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. 2021 Oct 29;16(10):e0259426. doi: 10.1371/journal.pone.0259426

ERBB3-dependent AKT and ERK pathways are essential for atrioventricular cushion development in mouse embryos

Kyoungmi Kim 1,*, Daekee Lee 2,*
Editor: Robert W Dettman3
PMCID: PMC8555822  PMID: 34714866

Abstract

ERBB family members and their ligands play an essential role in embryonic heart development and adult heart physiology. Among them, ERBB3 is a binding partner of ERBB2; the ERBB2/3 complex mediates downstream signaling for cell proliferation. ERBB3 has seven consensus binding sites to the p85 regulatory subunit of PI3K, which activates the downstream AKT pathway, leading to the proliferation of various cells. This study generated a human ERBB3 knock-in mouse expressing a mutant ERBB3 whose seven YXXM p85 binding sites were replaced with YXXA. Erbb3 knock-in embryos exhibited lethality between E12.5 to E13.5, and showed a decrease in mesenchymal cell numbers and density in AV cushions. We determined that the proliferation of mesenchymal cells in the atrioventricular (AV) cushion in Erbb3 knock-in mutant embryos was temporarily reduced due to the decrease of AKT and ERK1/2 phosphorylation. Overall, our results suggest that AKT/ERK activation by the ERBB3-dependent PI3K signaling is crucial for AV cushion morphogenesis during embryonic heart development.

Introduction

During mouse cardiogenesis, the linear heart tube from the cardiogenic mesoderm forms the outflow tract (OFT) and atrioventricular (AV) cushion after the looping, and endocardial cells initiate the endocardial to mesenchymal transformation (EndMT). After forming the endocardial cushion by the EndMT, mesenchymal cells in the cushion start to proliferate rapidly for valve formation. Furthermore, the superior and inferior AV cushions fuse to form a septum, dividing the ventricular inlet into right and left chambers. Meanwhile, the OFT cushion region gives rise to the arterial pole, and the myocardium of the ventricular becomes trabeculated.

Gene targeting experiments in mice reveal that ERBB family members and their ligands play an essential role in embryonic heart development and adult heart physiology [1–4]. The ERBB family includes ERBB1 (EGFR), ERBB2, ERBB3, and ERBB4, single transmembrane receptor tyrosine kinases that homodimerize or heterodimerize with other family members upon ligand binding to the extracellular domain. Subsequently, the phosphorylation of the cytoplasmic tail recruits adaptors to activate various downstream context-specific signaling pathways [5]. In contrast to the other members, ERBB3 has seven consensus binding sites to p85, the regulatory subunit of PI3K, which activates the downstream AKT pathway [6–8] and leads to the proliferation of a wide variety of cells. Despite its low kinase activity, ERBB3 is a preferential binding partner of ERBB2; the ERBB2/3 complex mediates downstream signals for cell proliferation [9, 10].

In this study, we generated a human ERBB3 knock-in mouse expressing a mutant ERBB3 whose seven YXXM p85 binding sites were replaced with YXXA. This human ERBB3 knock-in mouse exhibits a deficiency of seven consensus binding sites for p85 to uncouple the ERBB3-dependent PI3K pathway. Furthermore, we analyzed the ERBB3-dependent PI3K pathway for AV cushion morphogenesis during embryonic heart development in knock-in mouse.

Materials and methods

Expression vectors and site-directed mutagenesis

Full-length human ERBB2 and ERBB3 cDNAs were subcloned into pcDNA3.1- vector (Thermo Fisher Scientific) to obtain pERBB2 and pERBB3 expression vectors, respectively. Seven YXXM PI3K p85 consensus binding sites in human ERBB3 cDNA underwent site-directed mutagenesis using the QuickChange Multi Site-Directed mutagenesis kit as described by the manufacturer (Agilent Technologies). The pERBB37A vector was generated by changing methionine at positions 944, 1057, 1200, 1225, 1263, 1279, and 1292 to alanine [5], whereas the pERBB37F vector was generated by changing tyrosine at positions 941, 1054, 1197, 1222, 1260, 1276, and 1289 to phenylalanine [11]. DNA sequencing revealed that there was no change in the nucleotide sequences except at mutagenized sites. The flag was inserted to the c-terminal ERBB3 or ERBB37A expression vectors to obtain pERBB3-Flag or pERBB37A-Flag vectors.

Generation and establishment of Erbb3 mutant mice

The Erbb3 null allele without exon 2 (Erbb3–) was generated by pronuclear microinjection with 1 μg/mL of the circular pCreEGFP plasmid (expression of Cre recombinase fused with EGFP) into a heterozygous 1-cell embryo containing the conditional Erbb3 allele (Erbb3flox) [12]. To construct the ERBB37A-Neo knock-in vector, a 9-kb HindIII fragment of Erbb3 genomic DNA in 129SV BAC clone spanning exon 1 to exon 3 was subcloned into pBluescript II vector. The SacII site in exon 1 and BsrG1 in exon 2 was used for subcloning of the 5’ homology arm, and the 3’ homology arm was derived from the XhoI fragment of a conditional Erbb3 targeting vector described previously [12]. To add the polyA signal, the 0.7-kb 3’ untranslated region (UTR) of mouse Erbb3 was PCR cloned into the BsrG1 site right after the 5’ homology arm. Lastly, BsrG1 and NotI fragments of pERBB37A vector were subcloned between the 5’ homology arm and 3’UTR. So, the final fused Erbb3 gene consists of a mouse gene with the BsrG1 site in exon 2 and a human gene (from BsrG1 site to stop codon of ERBB37A cDNA) with mouse polyA signal. Targeting in 129/SvEv embryonic stem (ES) cells and generation of a chimeric mouse were described previously [12]. Germline transmission of the Erbb37A-Neo allele was verified with PCR. To remove the neomycin transphosphorylase (Neo) expression cassette, one-cell embryos derived from C57BL/6J (B6) females mated with Erbb3+/7A-Neo males were treated with 3 μM of purified HTNCre protein for 30 min as described previously [13]. Complete excision of the Neo selection marker was identified from the founder mice by PCR genotyping with primers p489, 5’-ACTGAACCCCACCTACATTGTC-3’ (sense, S) and p150, 5’-AAGCCTTCTCTATGGAAAGTG-3’ (antisense, AS). Accurate BsrGI junction between mouse and human ERBB3 cDNA was verified using RT-PCR product from the Erbb37A/7A embryo. The genotype of each mouse was determined by PCR using DNA extracts from the yolk sac or toe clip with the following primers: p149, 5’-TCCAGCGTGGAAAAGTTCAC-3’ (S); p150, 5’-AAGCCTTCTCTATGGAAAGTG-3’ (AS); p680, 5’-CCCTGACAGAATCTCGGTGA-3’ (AS). Erbb3+/- or Erbb3+/7A mice were backcrossed more than ten generations with the B6 background before intercross. Mice were housed in a specific-pathogen-free facility, and all experiments with mice were approved by the Institutional Animal Care and Use Committee at Ewha Womans University.

RNA preparation and quantitative real-time PCR (qRT-PCR)

Total RNA from an E10.5 embryo was prepared, and qRT-PCR and quantification of mRNA were performed as described previously [14]. The sequence of qRT-PCR primers used were: Egfr, 5’-GCAAAGTGCCTATCAAGTGGA-3’ (S), 5’-CCAGCACTTGACCATGATC-3’ (AS); Erbb2, 5’-CAGATTGCCAAGGGGATGA-3’ (S), 5’-TGCCCCCATCTGCATGGTA-3’ (AS); Erbb3, 5’-CCACAGCTGCTGCTCAACT-3’ (S), 5’-AATTGGAGTCTTGGCCTCAC-3’ (AS); Erbb4, 5’-ACTGCTGCCATCGAGAATG-3’ (S), 5’-CCAGTTGAAAGGTGGTTGG-3’ (AS); 18S rRNA, 5’-TCAACTTTCGATGGTAGTCGCC-3’ (S), 5’-GGCCTCGAAAGAGTCCTGTATTGT-3’ (AS). RT-PCR was performed as described previously [12] with primers p268, 5’-TTCCGAGATGGGCAACTCTC-3’ (S) and p665, 5’-ACTTCCCATCGTAGACCTGG-3’ (AS). Primer p268 is specific for the mouse Erbb3 sequence, whereas p665 is conserved in both human and mouse.

Histology and immunohistochemistry of embryonic samples

Embryos were fixed in 10% NBF at 4°C overnight and embedded in paraffin. Rehydrated paraffin sections were stained with hematoxylin and eosin Y (H&E) and mounted with Permount solution (Thermo Fisher Scientific). The embryo images were visualized with a light microscope (Eclipse 80i EPI, Nikon or Axiovert 200, Zeiss). For cryosection, the embryos were fixed in 4% PFA in 1× PBS at 4°C overnight, equilibrated in 30% sucrose in 1× PBS at 4°C, and embedded in OCT. Paraffin sections or cryosections were used for immunohistochemistry. For immunofluorescence staining, the embryo sections were blocked with 5% normal goat serum in TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20), then incubated overnight at 4°C with primary antibodies. For detecting a target with a mouse primary antibody, M.O.M kit (Vector Labs) was used according to the manufacturer’s protocol. The embryo sections were further treated with secondary antibodies conjugated with Alexa Fluor 488 or 568 (Life Technologies) for 1 h or treated with secondary antibody conjugated with biotin for 30 min, followed by treatment with fluorescein avidin DCS (Vector Labs) for 5 min to intensify the signal. The sections were counterstained with DAPI and mounted with Vectashield medium (Vector Labs). The images were visualized with an LSM 510 Meta confocal microscope (Zeiss) with the same exposure intensity value. The immunofluorescence images were quantified with Nikon NIS-Elements BR 3.2 imaging software. In a sample, each cell’s area and background were assigned, and their intensities were measured and calculated as average intensity, excluding background intensity.

BrdU staining

Embryos were labeled with BrdU (Sigma-Aldrich) for 2 h after intraperitoneal injection with 10 mM BrdU in PBS, pH 7.4, at 0.1 mL/10 g body weight into pregnant females and embedded as described in Methods. Paraffin sections were immunostained, as described previously [12].

Collagen gel analysis of EndMT in the embryo heart

The AVC region of the heart at the E9.5 embryo stage was dissected out and opened with fine iris scissors under a dissecting microscope. The endothelial surface was placed toward collagen gel containing 1 mg/mL collagen I from rat tail (Thermo Fisher Scientific) in Medium 199. The explant was allowed to attach at 37°C in a humidified atmosphere of 5% CO2 overnight and was cultured in 0.1 mL of Medium 199 supplemented with 1% FBS, 0.01% Insulin-Transferrin-Selenium (Thermo Fisher Scientific), and penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2 for 48 h [15]. Transformed cells showing stellate and spindle-shapes were counted under an inverted light microscope (Axiovert 40C,) as described previously [15–17]. PI3K inhibitor LY294002 (Merck KGaA) and MEK inhibitor PD0325901 (Selleckchem) were dissolved in DMSO, and an equal amount of stock solution was added to the culture media (final DMSO concentration, 0.2%).

