et al., (2020) has been written into the discussion. These can be found on page 21, line 501-503, and page 21-22, line 512-520.

Again, we thank the reviewer for taking the time to provide resources for us. These citations have been included on page 3, line 47 for PMID: 36011280, and page 20, line 483-486 for PMID: 39086770.

After reading through PMID: 39202774 and their citations, we have chosen not to include this in our manuscript. They rely on citations from Durbin et al., (2020), which we reference and discuss in greater detail.

We appreciate the recognition that this is the first lineage-specific knockout of Shroom3 in the heart. Please note that a retina-specific knockout of Shroom3 done by a collaborator using the same floxed line has recently been published (Herstine et al., 2025).

In terms of the n-vale discrepancy between Shroom3Gt and Shroom3fl experiments, we concede that there certainly is a significant difference. In the Shroom3Gt experiments, a maximum of 24 Shroom3+/+, 24 Shroom3+/Gt, and 33 Shroom3Gt/Gt E18.5 embryos were assessed for morphological defects. This contrasts with 8 control, 8 Nkx2-5-Cre;Shroom3+/fl, and 8 Nkx2-5-Cre;Shroom3fl/fl neonates that were assessed in Shroom3fl experiments. We agree that this was a concern and so we have added 30 animals to our Shroom3fl study, increasing the final n-values for the Shroom3fl experiment to 23, 8, and 23 neonates, respectively per the genotypes above. We currently maintain our Shroom3fl mouse line in a homozygous state, and breeding the heterozygous allele back into our colony would have taken too much time. In addition, the lack of phenotype in the homozygous floxed mice makes a phenotype in the heterozygous floxed mice very unlikely. The increased n-values for CHDs in the floxed line can be found in the methods section on page 7, line 157 and page 8, line 164-165. We still did not observe any CHDs in the floxed mice and have added more representative images in supplemental figure 3. Additionally, n-values for Shroom3fl ventricle wall thickness have been increased to 6, which is now the same as Shroom3Gt ventricle wall thickness at E18.5. This can be found in the methods section on page 8, line 178-179. The bar chart in Fig. 13 has been updated accordingly.

In the results section, we have explicitly written n-values in each experiment for morphological assessments. These changes can be found on page 12, line 270, 278, and 279. This will hopefully increase the transparency of the numbers assessed in these experiments. As well, for all morphology assessments, n-vales for each genotype are written in the figure legends.

We would also like to present a further description of our mice to support the lack of phenotypes in the Shroom3fl line. Nearly all the mice in our NkxCre;Shroom3fl colony live until a year of age. With the caveat that there may still be very low levels of functional protein in the heart based on the levels of recombination we see, the high levels of survival suggest no physiological implications from the lack of Shroom3.

The neural crest is the most obvious candidate for a cell lineage that could have roles in cardiac morphogenesis and wall thickness and still have Shroom3 expression based on the expression of the Nkx2-5 Cre driver. There has been evidence that cardiac neural crest cells integrate into the musculature of the heart and take on a myocardial identity. We highlight this in the discussion. However, from the available literature, there has been no evidence to suggest that neural crest cells are important in the size of cardiomyocytes, nor do they contribute to the thickening of the ventricle.

In the conditional knockout model we have created, we have eliminated the majority of myocardial Shroom3 expression and have found no phenotypes arising from this loss. That is why we suggest other cell populations with Shroom3 are responsible for the defects seen in the Shroom3Gt. We do agree that the cells which integrate into the musculature of the heart must be working in conjunction with other cell types which arise from the heart itself. While we do not have data ready to present in this manuscript, we are in the process of working on collaborations to follow these results with a Wnt1 Cre recombinase line.

While we understand the reasoning behind this suggestion, we hope that the amendments outlined in the response to comment 3 will give support to equally focus on both the established model and the new model. Since the time that we submitted this manuscript, our collaborators have published their findings of eye development using the same Shroom3fl line. This citation has been included on page 5, line 99-100 and page 15, line 354 and 363. They have found that Shroom3 is implicated in optic fissure closure and in regulating cellular polarity in these tissues. We believe that this adds credibility and validity of the new mouse model.

In addition, we strongly believe that this new model is essential to the story and intent of the manuscript. The conclusions we have made add to an emerging understanding of the cardiac neural crest cells and their role in heart development. They also provide a poignant example of non-cell autonomous influences on organogenesis and morphogenesis. This is valuable to the field of developmental biology as a whole.