CHO cell culture and establishment of ERBB2 stable cell line

CHO cells were maintained in DMEM medium supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO2. Stable colonies that expressed ERBB2 were established after transfection with the pERBB2 vector using LT1 reagent (Mirus), followed by selecting 250 μg/mL of Geneticin (Thermo Fisher Scientific) for 2 weeks. The ERBB2 expression level was examined with western blotting, and a representative stable cell line (CHO-ERBB2) was maintained.

Cell transfection

CHO-ERBB2 was seeded on 6-well plates in a density of 0.75 x 105 cells and was transiently transfected with pERBB3, pERBB37A, and pERBB37F vectors using LT1 17–24 h after seeding to express ERBB3, ERBB37A, and ERBB37F, respectively. After 30 h of transfection, transfected cells were starved for 18 h in DMEM containing 0.1% FBS then treated with 50 ng/mL of rhNRG1-β1 (R&D Systems) for 5 min 37°C. Cells were immediately washed with cold PBS and stored at -80°C until preparation of protein extract.

Western blot and immunoprecipitation

The protein extract of a whole embryo at E10.5 was prepared by homogenizing the embryo with a glass-Teflon homogenizer in tissue lysis buffer containing proteinase and phosphatase inhibitors. The protein extraction of the heart endocardial cushion and the trabecular region was performed by first dissecting the fetal heart at E11.5 with fine iris scissors and then freezing the samples in dry ice. A piece of tissue was homogenized with a microtube pellet pestle (Sigma-Aldrich) to prepare the cell extracts [12]. Centrifuging the cell lysate to separate the supernatant from the pellet, protein quantification, SDS-PAGE, western blotting, and quantification of blots were performed [12].

The detailed procedure for immunoprecipitation was described previously [18].

Antibodies

For immunostaining, the following antibodies were used: anti-p-AKT (S473, #4060) and anti-p-ERK1/2 antibodies (#4370) from Cell Signaling Technology; anti-BrdU antibody from Abcam (ab6326); anti-SNAIL antibody (sc28199) from Santa Cruz; and anti-CD144 (VE-cadherin) antibody from BD Pharmingen (550548). For western blotting or IP (Immunoprecipiation), the following antibodies were used: anti-ACTB (AC-15) and anti-Flag antibody (F3165) from Sigma-Aldrich; anti-AKT (#9272), anti-AKT(S473, #4060), anti-p-AKT(T308, #2965), anti-EGFR antibody (#2232), anti-p-ERBB3(Y1289, #4791), anti-ERK1/2 (#9102), anti-p-ERK1/2 (#9101) antibodies from Cell Signaling Technology; anti-ERBB2 (sc284-G), anti-ERBB3 (sc285-R) and anti-ERBB4 antibodies (sc283-R) from Santa Cruz Biotechnology; anti-GAPDH antibody from Abfrontier (LF-PA0018); and anti-p-Tyr antibodies from BD Biosciences (610024).

Statistical analysis

Experimental groups were compared with a two-tailed Student’s t-test or one-way ANOVA with Newman-Keuls multiple comparison test. Data represent mean ± SEM. P < 0.05 was considered statistically significant. Each letter (a, b, c or d) in the graph indicates a significant difference between experimental groups. Two letters that are the same (e.g., a and a) indicate a nonsignificant difference (ns), whereas different letters (e.g., a and b) indicate a significant difference. Two letters together (e.g., ab) indicates non-significant differences (ns) with either a or b, but a significant difference compared with c or d.

Results

Erbb37A/7A mutant embryos show embryonic lethality

All seven YXXM consensus binding motifs were changed to YXXA in the human ERBB3 cDNA to uncouple the PI3K downstream pathway from ERBB3. The mutant human ERBB3 cDNA was used to construct the Erbb37A knock-in mouse on the C57BL/6 background; the knock-in of the mutant ERBB3 was verified by measuring its mRNA level (Fig 1A–1E). Overall, the Erbb37A allele was found to express the human ERBB3 protein, whose N-terminus was substituted with the first 48 amino acids of the mouse ERBB3 protein. qRT-PCR revealed that the human ERBB3 mRNA level in the Erbb37A/7A embryos was comparable to the Erbb3 mRNA level in the wild-type embryos (Fig 1F). ERBB3 protein was not detected in Erbb3-/- embryo. However, the ERBB3 protein level in the Erbb37A/7A embryos, similar to Erbb3+/-, was only one-third of that in the wild-type embryos (Fig 1G). The protein levels of all other members were similar regardless of genotype. The transient transfection of Flag-tagged ERBB3 or Flag-tagged ERBB37A revealed that the reduced level of ERBB3 protein in the Erbb37A/7A embryo was not due to a possible epitope change in ERBB37A protein (Fig 1H).

Fig 1. Targeting strategy for Erbb3 gene and characterization of Erbb3 expression.

Fig 1

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(A) Erbb3 wild-type allele, targeting vector, and targeted Erbb37A-Neo allele are shown. The numbers 1–9 indicate exons. H indicates the HindIII site. ERBB37A, pA, and Neo in knock-in vectors represent human ERBB37A cDNA, the polyA sequence including the 3’UTR region of mouse Erbb3 gene and neomycin transphosphorylase expression cassette, respectively. Arrowheads indicate loxP sites. 2’ denotes the partial exon 2 of mouse gene. Arrows indicate PCR primers for genotyping. (B) Genomic DNA from ES cell clones was digested with HindIII and hybridized with a radiolabeled probe as shown in S1A Fig in S1 File, resulting in an 8.9-kb fragment from wild-type allele 7.5-kb fragment from the correctly targeted allele, respectively. (C) PCR genotyping using three primers (p149, p150, and p680 as shown in S1A Fig in S1 File) discerned the genotype of E10.5 embryos obtained from Erbb3+/7A-Neo intercrosses, and primers p149 and p150 resulted in a 354-bp PCR product for wild-type Erbb3 allele. In contrast, primers p149 and p680 resulted in a 507-bp PCR product for the Erbb37A-Neo allele. M, 1 kb Plus ladder. (D) PCR genotyping with indicated primers is shown using DNA from pups treated with HTNCre at the one-cell stage. Primers p489 and p150 resulted in a 309-bp PCR product specific for the Erbb37A allele. (E) Schematic drawing of cDNA from Erbb3 and Erbb37A allele and analysis of RT-PCR product. E1 to E4 indicates exon 1 to exon 4. Open-box and gray box derive from mouse Erbb3 and human ERBB3 cDNA, respectively. RT-PCR analysis, followed by BamHI digestion, confirmed human ERBB3 mRNA expression from the Erbb37A knock-in allele. Representative agarose gel electrophoresis of RT-PCR product from E10.5 embryos is shown on the right. BamHI digestion of a 287-bp RT-PCR product (primers with p268 and p665) resulted in 180-bp and 107-bp fragments in wild-type and heterozygous Erbb37A mutant embryos, respectively, whereas no fragments were shown in the Erbb37A/7A embryo. (F) Relative mRNA levels of Egfr family members were analyzed by qRT-PCR. (G) Protein levels of EGFR family members in a whole E10.5 embryo were analyzed by western blotting with indicated antibodies depending on genotypes (n = 5–6). (H) Western blotting was performed with cell lysates using the indicated antibodies. The transient expression of ERBB3 in CHO cells was achieved by transfecting cells with the indicated vectors. ACTB (Beta-Actin) was used as the loading control. Different letters on the bar graphs represent statistically significant differences, p < 0.05; ns, non-significant.

Next, we analyzed the viability of the Erbb37A/7A embryos because Erbb37A/7A pups were not found from the intercrosses of Erbb3+/7A mice (Fig 2A). The Mendelian ratio that 25% of the offspring were Erbb37A/7A mutant embryos was maintained until E11.5. Then, the proportion of Erbb37A/7A embryos decreased to 0% at E14.5. Similarly, no Erbb3-/- embryos on the C57BL/6 background survived to E14.5; these data were inconsistent with a previous finding that 21% of the Erbb3-/- embryos survived to term on a heterogeneous background [19]. These results suggest that the Erbb37A allele and the Erbb3- allele result in the same level of lethality. Microscopic and histological examination results of E12.5 embryos showed that both Erbb37A/7A and Erbb3-/- embryos have a small right ventricle (Fig 2B and 2C). Additionally, in the E13.5 Erbb3-/- and Erbb37A/7A embryos, hemorrhage was observed at the site of contact between the left superior intercostal vein and left superior vena cava; the site was filled with blood (Fig 2D and 2E).

Fig 2. Similar heart defects in Erbb37A/7A and Erbb3-/- embryos.

Fig 2

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(A) Genotype was determined by PCR analysis of embryos. The viability of Erbb3 mutant embryos from heterozygous intercrosses was determined through the heartbeat of embryos. The Mendelian ratio of homozygous mutant embryos is shown as a dotted line. The number denotes the total number of embryos genotyped. (B) Shown are E12.5 hearts of wild-type, Erbb3+/-, Erbb3-/- and Erbb37A/7A embryos observed under a dissecting microscope. Arrows indicate a small right ventricle of the mutant embryos. (C) Representative H&E staining of the E12.5 heart sections. Asterisks indicate the right ventricle, arrows indicate the opening of the interventricular septum, and arrowheads indicate the non-fused superior and inferior endocardial cushion of mutant embryos, respectively. (D) Shown are E13.5 wild-type, Erbb3-/- and Erbb37A/7A embryos observed under a dissecting microscope. Arrows indicate bleeding in the mutant embryos. (E) Representative H&E staining of the E13.5 heart sections. Asterisks indicate the right and left AV valve, respectively. Arrowheads indicate bleeding in the pericardial cavity, and pluses (+) mark the site of communication between the left superior intercostal vein and left superior vena cava filled with blood in the mutant embryos, respectively. IC, inferior endocardial cushion; IVS, interventricular septum; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; SC, superior endocardial cushion.

Reduced EndMT in Erbb3 mutant embryos is associated with aberrant SNAIL and VE-cadherin protein levels

Erbb3-/- embryos lacked AV cushion mesenchymal cells at E9.5-E10, and AVC-explant culture demonstrated that the Erbb3-/- embryos had a defective endothelial to mesenchymal transition (EndMT) [10, 17, 20]. We first identified ERBB3 expression in the hearts of E10.5 stage embryos. ERBB3 was expressed in endothelial cells and mesenchymal cells of the AV cushion (S1 Fig in S1 File). Also, we performed histological analysis of the AV cushion in Erbb37A/7A embryos because Erbb37A/7A embryos have heart defects similar to Erbb3-/- embryos. In E10.5 embryos of Erbb37A/7A and Erbb3-/-, the number of mesenchymal cells in the AV cushion was reduced compared to the wild type (Fig 3A and 3B). The number of mesenchymal cells continued to increase in both types of mutant embryos, but at E11.5 their numbers were still lower than in wild-type embryos (Fig 3A and 3B). The number of endocardial cells per area showed no difference regardless of genotype (Fig 3C).

Fig 3. The attenuation of EndMT in the heart of Erbb3 mutant embryos.

Fig 3

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(A) The atrioventricular (AV) cushion in the heart at E10.5 and E11.5 was examined with H&E staining. Scale bars indicate 200 μm and 50 μm, respectively. (B) The number of mesenchymal cells in the AV cushion in the samples was quantified (n = 5–8). (C) The endocardial cells in the AV cushion in the samples were quantified (n = 4–7). (D) EndMT analysis via the collagen gel method of the AVC-explants at E9.5. Arrows indicate the transformed mesenchymal cells. The bottom graph displays the quantification of the transformed cells. (E) Immunofluorescence staining of VE-cadherin and SNAIL in the AV cushion at E9.5 heart. Arrows indicate endocardial cells showing the SNAIL protein mainly located in the nucleus, whereas arrowheads indicate SNAIL proteins in the cytoplasm. Nuclei were counterstained with DAPI. RBC, red blood cell. The bar graphs represent the mean ± SEM, and different letters on the bar represent statistically significant differences, p < 0.05; ns, non-significant. Each letter of a or b in the bar graph means a significant difference (p < 0.05) for each experimental group, and ab means a non-significant difference for experimental groups.

In wild-type AVC-explants, the outgrowths of endocardial cells appeared widely spread around the collagen gel surface, but the both Erbb37A/7A and Erbb3-/- mesenchymal cells did not (Fig 3D). The AVC-explants in both mutant embryos had a reduced number of mesenchymal cells by 40%–60% as compared to wild-type embryos (Fig 3D). The VE-cadherin, a key player of EndMT [21], and SNAIL, one of the transcriptional repressors of VE-cadherin [21–23], were analyzed using immunofluorescence staining in AVC of E9.5 embryos to determine the cause of the reduced EndMT (Fig 3E). Overall, the VE-cadherin protein was distributed more broadly in both types of ErbB3 mutant embryos than wild-type embryos (Fig 3E).

In contrast to VE-cadherin’s weak immunostaining between the endothelial and delaminated cells in wild-type embryos, we more frequently detected intense VE-cadherin immunostaining between the cell types in mutant embryos. These results suggest the possibility that attenuation of EndMT is related to high VE-cadherin levels (Fig 3E). Moreover, round and oval nuclei were found in the AVC of the mutant embryos, whereas thin and long nuclei were found in the wild-type endocardial cells. The SNAIL protein was mainly found in the endocardial cells with thin and long-shaped nuclei and less frequently in the endocardial cells with round and oval nuclei (Fig 3E).

Proliferation of mesenchymal cells is transiently attenuated in the AV cushion of Erbb3 mutant embryos

After EndMT, the transformed mesenchymal cells undergo extensive proliferation in the AV cushion before AVC morphogenesis [24, 25]. We performed BrdU immunostaining to investigate cell proliferation in the heart (Fig 4A). At E10.5, approximately 43% of mesenchymal cells in the wild-type embryos were BrdU-positive. However, the BrdU-positive cells were reduced to approximately 31% in mutant embryos (Fig 4B). BrdU-positive mesenchymal cells were temporarily weakened at E10.5 of the two mutant embryos, but there was no difference at E11.5 stage. In contrast, the number of BrdU-positive endocardial cells in the wild-type and mutant embryos at E10.5 were similar. However, their numbers decreased in both mutant embryos at E11.5 (Fig 4C).

Fig 4. Reduced proliferation of endocardial and mesenchymal cells of AV cushion in Erbb3 mutant embryos.

Fig 4

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(A) AV cushion was immunostained with anti-BrdU antibody. Scale bar indicates 50 μm. (B) The number of BrdU-positive mesenchymal cells in the AV cushion was counted and quantified (n = 5–7). (C) The number of BrdU-positive endocardial cells in the AV cushion was counted and quantified (n = 5–7). Different letters on the bar graphs represent statistically significant differences, p < 0.05; ns, non-significant.

AKT and ERK1/2 phosphorylation is attenuated in the AV cushion of Erbb3 mutant embryos

Western blot analysis was performed with protein extracts from individual AV cushions at E11.5 to identify the molecular mechanism of Nrg1-ERBB3 signaling for embryonic heart development. AKT and ERK pathways are two major downstream pathways of ERBB3 signaling [26, 27]. The relative levels of phosphorylated AKT (p-AKT(S473)) and ERK1/2(p-ERK1/2) were lower in both mutant embryos than in the wild-type, although their total protein levels did not differ (Fig 5A and S2 Fig in S1 File). Next, we performed immunostaining with phospho-specific antibodies in the AV cushion at E10.5 and E11.5. AKT and ERK1/2 were mainly phosphorylated in the endocardial and mesenchymal cells of the AV cushion. However, the phosphorylation levels were decreased in both mutant embryos (Fig 5B and 5C; Top). The quantification of phosphorylation intensity in the endocardial or mesenchymal cells of the AV cushion revealed that p-AKT or p-ERK1/2 was significantly reduced in both mutant embryos compared to the wild-type (Fig 5B and 5C; Bottom).

Fig 5. Reduction of AKT and ERK phosphorylation in the heart of Erbb3 mutant embryos.

Fig 5

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(A) (Left) The relative levels of AKT and ERK phosphorylation in the endocardial cushion as determined by western blotting. GAPDH was used as the loading control. (Right) The band intensities of p-AKT(S473) and p-ERK1/2 were quantified and compared to the wild-type (n = 6–10). (B) p-AKT or (C) p-ERK1/2 in the AV cushion at E11.5 was examined by immunostaining with the indicated antibodies. The immunostained images in mesenchymal and endocardial cells were quantified using Nikon NIS-Elements BR3.2 imaging software (n = 4–9) to analyze the relative level of AKT or ERK1/2 phosphorylation. Different letters on the bars represent a statistically significant difference between groups, p < 0.05. Each letter of a or b or c on the bar graph means a significant difference (p < 0.05) for each experimental group, and ab means a non-significant difference for experimental groups.

PI3K and ERK pathways inhibitors blocked the EndMT in AVC-explant culture [28, 29]. We tested co-treatment of the AVC explant with PI3K and MEK inhibitors to determine whether the reduced expression of the PI3K and MEK pathways by ERBB37A mutation affected early EndMT. The co-treatment of the AVC explant with PI3K and MEK inhibitors achieved higher EndMT inhibition within the AV cushion than did treatment with one inhibitor (S3A, S3B Fig in S1 File). These results indicate that the embryonic heart defect in Erbb3 mutant embryos was due to the reduction in early EndMT and attenuation of both AKT and ERK phosphorylation.

AKT and ERK pathways are attenuated by ERBB37A, but not by ERBB37F in CHO cells

Lahlou H et al. reported the generation of a Erbb3Δ85 knock-in mouse, in which the seven YXXM PI3K p85 consensus binding sites of human ERBB3 were replaced with FXXM. In contrast to Erbb37A mice, some mice carrying the homozygous Erbb3Δ85 allele survived into adulthood [11]. This indicates that their ERBB3 mutant proteins may induce differential downstream pathways. However, both proteins have mutations in the p85 binding sites; therefore, we set out to identify their different effects on downstream signaling. We generated a pERBB37F (YXXM to FXXM) vector by replacing the wild-type amino acid sequences of human ERBB3 with the corresponding mutant sequence in the Erbb3Δ85 allele. We transfected the mutant construct into an ERBB2-expressing CHO cell line (CHO-ERBB2) [30].

Western blotting using the anti-phosphorylated ERBB3(p-ERBB3) antibody detects the phosphorylation of ERRB3 at Tyr 1289, located in the far end of the p85-binding site of the C-terminus. The level of NRG1β-induced ERBB3 phosphorylation and subsequent AKT and ERK1/2 phosphorylation were dramatically increased in the cells transfected with the pERBB3 vector (Fig 6A and 6B). In contrast, the level of NRG1β-induced p-AKT and p-ERK1/2 was profoundly attenuated in pERBB37A-transfected cells despite a slight increase in phosphorylation by NRG1β treatment. Despite the absence of phosphorylated ERBB3 (p-ERBB3) in FXXM mutation human ERBB3 (pERBB37F)-transfected cells, NRG1β-induced p-AKT and p-ERK1/2 did not differ from wild-type human ERBB3 (pERBB3)-transfected cells. Following NRG1β induction, the molecular weight of the phosphorylated protein at the tyrosine residue was approximately 190-kD, which corresponded to the ERBB2 and ERBB3 in pERBB3-transfected cells. The level of phosphorylated protein at tyrosine residues was subdued in pERBB37F-transfected cells compared to pERBB3. pERBB37A-transfected cells particularly were more severely attenuated compared to others (Fig 6A).

Fig 6. Differential induction of downstream signaling by ERBB3 mutant proteins in CHO cells.

Fig 6

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(A) CHO cells stably expressing ERBB2 (CHO-ERBB2) were transiently transfected with pcDNA3.1 (Ctrl), pERBB3, pERBB37A, or pERBB37F vectors. Transfected cells were treated with NRG1β (+) or vehicle (-), and the whole-cell lysates were subjected to western blotting using the indicated antibodies. (B) The relative intensity of the bands in western blots was compared to the Ctrl vector without NRG1β treatment. Each different letter on the bars represents statistically significant differences between groups, p < 0.05, and bc means a non-significant difference for experimental groups. (C) The cell lysates were immunoprecipitated (IP) with the anti-ERBB2 or anti-ERBB3 antibodies, and IP samples were further analyzed by western blotting (IB) using an anti-p-Tyr antibody.

After immunoprecipitation (IP) using anti-ERBB2 antibody or anti-ERBB3 antibody, western blot with anti-p-Tyr antibody was performed to identify the phosphorylation sites in ERBB3 other than the p85 binding sites (Fig 6C). After treatment with NRG1β, the level of p-Tyr (phosphorylation of Tyrosine) after IP with anti-ERBB2 antibody was slightly increased only in the pERBB3-transfected cells. Similar to the previous p-Tyr blot results, p-Tyr expression in IP using an anti-ERBB3 antibody revealed the NRG1β-dependent elevation of p-ERBB3 in pERBB37F-transfected cells. However, the elevation of p-ERBB3 was lower than in pERBB3-transfected cells. In contrast, the basal and elevated p-ERBB3 was barely detected in pERBB37A-transfected cells. Overall, these data suggest that the tyrosine phosphorylation of the p85 binding sites in ERBB3 is predominant but not exclusive; NRG1β-dependent ERBB3 phosphorylation of other sites may also activate the AKT and ERK pathways.