Similar to the above comment, we strongly believe that both data sets together tell a more complete and novel story. We hope that the amendments to the n-values in the Shroom3fl line lend credence to this. A title that focusses on the Shroom3Gt data set may be too similar to Durbin et al., (2020). As an alternative, we have opted for a more generic title: Understanding the role of Shroom3 in the developing mouse myocardium

7. The abstract also needs to be modified to highlight the novel findings obtained from a robust dataset versus the negative conclusions drawn from a much smaller number of embryos.

In the same vein as both above responses, we believe that both mouse lines highlight important findings which should be presented. Once again, we are hopeful that the additional n-values will support our decision.

Again, we thank the reviewers for their very timely responses and thorough feedback.

Sincerely,

Jennifer Carleton

PhD candidate, Drysdale lab

Department of Physiology and Pharmacology, Developmental Biology specialization

Schulich School of Medicine and Dentistry

The University of Western Ontario

Attachment

Submitted filename: Response to reviewers.docx

pone.0331583.s004.docx^{(120.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0331583.r003

Decision Letter 1

Federica Limana

31 Jul 2025

PONE-D-25-16398R1Understanding the role of Shroom3 in the developing mouse myocardiumPLOS ONE

Dear Dr. Carleton,

==============================There are still concerns about the presence of Shroom 3 in the outflow tract

==============================

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Reviewer #2: (No Response)

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Reviewer #2: Yes

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Reviewer #2: The increased number of embryos analyzed, clarification of the number of embryos used in each analysis, and the revised title, abstract and discussion satisfy previous concerns.

One minor point that needs to be addressed before acceptance.

The manuscript describes the Shroom3 expression pattern, which is robust and an important contribution to the manuscript. The manuscript states: “This expression was specific to the myocardium of the atria and ventricles of the heart and was not seen in the outflow tracts or great arteries….” and then later …” Shroom3 expression was seen in the base of the outflow tract“

The staining pattern is clear and there is clearly a sharp drop-off and absence in the great arteries. However, it seems contradictory to say staining is not present in the outflow tract and then later that it is present in the base of the outflow tract. It seems more accurate to state that staining was localized to the base of the outflow tract and absent from the great arteries.

This is an important point given that cardiac neural crest cells are responsible for outflow tract, aortopulmonary, and ventricular septation, and the article hypothesizes (logically) that neural crest cells may be responsible for the septal defects seen in whole body loss of function embryos.

Clarification of this minor point would make the manuscript acceptable for publication.

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Reviewer #2: No

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PLoS One. 2025 Sep 8;20(9):e0331583. doi: 10.1371/journal.pone.0331583.r004

Author response to Decision Letter 2

11 Aug 2025

Dear Dr. Chenette:

We are happy to respond to Reviewer #2 and address their concerns about the description of Shroom3 expression in the outflow tract.

Reviewer #2

1. The manuscript describes the Shroom3 expression pattern, which is robust and an important contribution to the manuscript. The manuscript states: “This expression was specific to the myocardium of the atria and ventricles of the heart and was not seen in the outflow tracts or great arteries….” and then later …” Shroom3 expression was seen in the base of the outflow tract“

Clarification of this minor point would make the manuscript acceptable for publication

In saying “the base of the outflow tracts”, we were referring to the portion of the heart which surrounds the base of the outflow tracts, rather than the most basal portion of the outflow tract itself. We understand that our description was unclear and we thank you for bringing it to our attention. To make this clearer, we have amended the sentences to read “the myocardium surrounding the base of the outflow tracts”. These changes can be found on page 10, line 219-220; page 11, line 237-238, 254; and page 20, line 475.

We once again thank the reviewers for their attention to detail and their suggestions and insights which have improved the quality of the manuscript.

Sincerely,

Jennifer Carleton

PhD candidate, Drysdale lab

Department of Physiology and Pharmacology, Developmental Biology specialization

Schulich School of Medicine and Dentistry

The University of Western Ontario

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Submitted filename: Response_to_reviewers_auresp_2.docx

pone.0331583.s005.docx^{(103.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0331583.r005

Decision Letter 2

Federica Limana

19 Aug 2025

Understanding the role of Shroom3 in the developing mouse myocardium

PONE-D-25-16398R2

Dear Dr. Carleton,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0331583.r006

Acceptance letter

Federica Limana

PONE-D-25-16398R2

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Schematic of Shroom3 gene trap allele and novel floxed Shroom3 allele.