Conclusions

The anomalies during heart development observed in Erbb3 mutants suggest the role of ERBB3 in developmental processes. ERBB3 is mainly expressed in the endocardial and mesenchymal cells of the AV cushion region at E9.5 to E10.5 during the EndMT. ERBB3 was only expressed in mesenchymal cells during E11.5 to E12.5 [10]. Erbb3 null and 7A mutations caused embryonic lethality. In both mutations, mesenchymal cell proliferation was temporarily reduced at E10.5 and endocardial cell proliferation was significantly reduced at E11.5. In both mutants, the right ventricle was smaller than the left ventricle, and the myocardial thickness was reduced. Our results suggest that the heart defects in both the Erbb3-/- and Erbb37A/7A mutants were caused by overall growth retardation.

VE-cadherin is specifically expressed in endocardial cells, and the regulation of VE-cadherin expression by Notch signaling directly affects EndMT [21–23]. As EndMT proceeds, the level of VE-cadherin is reduced in the endocardial cells, whereas the level of SNAIL is significantly increased. Our results suggest that the ERBB3-dependent signaling appears connected to the SNAIL-VE-cadherin pathway in regulating the EndMT for AV cushion morphogenesis. Also, the defects of Erbb3 mutants have been implicated in the dysregulation of proliferation and EndMT during endocardial cushion development [20]. EndMT is essential for the development of the endocardial cushion. The previous report on ERBB2-ERBB3 knockouts showed that EndMT events were attenuated in heart explant cultures [10]. The EndMT of Erbb3 mutants was also attenuated in our AVC-explant experiments.

The main pathways involved in Nrg1-ERBB3 signaling are PI3K and MAPK. Treatment with PI3K or MAPK inhibitors decreased EndMT in the heart explant culture. Moreover, the inhibition of PI3K and MAPK signaling dramatically reduced the transformation of endothelial cells to mesenchymal cells. In these results, both the PI3K and MAPK signals were essential for the EndMT event during the endocardial cushion formation, and Erbb3 mutations inhibited EndMT due to the decline in PI3K and MAPK signaling (S4 Fig in S1 File). We additionally identified a reduction in the number of mesenchymal cells in the AV cushion due to failed EndMT event and decreased cell proliferation. These results suggest that the proliferation of mesenchymal cells in the AV cushion of Erbb3 mutant embryos was transiently reduced due to attenuated AKT and ERK phosphorylation.

Previously, an Erbb3 mutant with YXXM-to-FXXM mutations (Erbb37F) was analyzed in a mouse breast cancer model and revealed no embryonic lethality [11]. A possible explanation for this would be due to the differential NRG1-ERBB3 signaling between Erbb37A and Erbb37F mutations. NRG1-dependent p-AKT(T308) and p-AKT(S473) were clearly reduced in the ERBB37A, but not in ERBB37F, according to our results in CHO cells. ERBB37F does not exhibit a decreased level of p-ERK1/2 contrary to ERBB37A, which is also likely to contribute to the survival of Erbb37F embryos. Overall, our results suggest that the YXXA mutation blocks the activation of ERBB3-dependent PI3K signaling more than does the FXXM mutation of ERBB3.

Supporting information

S1 File

(DOCX)

Click here for additional data file. (933.4KB, docx)

Acknowledgments

We would like to thank Dr. Woong Sun for the helpful discussion.

Data Availability

All relevant data, including original uncropped and unadjusted images underlying all blot or gel results, is within the paper and Supplementary data files.

Funding Statement

This work was supported by the National Research Foundation of Korea (2011-0009719, 2020M3A9D5A01082439, and 2019R1A2C2087198).

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Decision Letter 0

Robert W Dettman

23 Feb 2021

PONE-D-21-03408

ERBB3-dependent AKT and ERK pathways are essential for atrioventricular cushion development and valve formation in the mouse embryo

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Reviewer #1: This study by Kim et al., examines the endocardial cushion phenotype in mice harboring a mutant form of human ERBB3, whereby YXXM p85 binding sites were replaced with YXXA. Erbb3 knock-in embryos were embryonically lethal around E12.5-E13.5 with hypoplastic endocardial cushions, attributed to decreased transformation and proliferation; likely due to decreased pAkt and pErk. These findings are consistent with previous studies utilizing global Erbb3 null mice. The manuscript introduces a new, mouse model to the field that allows for studies focused on the role of ERBB3-dependent PI2K signaling during development. However, findings from the manuscript provide incremental insights into the field, and the data does now distinguish between cause, and effect of the ERBB3 mutation. In addition, without the inclusion of quantitative data, the conclusions might be considered overinterpreted.

1. Line 71 is not clear – what is meant by “ERBB3-dependent pathways are incorrectly activated for proliferation”?

2. Where is Erbb3 expressed during endocardial cushion development?

3. The cardiac phenotype of both Erbb3 mutants is very interesting. On line 218 the authors comment on a small right ventricle, and this is further expanded upon on lines 330-333, which include reports of “a small tricuspid valve, comparable thickness of the myocardium, thin myocardial wall, HRHS”. The authors should quantify these phenotypes based on histological sections; myocardial thickness, AV cushion size. In addition the penetrance of these phenotypes should be included.

4. Did the authors examine outflow tract valves?

5. A consistent concern throughout the manuscript is the number of studies and interpretations drawn from E13.5 embryos. The authors report 0% survivability by E14.5, and a significant loss at E13.5, and therefore there is a concern that phenotypes are being observed due to lethality and not directly related to the function of Erbb3 in these process. In Figure 2D, E, viability of the mutant embryos is clearly a problem as there is a lack of blood circulation.

6. Line 230-231, what is meant by AV cushions of mutant embryos were comparable with wild-type?

7. The authors comment several times on differences between endothelial, versus mesenchyme cells. How were the cell types determine?

8. Figure 3E is interesting. The quantitation of VE-cadherin and Snai1 is critical here, in addition to the number of long-shaped, versus rounded nuclei. The authors should also be cautious that the EMT defect may not be caused by changes in VE-cadherin and Snai1 expression, this may be a molecular readout of impaired EMT (and not the cause). There is certainly no evidence to suggest that the attenuation of EMT is directly related to a high level of VE-cadherin.

9. In Figure 5, the concern over embryo viability remains. Another point to consider is that valve elongation is not occurring due to the initial EMT defect. Following on, the reduced expression of Nfatc1 is likely attribute to survivability (or lack of), and EMT impairment and independent of ERBB3. The orientation of tissue sections in this figure are also not clear. Figure 5 is a major concern.

Reviewer #2: This article describes atrioventricular (AV) defects and deficient P13K/AKT and ERK pathways in Erbb3-null and knock-in mutant mouse embryos.

The authors provide detailed description of experimental procedures and present interesting observations of AV endocardial cushion/AV canal defects in Erbb3-null and knock-in mutant mouse embryos

Several major concerns are raised.

1. Page 13, Line 326- 328

The authors describe that ERBB3 is mainly expressed in the endocardial and mesenchymal cells in the AV cushion from E9.5 to E10 during AV endocardial mesenchymal transformation (EndMT) and ERBB3 is only expressed in mesenchymal cells from E11.5 to E12.5 by citing Camenisch et al., 2002 (ref. 10 in this paper). However, intense expression of ERBB3 is seen in AV myocardium in Figure 2 in Camenisch et al. (2002). Since the authors use conventional knock-in mice, the notion that ERBB3 is expressed both in endocardial and myocardial cells in the AV canal is critical to understand the nature of AV canal defects found in the mutant mice.

2. Page 9, Line 216-221 and Page 13, line 331-333.

The authors describe that both Erbb3-null and 7A mutant exhibit small right ventricle and AV septal defects at E12.5. However, cardiac septation is known to be established by E15.5 in the normal condition and therefore, defects found in the null and mutant hearts may not be thoroughly attributed to the AV septal defects. Because of the growth defects of the embryos seen at E13.5 (Fig.1D), these heart defects can be caused by overall growth retardation.

3. Page 9, Line 227-235.

The authors describe that Erbb3-null and 7A mutant have fewer mesenchymal cells in the AV endocardial cushion. However, at E11.5, the 7A mutant (Fig. 3A lower right panel) shows more mesenchymal cells in AV cushions as compared to the wild type (Fig. 3A lower left). The histogram (Fig. 3B) provided by the authors does not appear to represent actual cell density seen in Fig. 3A.

4. Page 10, Line 252-261.

The authors describe that proliferation ratio of mesenchymal cells is reduced in the null and 7A mutant at E10.5

If the mesenchymal cell proliferation ratio is decreased, that itself can directly cause reduction of cell density of mesenchymal cells in the AV cushion (Fig. 3B) without deficient endocardial cell transformation that they claimed in Fig. 3D. EndMT commences at around E9.5 but continues for a while until fusion of the superior and inferior AV cushions. Mesenchymal cell formation by EndMT and proliferation of mesenchymal cells occur in the same AV cushions at E10.5. Therefore, if there is significant reduction in proliferation of mesenchymal cells in the AV cushions at ED10.5, it is not possible to attribute reduction of cell density to reduction of the EndMT.

5. Page 11, Line 263-279 & Page 7, Line 172-178.

Authors claim that they collected AV endocardial cushions from E11.5 mouse embryos for protein extraction. However, at E11.5, it is technically very challenging to dissect out AV endocardial cushions without contamination with AV myocardium that associate with the AV cushion. Especially because the null and 7A mutants have smaller defective hearts, it is extremely difficult to correctly dissect out endocardial cushions.

The data presented here could likely represent protein extraction from E11.5 AV canal, that include endocardial cushions and associated myocardium. Also, it is important that AV myocardium intensely expresses ERBB3 (Camenisch et al., 2002, Ref. 10, Fig. 2). Therefore, the data obtained here would be largely coming from deficiencies of ERBB3 expression in the myocardium.

6. Page 11, line 281-Page 12, Line 239.

Here is the authors’ fundamental misunderstanding of AV valvulogenesis. AV valves are not directly formed from superior and inferior AV endocardial cushions. Superior and inferior AV endocardial cushions fuse in the midline of the heart to form the septal AV cushion and from the septal AV cushion, septal leaflets of AV valves are formed. Parietal leaflets of the AV valves are derived from lateral AV cushions not from superior and inferior AV cushions (Snarr et al., Dev Dyn 236, 1287-1294, 2007; Snarr et al., Dev Dyn 237, 2804-2819, 2008).

Therefore, arrows shown in Fig. 5A-B have nothing to do with the formation of AV valves. Figure 5C is showing fusion points of superior and inferior AV cushions.

The data presented here do not provide any evidence for interaction of NFATc1 and ERBB3 in AV valvulogenesis.

Other concerns:

The authors show histograms with different letters, a, ab and b. They indicate in the figure legends that the different letters on the bar graphs represent the statistically significant differences. It is not clear what was truly compared.

Reviewer #3: In this paper the authors analyze knock-in mice that express a mutant ERBB protein whose seven p85 binding sites have been altered to determine the role of ERBB3-dependent PI3K signaling in AV cushion morphogenesis.

1) For some readers the level of detail provided by the authors may be sufficient. However, as a relative novice to this particular protein and its downstream targets, I found it difficult to follow the reasoning of the authors and how they came to some of their conclusions. As a first step, I think it would be helpful for the authors to provide the reader with a clear understanding of the pathways discussed in this manuscript and how they intersect. Personally, I found it very hard to link some of the pathways being discussed and Figure S4 is not sufficiently detailed to provide much help.

2) The authors should also address the following concerns.

3) How do the authors account for the significant drop in expression of the the ERBB3 protein level in the Erbb37A/7A embryos to only one-third of that in the wild-type embryos? This significant drop could independently affect development. At a minimum, comparisons should be made with Erbb3+/- embryos whose expression level is similar to that of Erbb37A/7A embryos.

4) The data in Figure 1H should be quantified for comparison to the data in Figure 1G. Was this experiment repeated? What was the n (number of repeats)?

5) The interventicular spetum is not always complete at E12.5 in wild type embryos. In figure 2C the authors show what appears to be a complete septum in a wild type embryo, but this section does not correspond to the sections shown for the null and 7A/7A embryos. The authors should provide counts (how many embryos have complete septums) from all genotype and show comparable sections. Without this, it is very difficult to see whether there is a real difference at this stage.

6) The authors state that, “In contrast to the weak immunostaining of VE-cadherin between the endothelial and delaminated cells in wild-type embryos, the intense immunostaining of VE-cadherin between the cell types was detected more frequently in mutant embryos, indicating that the attenuation of EndMT is directly related to a high level of VE-cadherin.” The term directly related suggest causality which is not proved. These things may be correlated, but not causal. The authors will need to indicate that more clearly.

7) There appears to be a distinct difference in the level of SNAIL protein in the 7A/7A sections than in the -/- sections (figure 3E). Nuclei also look rounder and the VE-cadherin level looks different. What is the n of this experiment? Are these sections representative?

8) Supplemental figure 1 should be included in the manuscript since it contains important data.

9) For Figure 4A, the authors should provide graphs of AKT and ERK levels as well. Saying “they all had comparable levels of total protein” is ambiguous since we don’t know what that refers to

10) Figure 4B, no images are provided for E10.5, or did I miss them?

11) It is not obvious why the authors observation that “co-treatment of the AVC-explant with PI3K and MEK inhibitors achieved higher inhibition of the EndMT of the AV cushion than treatment with one inhibitor (Fig. S2A, B)” help us to conclude that, “the embryonic heart defect in the Erbb3 mutant embryos is likely due to the attenuation of both AKT and ERK phosphorylation.” If this does help us come to that conclusion, the authors need to help the reader to make that connection.

12) The authors state, “at E11.5 (Fig. S3B) and E12.5 (Fig. 5B, C), the nuclear localization of NFATC1 in endocardial cells where valve formation occurred was reduced in mutant embryos.” At E11.5, the images do not suggest that NFATC1 is in the cytoplasm of these cells (having failed to be localized to the nucleus). Rather that fewer cells are expressing NFATC1. Perhaps this is because we can’t see where the nuclei are in the double stained images. The authors should provide images with DAPI alone to help us visualize the nucleus of each cell.

13) The authors introduce the delta85 allele into the paper in the results section with no introduction (just a reference). What is this mutation? Why is it of interest?

14) If the authors wish to comment on the level of Nrg1β-induced p-AKT levels, they should normalize not only to AKT but to the Nrg1β negative levels. Without that normalization, it is very difficult to determine if there is attenuation of the Nrg1β response.

15) The authors state, “despite the absence of p-ERBB3 in pERBB37F309 -transfected cells, Nrg1β-induced p-AKT and p-ERK1/2 increased compared to that I the pERBB3-transfected cells.” Not sure what they meant by “…that I the…” Assuming they meant “….to that see in the…” it would not appear that this si the case for p-ERK whose response was similar to that of pERBB3 (a to c vs. a to c).

16) The authors state, “Erbb3 null and 7A mutations caused…thin myocardial wall.” Then they say that, “the thickness of the myocardium in the right and left ventricles was similar.” That is inconsistent.

17) The authors state, “the heart malformation of both Erbb3-/- and Erbb3331 7A/7A mutants was related with hypoplastic right heart syndrome (HRHS) and atrioventricular septal defect (AVSD).” This will require a better comparison of E12.5 heart sections in Figure 2C.

18) I don’t feel that the authors provided sufficient evidence to suggest that the “tricuspid valves of the mutants was smaller.

19) It would be inaccurate to say, “EndMT events failed in the heart explant culture” if you have any level of mesenchymal cell production. “Fail” suggest no EndMT. Words like, “reduced” or “attenuated” are a more accurate description of their data.

20) The authors show that their mutations are associated with decreases in p-AKT and p-ERK1/2 levels. I do not follow how this data and a supplemental experiment showing that PI3K and MEK inhibitors additively affect EndMT leads them to conclude that their “Erbb3 mutations inhibited EndMT due to the decline in… MAPK signaling (Fig. S4).” Perhaps I am just not familiar with these pathways, but the authors don’t provide enough explanation for me—a simple reader—to understand how they have proven an effect on MAPK signaling (which by the way is never mentioned in the abstract).

Minor

1) Proteins should be written in all capital letters.

2) The paper needs to be reviewed by a native English speaker.

3) The quality of the figures provided for review is very poor. It is really hard to see details. In revision, high resolution images should be submitted (300 dpi or higher).

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PLoS One. 2021 Oct 29;16(10):e0259426. doi: 10.1371/journal.pone.0259426.r002

Author response to Decision Letter 0

18 Aug 2021

Reviewer #1:

This study by Kim et al., examines the endocardial cushion phenotype in mice harboring a mutant form of human ERBB3, whereby YXXM p85 binding sites were replaced with YXXA. Erbb3 knock-in embryos were embryonically lethal around E12.5-E13.5 with hypoplastic endocardial cushions, attributed to decreased transformation and proliferation; likely due to decreased pAkt and pErk. These findings are consistent with previous studies utilizing global Erbb3 null mice. The manuscript introduces a new, mouse model to the field that allows for studies focused on the role of ERBB3-dependent PI2K signaling during development. However, findings from the manuscript provide incremental insights into the field, and the data does now distinguish between cause, and effect of the ERBB3 mutation. In addition, without the inclusion of quantitative data, the conclusions might be considered overinterpreted.

1. Line 71 is not clear – what is meant by “ERBB3-dependent pathways are incorrectly activated for proliferation”?

Response: We agree that this line was not clear and did not fit appropriately in the context. Accordingly, we have deleted this sentence.

2. Where is Erbb3 expressed during endocardial cushion development?

Response: ERBB3 is expressed at E10.0 in the endocardial and mesenchymal cells in the AV cushion of the embryonic heart (Todd D. Camenisch et al., Nat Med., 2002). We also identified ERBB3 expression in the heart of an E10.5 embryo using ERBB3 antibody immunohistochemistry. At E10.5, ERBB3 is expressed only in the endocardial and mesenchymal cells in the AV cushion of the embryonic heart. These data can be found below, and have also been added to Fig. S1.

Figure S1. Histological ERBB3 expression in the heart of the wild-type E10.5. Section 1 represents the trabeculated myocardium, and section 2 represents the AV cushion.

3. The cardiac phenotype of both Erbb3 mutants is very interesting. On line 218 the authors comment on a small right ventricle, and this is further expanded upon on lines 330-333, which include reports of “a small tricuspid valve, comparable thickness of the myocardium, thin myocardial wall, HRHS”. The authors should quantify these phenotypes based on histological sections; myocardial thickness, AV cushion size. In addition the penetrance of these phenotypes should be included.

Response: Thank you for your comment. We have measured the myocardial thickness of E12.5 embryos. Both Erbb3-/- and Erbb37A/7A had reduced myocardial wall thickness compared to the wild type. These data can be found below and have been added to Fig. S4.

In Figure 3C, we had previously measured the number of mesenchymal cells in the AV cushion. These data showed that the number of mesenchymal cells in the AV cushion was reduced in both mutant embryos. This reduction of mesenchymal cells is expected to eventually affect the size of the AV cushion.

Figure. S4. Myocardial wall thickness of E12.5 embryos. (A) At E12.5, the myocardial wall of the heart was examined by hematoxylin and eosin (H&E) staining. Scale bar = 50 µm. (B) Quantification of myocardial wall thickness in each samples of Erbb3+/+, Erbb3-/-, and Erbb37A/7A.

4. Did the authors examine outflow tract valves?

Response: We focused only on AV cushion formation.

5. A consistent concern throughout the manuscript is the number of studies and interpretations drawn from E13.5 embryos. The authors report 0% survivability by E14.5, and a significant loss at E13.5, and therefore there is a concern that phenotypes are being observed due to lethality and not directly related to the function of Erbb3 in these process. In Figure 2D, E, viability of the mutant embryos is clearly a problem as there is a lack of blood circulation.

Response: We agree with your opinion. Most phenotypes were analyzed at the E10.5 to E12.5 stages before lethality occurred. The E13.5 embryo phenotypes were investigated to determine the direct reason for the lethality.

6. Line 230-231, what is meant by AV cushions of mutant embryos were comparable with wild-type?

Response: We corrected the phrase on p.9, line 234-235, as follows: “In E10.5 embryos of Erbb37A/7A and Erbb3-/-, the number of mesenchymal cells in the AV cushion was reduced compared to the wild type (Fig. 3A, B).” We have also marked this sentence with a yellow highlight in the manuscript.

7. The authors comment several times on differences between endothelial, versus mesenchyme cells. How were the cell types determine?

Response: Todd D. Camenisch et al. (Nat Med., 2002/reference 10) and Ching-Pin Chang et al. (Cell, 2004/reference 20) showed EMT analysis using the E9.0 endocardial cushion to study the role of calcineurin signaling during the transformation process. We distinguished the difference between endothelial and mesenchymal cells by referring to the findings of these studies.

8. Figure 3E is interesting. The quantitation of VE-cadherin and Snai1 is critical here, in addition to the number of long-shaped, versus rounded nuclei. The authors should also be cautious that the EMT defect may not be caused by changes in VE-cadherin and Snai1 expression, this may be a molecular readout of impaired EMT (and not the cause). There is certainly no evidence to suggest that the attenuation of EMT is directly related to a high level of VE-cadherin.