(TIF)

pone.0331583.s001.tif^{(504.4KB, tif)}

S2 Fig. Semi-quantitative PCR for Shroom3^fl recombination and band quantification with densitometry.

(TIF)

pone.0331583.s002.tif^{(3.1MB, tif)}

S3 Fig. Additional images of Nkx2–5-Cre;Shroom3^fl/fl neonate semilunar valve morphologies.

(TIF)

pone.0331583.s003.tif^{(25.4MB, tif)}

Attachment

Submitted filename: Response to reviewers.docx

pone.0331583.s004.docx^{(120.1KB, docx)}

Attachment

Submitted filename: Response_to_reviewers_auresp_2.docx

pone.0331583.s005.docx^{(103.7KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting information files.

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PERMALINK

Understanding the role of Shroom3 in the developing mouse myocardium

Jennifer L Carleton

Rami R Halabi

Jessica A Willson

Timothy F Plageman Jr

Darren Bridgewater

Qingping Feng

Thomas A Drysdale

Roles

Abstract

Introduction

Methods

Mouse lines

Genotyping

mRNA isolation, cDNA synthesis, RT-PCR

Wholemount X-gal staining and clearing

Hematoxylin and eosin staining

Congenital and postnatal heart morphology assessment

Immunofluorescence

Cardiomyocyte area measurements

Statistical analysis

Results

Shroom3 is expressed throughout the embryonic and adult myocardium

Fig 1. Shroom3 is expressed in the mouse heart during cardiogenesis.

Fig 2. Shroom3 is expressed throughout the mouse myocardium during cardiogenesis.

Fig 3. Shroom3 is expressed in the postnatal mouse heart.

Fig 4. Shroom3 is expressed throughout the mouse myocardium postnatally.

Full-body loss of Shroom3 during development shows congenital heart defects in embryos and ventricular thinning and decreased cardiomyocyte size in adults

Fig 5. Embryonic Shroom3Gt/Gt hearts show ventricular septal defects.

Fig 6. Embryonic Shroom3Gt/Gt hearts show left ventricular thinning.

Fig 7. Embryonic Shroom3Gt/Gt hearts show abnormal semilunar valves.

Fig 8. Adult Shroom3+/Gt mouse hearts show left ventricular thinning.

Fig 9. Adult Shroom3+/Gt hearts show decreased cardiomyocyte cross-sectional area.

Floxed allele recombination and verification

Fig 10. Embryos with compound Shroom3Gt/fl alleles phenocopy Shroom3Gt/Gt embryos.

Myocardial Shroom3 loss during development does not produce congenital heart defects in embryos and does not impact adult cardiac or cardiomyocyte morphology

Fig 11. Myocardial Shroom3 loss during development does not show ventricular septal defects.

Fig 12. Myocardial Shroom3 loss during development does not show abnormal semilunar valves.

Fig 13. Myocardial Shroom3 loss during development does not alter ventricular wall thickness.

Fig 14. Shroom3 myocardial specific loss does not alter postnatal cardiac morphology.

Fig 15. Developmental loss of Shroom3 in the myocardium does not affect cardiomyocyte cross sectional area in adults.

Discussion

Shroom3 expression begins early in heart development and continues in the adult heart

SHROOM3, but not myocardial SHROOM3, is required for normal cardiac morphogenesis

Shroom3 loss causes left ventricular thinning and decreased cardiomyocyte size

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Federica Limana

Roles

Author response to Decision Letter 1

Decision Letter 1

Federica Limana

Roles

Author response to Decision Letter 2

Decision Letter 2

Federica Limana

Roles

Acceptance letter

Federica Limana

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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Fig 5. Embryonic Shroom3^Gt/Gt hearts show ventricular septal defects.

Fig 6. Embryonic Shroom3^Gt/Gt hearts show left ventricular thinning.

Fig 7. Embryonic Shroom3^Gt/Gt hearts show abnormal semilunar valves.

Fig 8. Adult Shroom3⁺^/Gt mouse hearts show left ventricular thinning.

Fig 9. Adult Shroom3⁺^/Gt hearts show decreased cardiomyocyte cross-sectional area.

Fig 10. Embryos with compound Shroom3^Gt/fl alleles phenocopy Shroom3^Gt/Gt embryos.