Response: We tried but were unable to obtain ERBB3 mutant embryos for quantification of VE-cadherin and SNAIL during the revision period. We agree with your opinion that alterations in VE-cadherin and Snai1 expression may not be the cause of impaired EndMT. However, many previous studies reported that the regulation of SNAIL-VE-cadherin expression has a direct effect on EndMT (Luika A. Timmerman et al., Genes Dev. 2004; Sonsoles Piera-Velazquez et al., Physiol Rev. 2019). We found that whereas EndMT progression reduced the level of VE-cadherin in the endocardial cells, it significantly increased the level of Snai1 as an EndMT transcription factor. Overall, ERBB3-dependent signaling during AV cushion morphogenesis appears to be connected to the SNAIL-VE-cadherin pathway for regulating EndMT.

9. In Figure 5, the concern over embryo viability remains. Another point to consider is that valve elongation is not occurring due to the initial EMT defect. Following on, the reduced expression of Nfatc1 is likely attribute to survivability (or lack of), and EMT impairment and independent of ERBB3. The orientation of tissue sections in this figure are also not clear. Figure 5 is a major concern.

Response: We conducted experiments using only live E12.5 embryos. We agree that valve elongation did not occur due to the initial EMT defect. These heart defects can be caused by overall growth retardation. We also identified that AV valves are not directly formed from the superior and inferior AV endocardial cushions. Rather, the superior and inferior AV endocardial cushions fuse in the heart’s midline to form the septal AV cushion and septal leaflets of the AV valves. The parietal leaflets of the AV valves are derived from the lateral AV cushions, and not from the superior and inferior AV cushions (Brian S. Snarr et al., Dev Dyn. 2007; Brian S. Snarr et al., Dev Dyn. 2008). Therefore, Figure 5 is in error and has been removed from the manuscript.

Reviewer #2:

This article describes atrioventricular (AV) defects and deficient P13K/AKT and ERK pathways in Erbb3-null and knock-in mutant mouse embryos.

The authors provide detailed description of experimental procedures and present interesting observations of AV endocardial cushion/AV canal defects in Erbb3-null and knock-in mutant mouse embryos

Several major concerns are raised.

1. Page 13, Line 326- 328

The authors describe that ERBB3 is mainly expressed in the endocardial and mesenchymal cells in the AV cushion from E9.5 to E10 during AV endocardial mesenchymal transformation (EndMT) and ERBB3 is only expressed in mesenchymal cells from E11.5 to E12.5 by citing Camenisch et al., 2002 (ref. 10 in this paper). However, intense expression of ERBB3 is seen in AV myocardium in Figure 2 in Camenisch et al. (2002). Since the authors use conventional knock-in mice, the notion that ERBB3 is expressed both in endocardial and myocardial cells in the AV canal is critical to understand the nature of AV canal defects found in the mutant mice.

Response: Todd D Camenisch et al., (Nat Med., 2002) noted as follows: “ErbB3 is present in the invading mesenchyme and endocardium. Although ErbB3 is detected in myocardium, it may be nonspecific as some staining is observed in the myocardium of ErbB3–/– embryos.” In conclusion, ERBB3 is expressed only in the endocardial and mesenchymal cells in the embryonic heart’s AV cushion at E10.0.

We also identified ERBB3 expression in the heart of E10.5 embryos using ERBB3 antibody immunohistochemistry. ERBB3 was expressed only in the endocardial and mesenchymal cells in the embryonic heart AV cushion at E10.5. These data can be found below and have been added to Fig. S1.

Figure S1. Histological ERBB3 expression in the heart of the wild-type E10.5. Section 1 represents the trabeculated myocardium, and section 2 represents the AV cushion.

2. Page 9, Line 216-221 and Page 13, line 331-333.

The authors describe that both Erbb3-null and 7A mutant exhibit small right ventricle and AV septal defects at E12.5. However, cardiac septation is known to be established by E15.5 in the normal condition and therefore, defects found in the null and mutant hearts may not be thoroughly attributed to the AV septal defects. Because of the growth defects of the embryos seen at E13.5 (Fig.1D), these heart defects can be caused by overall growth retardation.

Response: We agree with your opinion and have modified the phrase on p.13, lines 327-329, as follows: “Overall, our results suggest that heart defects of both Erbb3-/- and Erbb37A/7A mutants were caused by overall growth retardation.” We have also marked this sentence with a yellow highlight in the manuscript.

3. Page 9, Line 227-235.

The authors describe that Erbb3-null and 7A mutant have fewer mesenchymal cells in the AV endocardial cushion. However, at E11.5, the 7A mutant (Fig. 3A lower right panel) shows more mesenchymal cells in AV cushions as compared to the wild type (Fig. 3A lower left). The histogram (Fig. 3B) provided by the authors does not appear to represent actual cell density seen in Fig. 3A.

Response: We quantified the number of mesenchymal cells in the AV cushion up to n=8 in E11.5-stage embryos. Our results identified that the number of mesenchymal cells differed significantly between Erbb3 mutants and the wild type.

4. Page 10, Line 252-261.

The authors describe that proliferation ratio of mesenchymal cells is reduced in the null and 7A mutant at E10.5. If the mesenchymal cell proliferation ratio is decreased, that itself can directly cause reduction of cell density of mesenchymal cells in the AV cushion (Fig. 3B) without deficient endocardial cell transformation that they claimed in Fig. 3D. EndMT commences at around E9.5 but continues for a while until fusion of the superior and inferior AV cushions. Mesenchymal cell formation by EndMT and proliferation of mesenchymal cells occur in the same AV cushions at E10.5. Therefore, if there is significant reduction in proliferation of mesenchymal cells in the AV cushions at ED10.5, it is not possible to attribute reduction of cell density to reduction of the EndMT.

Response: Thank you for your comment. We agree that the decreased mesenchymal cell proliferation rate may itself decrease the mesenchymal cell density in the AV cushion. However, we found a low number of mesenchymal cells in the AV cushion at E10.5. This result suggests that the formation of mesenchymal cells by early EndMT was also defective. Overall, the defects in the AV cushion are due both to the lack of mesenchymal cell formation by early EndMT, and the decreased proliferation of mesenchymal cells in the AV cushion.

5. Page 11, Line 263-279 & Page 7, Line 172-178.

Authors claim that they collected AV endocardial cushions from E11.5 mouse embryos for protein extraction. However, at E11.5, it is technically very challenging to dissect out AV endocardial cushions without contamination with AV myocardium that associate with the AV cushion. Especially because the null and 7A mutants have smaller defective hearts, it is extremely difficult to correctly dissect out endocardial cushions.

The data presented here could likely represent protein extraction from E11.5 AV canal, that include endocardial cushions and associated myocardium. Also, it is important that AV myocardium intensely expresses ERBB3 (Camenisch et al., 2002, Ref. 10, Fig. 2). Therefore, the data obtained here would be largely coming from deficiencies of ERBB3 expression in the myocardium.

Response: We carefully dissected the heart’s AVC region at the E11.5 stage using fine iris scissors on a dissecting microscope. We cannot completely exclude the possibility of contamination with AV myocardium associated with the AV cushion. However, as mentioned earlier, ERBB3 is expressed in the endocardial and mesenchymal cells in the AV cushion of the embryonic heart. Therefore, the data obtained here would largely come from deficiencies of ERBB3 expression in the endocardial and mesenchymal cells in the embryonic heart’s AV cushion.

6. Page 11, line 281-Page 12, Line 239.

Here is the authors’ fundamental misunderstanding of AV valvulogenesis. AV valves are not directly formed from superior and inferior AV endocardial cushions. Superior and inferior AV endocardial cushions fuse in the midline of the heart to form the septal AV cushion and from the septal AV cushion, septal leaflets of AV valves are formed. Parietal leaflets of the AV valves are derived from lateral AV cushions not from superior and inferior AV cushions (Snarr et al., Dev Dyn 236, 1287-1294, 2007; Snarr et al., Dev Dyn 237, 2804-2819, 2008). Therefore, arrows shown in Fig. 5A-B have nothing to do with the formation of AV valves. Figure 5C is showing fusion points of superior and inferior AV cushions. The data presented here do not provide any evidence for interaction of NFATc1 and ERBB3 in AV valvulogenesis.

Response: Thank you very much for your comments. We agree that AV valves are not directly formed from the superior and inferior AV endocardial cushions. Therefore, Figure 5 is in error and has been removed from the manuscript. We have accordingly revised the manuscript and figures for AV valvulogenesis.

Other concerns:

The authors show histograms with different letters, a, ab and b.

They indicate in the figure legends that the different letters on the bar graphs represent the statistically significant differences. It is not clear what was truly compared.

Response: We revised the phrase on p.8, lines 195-199, as follows: “Each letter (a, b, c or d) in the graph indicates a significant difference between experimental groups. Two letters that are the same (e.g., a and a) indicate a nonsignificant difference (ns), whereas different letters (e.g., a and b) indicate a significant difference. Two letters together (e.g., ab) indicates non-significant differences (ns) with either a or b, but a significant difference compared with c or d.” We have also marked this revised phrase with yellow highlighting in the manuscript.

Reviewer #3:

In this paper the authors analyze knock-in mice that express a mutant ERBB protein whose seven p85 binding sites have been altered to determine the role of ERBB3-dependent PI3K signaling in AV cushion morphogenesis.

1) For some readers the level of detail provided by the authors may be sufficient. However, as a relative novice to this particular protein and its downstream targets, I found it difficult to follow the reasoning of the authors and how they came to some of their conclusions. As a first step, I think it would be helpful for the authors to provide the reader with a clear understanding of the pathways discussed in this manuscript and how they intersect. Personally, I found it very hard to link some of the pathways being discussed and Figure S4 is not sufficiently detailed to provide much help.

Response: We agree with your opinion. We admit that there is a lack of direct evidence to explain Figure S4. As such, Figure S4 has been removed and its details have been added to the manuscript’s conclusion.

2) The authors should also address the following concerns.

3) How do the authors account for the significant drop in expression of the the ERBB3 protein level in the Erbb37A/7A embryos to only one-third of that in the wild-type embryos? This significant drop could independently affect development. At a minimum, comparisons should be made with Erbb3+/- embryos whose expression level is similar to that of Erbb37A/7A embryos.

Response: We agree with your opinion. In Figure 1G, we confirmed that ERBB3 protein expression was similar in Erbb3+/- and Erbb37A/7A. For this reason, we also compared Erbb3+/- embryos in most experiments. However, Erbb3+/- showed a phenotype close to Erbb3+/+ (wild type).

4) The data in Figure 1H should be quantified for comparison to the data in Figure 1G. Was this experiment repeated? What was the n (number of repeats)?

Response: This experiment was repeated three times. Herein, we have quantified the western blot data and added it to Figure 1H.

5) The interventicular spetum is not always complete at E12.5 in wild type embryos. In figure 2C the authors show what appears to be a complete septum in a wild type embryo, but this section does not correspond to the sections shown for the null and 7A/7A embryos. The authors should provide counts (how many embryos have complete septums) from all genotype and show comparable sections. Without this, it is very difficult to see whether there is a real difference at this stage.

Response: Under normal conditions, cardiac septation is known to be established by E15.5. Therefore, the defects found in the null and mutant hearts may not be thoroughly attributed to AV septal defects. These heart defects may be caused by overall growth retardation owing to the embryo growth defects seen at E13.5 (Fig. 1D).

6) The authors state that, “In contrast to the weak immunostaining of VE-cadherin between the endothelial and delaminated cells in wild-type embryos, the intense immunostaining of VE-cadherin between the cell types was detected more frequently in mutant embryos, indicating that the attenuation of EndMT is directly related to a high level of VE-cadherin.” The term directly related suggest causality which is not proved. These things may be correlated, but not causal. The authors will need to indicate that more clearly.

Response: We agree with your opinion. We have modified the phrase on p.10, lines 247-250, as follows: “In contrast to VE-cadherin’s weak immunostaining between the endothelial and delaminated cells in wild-type embryos, we more frequently detected intense VE-cadherin immunostaining between the cell types in mutant embryos. These results suggest the possibility that attenuation of EndMT is related to high VE-cadherin levels (Fig. 3E).” We have also marked this passage with a yellow highlight in the manuscript.

7) There appears to be a distinct difference in the level of SNAIL protein in the 7A/7A sections than in the -/- sections (figure 3E). Nuclei also look rounder and the VE-cadherin level looks different. What is the n of this experiment? Are these sections representative?

Response: The sample size used in the experiment was n=3, and this figure is a representative image.

8) Supplemental figure 1 should be included in the manuscript since it contains important data.

Response: We have moved these data from Supplemental figure 1 to main Figure 4.

9) For Figure 4A, the authors should provide graphs of AKT and ERK levels as well. Saying “they all had comparable levels of total protein” is ambiguous since we don’t know what that refers to

Response: The quantified AKT and ERK data were added to Fig. S2 A and B. We also corrected the phrase on p.11, line 269-271, as follows: “The relative levels of phosphorylated AKT (p-AKT(S473)) and ERK1/2(p-ERK1/2) were lower in both mutant embryos than in the wild type, although their total protein levels did not differ (Fig. 5A, S2).” We have also marked this sentence with a yellow highlight in the manuscript.

10) Figure 4B, no images are provided for E10.5, or did I miss them?

Response: It is correct that there is no image via immunohistochemistry staining of E10.5. We performed immunofluorescence staining in E10.5 and E11.5 embryos to measure the relative intensities of the P-AKT and P-ERK proteins. Immunohistochemistry staining was performed and imaged only at the representative E11.5 stage.

11) It is not obvious why the authors observation that “co-treatment of the AVC-explant with PI3K and MEK inhibitors achieved higher inhibition of the EndMT of the AV cushion than treatment with one inhibitor (Fig. S2A, B)” help us to conclude that, “the embryonic heart defect in the Erbb3 mutant embryos is likely due to the attenuation of both AKT and ERK phosphorylation.” If this does help us come to that conclusion, the authors need to help the reader to make that connection.

Response: We agree with your opinion. We have added the phrase on p.11, lines 278-284, as follows: “We tested co-treatment of the AVC explant with PI3K and MEK inhibitors to determine whether the reduced expression of the PI3K and MEK pathways by ERBB37A mutation affected early EndMT. The co-treatment of the AVC explant with PI3K and MEK inhibitors achieved higher EndMT inhibition within the AV cushion than did treatment with one inhibitor (Fig. S3A, B). These results indicate that the embryonic heart defect in Erbb3 mutant embryos was due to the reduction in early EndMT and attenuation of both AKT and ERK phosphorylation.” We have marked this passage with a yellow highlight in the manuscript.

12) The authors state, “at E11.5 (Fig. S3B) and E12.5 (Fig. 5B, C), the nuclear localization of NFATC1 in endocardial cells where valve formation occurred was reduced in mutant embryos.” At E11.5, the images do not suggest that NFATC1 is in the cytoplasm of these cells (having failed to be localized to the nucleus). Rather that fewer cells are expressing NFATC1. Perhaps this is because we can’t see where the nuclei are in the double stained images. The authors should provide images with DAPI alone to help us visualize the nucleus of each cell.

Response: We agree with your opinion. The data presented here do not provide any evidence for the interaction of NFATc1 and ERBB3 in AV valvulogenesis. Accordingly, we have removed Fig. S3B and Fig. 5C.

13) The authors introduce the delta85 allele into the paper in the results section with no introduction (just a reference). What is this mutation? Why is it of interest?

Response: The ErbB3△85 allele refers to a mouse in which the ERBB3 and PI3K pathways were isolated from YXXM to FXXM by replacing the Erbb3 gene with human ERBB3. Tyrosine-encoding codons at the positions 941, 1054, 1197, 1222, 1260, 1276, and 1289 within the YXXM PI3K p85 consensus binding sites were mutated at the second position (A to T) into phenylalanine-encoding codons. To reflect this information, we have added the phrase on p.11, lines 287-288, as follows: “Lahlou H et al. reported the generation of a Erbb3Δ85 knock-in mouse, in which the seven YXXM PI3K p85 consensus binding sites of human ERBB3 were replaced with FXXM.” We have also marked this sentence with a yellow highlight in the manuscript.

14) If the authors wish to comment on the level of Nrg1β-induced p-AKT levels, they should normalize not only to AKT but to the Nrg1β negative levels. Without that normalization, it is very difficult to determine if there is attenuation of the Nrg1β response.

Response: Thank you for your comment. However, this opinion has been sufficiently confirmed in the Figure 7 data.

15) The authors state, “despite the absence of p-ERBB3 in pERBB37F309 -transfected cells, Nrg1β-induced p-AKT and p-ERK1/2 increased compared to that I the pERBB3-transfected cells.” Not sure what they meant by “…that I the…” Assuming they meant “….to that see in the…” it would not appear that this si the case for p-ERK whose response was similar to that of pERBB3 (a to c vs. a to c).

Response: we revised the phrase on p.12, lines 301-304, as follows: “Despite the absence of phosphorylated ERBB3 (p-ERBB3) in FXXM mutation human ERBB3 (pERBB37F)-transfected cells, NRG1β-induced p-AKT and p-ERK1/2 did not differ from wild-type human ERBB3 (pERBB3)-transfected cells.” We have also marked this sentence with a yellow highlight in the manuscript.

16) The authors state, “Erbb3 null and 7A mutations caused…thin myocardial wall.” Then they say that, “the thickness of the myocardium in the right and left ventricles was similar.” That is inconsistent.

Response: We agree with your opinion. We have revised the phrase on p.13, lines 324-328, as follows: “The Erbb3 null and 7A mutations caused embryonic lethality. In both mutations, mesenchymal cell proliferation was temporarily reduced at E10.5 and endocardial cell proliferation was significantly reduced at E11.5. In both mutants, the right ventricle was smaller than the left ventricle, and the myocardial thickness was reduced. Our results suggest that the heart defects in both the Erbb3-/- and Erbb37A/7A mutants were caused by overall growth retardation.” We have also marked this passage with a yellow highlight in the manuscript.

17) The authors state, “the heart malformation of both Erbb3-/- and Erbb37A/7A mutants was related with hypoplastic right heart syndrome (HRHS) and atrioventricular septal defect

(AVSD).” This will require a better comparison of E12.5 heart sections in Figure 2C.

Response: We judged that this sentence was over-interpreted in the conclusion. Cardiac septation is known to be established by E15.5 in the normal condition, and therefore, defects found in the null and mutant hearts may not be thoroughly attributed to AV septal defects. These heart defects may be caused by overall growth retardation due to the growth defects in the E13.5 embryos (Fig. 1D). Hence, we have revised the phrase on p.13, lines 327-328, as follows: “Our results suggest that the heart defects in both the Erbb3-/- and Erbb37A/7A mutants were caused by overall growth retardation.” We have also marked this sentence with a yellow highlight in the manuscript.

18) I don’t feel that the authors provided sufficient evidence to suggest that the “tricuspid valves of the mutants was smaller.

Response: We agree with your opinion. We identified that AV valves are not directly formed from the superior and inferior AV endocardial cushions. Rather, the superior and inferior AV endocardial cushions fuse in the heart’s midline to form the septal AV cushion and septal leaflets of the AV valves. The parietal leaflets of the AV valves are derived from the lateral AV cushions, and not from the superior and inferior AV cushions (Brian S. Snarr et al., Dev Dyn. 2007; Brian S. Snarr et al., Dev Dyn. 2008). Therefore, Figure 5 is in error and has been removed from the manuscript.

19) It would be inaccurate to say, “EndMT events failed in the heart explant culture” if you have any level of mesenchymal cell production. “Fail” suggest no EndMT. Words like, “reduced” or “attenuated” are a more accurate description of their data.

Response: We agree with your opinion. We corrected the phrase on p.13, lines 335-337, as follows: “The previous report on ERBB2-ERBB3 knockouts showed that EndMT events were attenuated in heart explant cultures. The EndMT of Erbb3 mutants was also attenuated in our AVC-explant experiments.” We have also marked this passage with a yellow highlight in the manuscript.

20) The authors show that their mutations are associated with decreases in p-AKT and p-ERK1/2 levels. I do not follow how this data and a supplemental experiment showing that PI3K and MEK inhibitors additively affect EndMT leads them to conclude that their “Erbb3 mutations inhibited EndMT due to the decline in… MAPK signaling (Fig. S4).” Perhaps I am just not familiar with these pathways, but the authors don’t provide enough explanation for me—a simple reader—to understand how they have proven an effect on MAPK signaling (which by the way is never mentioned in the abstract).

Response: We agree with your opinion. We have added the phrase on p.13-14, lines 338-346, as follows: “The main pathways involved in Nrg1-ERBB3 signaling are PI3K and MAPK. Treatment with PI3K or MAPK inhibitors decreased EndMT in the heart explant culture. Moreover, the inhibition of PI3K and MAPK signaling dramatically reduced the transformation of endothelial cells to mesenchymal cells. In these results, both the PI3K and MAPK signals were essential for the EndMT event during the endocardial cushion formation, and Erbb3 mutations inhibited EndMT due to the decline in PI3K and MAPK signaling (Fig. S4). We additionally identified a reduction in the number of mesenchymal cells in the AV cushion due to failed EndMT event and decreased cell proliferation. These results suggest that the proliferation of mesenchymal cells in the AV cushion of Erbb3 mutant embryos was transiently reduced due to attenuated AKT and ERK phosphorylation.” We have also marked this passage with a yellow highlight in the manuscript.

Minor

1) Proteins should be written in all capital letters.

Response: We corrected all protein names to capital letters (Snail to SNAIL).

2) The paper needs to be reviewed by a native English speaker.

Response: Our manuscript was revised by a professional native English editor.

3) The quality of the figures provided for review is very poor. It is really hard to see details. In revision, high resolution images should be submitted (300 dpi or higher).

Response: We changed our figures to 300 dpi to increase the resolution.

Attachment

Submitted filename: Response to Reviewers_20210816.pdf

Click here for additional data file. (437.2KB, pdf)

Decision Letter 1

Robert W Dettman

22 Sep 2021

PONE-D-21-03408R1ERBB3-dependent AKT and ERK pathways are essential for atrioventricular cushion development in mouse embryosPLOS ONE

Dear Dr. Kim,

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Reviewers' comments:

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Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

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Reviewer #2: Partly

Reviewer #3: Partly

**********

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Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #3: Yes

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Reviewer #2: The authors addressed several concerns raised by this reviewer. Specifically, the authors admit their misunderstanding of AV valve morphogenesis and corrected their statements in the manuscript and removed related-figures. The authors also corrected their statements for AV septal morphogenesis.

However, there are some other concerns remained to be addressed.

The authors also agreed that low cell density in the mutant AV cushions can be explained by the low proliferation ratio of endocardial mesenchymal cells in the mutant AV cushions without having EndMT failure.

The AV explants were dissected out from the embryos at E9.5 and cultured on the collagen gels for 48 hours. The authors show a decrease in mesenchymal cell numbers in the mutant AV cushions cultured on the collagen gels (Fig. 3D). During the 48 hours, newly formed mesenchymal cells proliferate. Proliferation ratio of mesenchymal cells in mutant mice is significantly lower than that of wild type mesenchymal cells (Fig. 4B) at E10.5. Lower cell numbers of mesenchymal cells derived from mutant AV explants and lower cell densities in mutant AV cushion mesenchyme can be simply explained by lower proliferation ratio of mutant AV cushion mesenchymal cells.

In case cell numbers and densities are low but there is no significant difference in cell proliferation ratio between mutant and wild type AV cushion mesenchymal cells, one can claim that there is EndMT defect.

In this paper, the authors do not present clear evidence that there are EndMT defects in the mutant AV cushions. The data the authors present in this manuscript show that the mutant mice exhibit defects in AV endocardial cushion development. The abstract should be revised to reflect their findings.

Abstract, Page 2, Line 33, Erbb3 knock-in-embryos showed a decrease in the endocardial mesenchymal transformation (EndMT)

The following description is suggested:

Erbb3 knock-in-embryos showed a decrease in mesenchymal cell numbers and density in AV cushions.

Minor issue:

As the reviewer #3 suggests, the authors have to improve qualities of their figures. According to the figures this reviewer can see through the web, figures appear still very vague and not sharp enough for actual publication.

Reviewer #3: I appreciate the authors’ efforts to address the changes requested by the reviewers. I have read the new manuscript and there are several issues that still need to be addressed.

1) The sentence, “were caused by overall (Fig. 2C).” on line 223 appears to have been truncated.

2) Remove the word “Expected” in Figure 2A

3) If the number of mesenchymal cells is lower in WT and 7a/7a embryos, but the density is the same, then that indicates that the endocardial cushion size in 7A/7A embryos is smaller. Correct?

4) The authors state that, VE-cadherin is a “key player” in EMT. They should state specifically its role in EMT. I assume that in this context, VE-cadherin represses EMT. Correct?

5) For Fig 5A it appears that the authors selected differnet p-AKT and AKT lanes for their analysis of wild-type, heterozygous and null embryos. The GAPDH control lanes were also selected form alternative lanes as well (see Supplemental Material). This is unacceptable.

6) Fig. 5B. Show the E10.5 immunohistochemistry.

7) Fig. 5C. Show the E10.5 immunohistochemistry.

8) I agree that the authors show that, “In both mutants, the right ventricle was smaller than the left ventricle, and the myocardial thickness was reduced.” I however, disagree that these results, “suggest that the heart defects in both the Erbb3-/- and Erbb37A/7A mutants were caused by overall growth retardation.” If that were true, then you would expect an overall reduction in heart size, not in defects in only one ventricle. They may not have a good explanation, but no explanation is better than one that can’t be supported by the data.

9) I also note that the change in the right ventricle size compared to the left ventricle is easy to see at E12.5, The authors show no embryo photos at that stage. Instead, the small embryo size is seen at E13.5 when the hearts in section also seem to be overall smaller than wild-type.

10) Was the myocardial thickness of the left ventricle affected?

11) The authors state, “Overall, our results suggest that the YXXA mutation completely blocks the activation of ERBB3-dependent PI3K signaling than does the FXXM mutation of ERBB3.” This is not grammatically correct. Did they mean to say, “Overall, our results suggest that the YXXA mutation blocks the activation of ERBB3-dependent PI3K signaling more so than does the FXXM mutation of ERBB3.”

12) The authors also state, “Furthermore, our data suggest that ERBB3-dependent PI3K signaling is essential for AV cushion development and valve formation.” The AV cushions develop in ERBB3 null mice, right? Hence, ERRB3 can’t be essential for AV cushion development. In addition, I have seen no evidence of any effect on valve formation in this paper.

**********

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Reviewer #3: No

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PLoS One. 2021 Oct 29;16(10):e0259426. doi: 10.1371/journal.pone.0259426.r004

Author response to Decision Letter 1

6 Oct 2021

Point-by-point Responses

We thank the reviewers for their positive and constructive comments and suggestions. In response to their comments, we have accordingly revised and rearranged the manuscript and figures. Please find our point-by-point responses to the reviewers’ constructive criticisms below.

Reviewers' comments:

Reviewer #2:

The authors addressed several concerns raised by this reviewer. Specifically, the authors admit their misunderstanding of AV valve morphogenesis and corrected their statements in the manuscript and removed related-figures. The authors also corrected their statements for AV septal morphogenesis. However, there are some other concerns remained to be addressed. The authors also agreed that low cell density in the mutant AV cushions can be explained by the low proliferation ratio of endocardial mesenchymal cells in the mutant AV cushions without having EndMT failure.

The AV explants were dissected out from the embryos at E9.5 and cultured on the collagen gels for 48 hours. The authors show a decrease in mesenchymal cell numbers in the mutant AV cushions cultured on the collagen gels (Fig. 3D). During the 48 hours, newly formed mesenchymal cells proliferate. Proliferation ratio of mesenchymal cells in mutant mice is significantly lower than that of wild type mesenchymal cells (Fig. 4B) at E10.5. Lower cell numbers of mesenchymal cells derived from mutant AV explants and lower cell densities in mutant AV cushion mesenchyme can be simply explained by lower proliferation ratio of mutant AV cushion mesenchymal cells.

In case cell numbers and densities are low but there is no significant difference in cell proliferation ratio between mutant and wild type AV cushion mesenchymal cells, one can claim that there is EndMT defect. In this paper, the authors do not present clear evidence that there are EndMT defects in the mutant AV cushions. The data the authors present in this manuscript show that the mutant mice exhibit defects in AV endocardial cushion development. The abstract should be revised to reflect their findings. Abstract, Page 2, Line 33, Erbb3 knock-in-embryos showed a decrease in the endocardial mesenchymal transformation (EndMT).

The following description is suggested: Erbb3 knock-in-embryos showed a decrease in mesenchymal cell numbers and density in AV cushions.

Response: We agree with your proposed suggestion and have modified the phrase on p.2, lines 32–34, as follows: “Erbb3 knock-in embryos exhibited lethality between E12.5 to E13.5, and showed a decrease in mesenchymal cell numbers and density in AV cushions.”

Minor issue:

As the reviewer #3 suggests, the authors have to improve qualities of their figures. According to the figures this reviewer can see through the web, figures appear still very vague and not sharp enough for actual publication.

Response: Thank you for the helpful feedback. We replaced the original figures with revised figures using PACE to improve their quality.

Reviewer #3:

I appreciate the authors’ efforts to address the changes requested by the reviewers. I have read the new manuscript and there are several issues that still need to be addressed.

1) The sentence, “were caused by overall (Fig. 2C).” on line 223 appears to have been truncated.

Response: Our apologies for the unintended omission. We have modified the sentence on p.9, lines 221–222, as follows: “Microscopic and histological examination results of E12.5 embryos showed that both Erbb37A/7A and Erbb3-/- embryos have a small right ventricle (Fig. 2B, C).”

2) Remove the word “Expected” in Figure 2A

Response: We have removed the word "Expected" from Figure 2A.

3) If the number of mesenchymal cells is lower in WT and 7a/7a embryos, but the density is the same, then that indicates that the endocardial cushion size in 7A/7A embryos is smaller. Correct?

Response: Yes, that is correct.

4) The authors state that, VE-cadherin is a “key player” in EMT. They should state specifically its role in EMT. I assume that in this context, VE-cadherin represses EMT. Correct?

Response: Thank you for your query. Midgett et al. (Frontiers in Physiology, 2017, doi: 10.3389/fphys.2017.00056.) reported that VE-cadherin connects the junctions between endocardial cells and it is lost when EndMT occurs. Furthermore, VE-cadherin protein expressed in endocardial cells is a recognized endocardial cell marker. Niessen et al. (Circulation Research, 2008, doi: 10.1161/CIRCRESAHA.108.174318.) described a mechanism in which the function of Slug and Snail is similar, both involving the negative regulation of VE-cadherin expression and the initiation of EndMT. In this context, we agree with the reviewer’s position that VE-cadherin is a key player in EndMT and represses EndMT.

5) For Fig 5A it appears that the authors selected differnet p-AKT and AKT lanes for their analysis of wild-type, heterozygous and null embryos. The GAPDH control lanes were also selected form alternative lanes as well (see Supplemental Material). This is unacceptable.

Response: We agree with your opinion. We use Western blot data from the same lane in the revised Fig. 5A.

6) Fig. 5B. Show the E10.5 immunohistochemistry.

Response: Fig. 5B and C only show the E11.5 immunohistochemistry and there are no images of E10.5 immunohistochemical staining. We performed immunofluorescence staining in both E10.5 and E11.5 embryos to measure the relative intensities of P-AKT and P-ERK proteins, but immunohistochemical staining was performed and imaged only at the representative E11.5 stage.

7) Fig. 5C. Show the E10.5 immunohistochemistry.

Response: Although immunofluorescence staining was performed in E10.5 and E11.5 embryos to measure P-AKT and P-ERK protein intensity, immunohistochemical staining was performed and imaged only at the representative E11.5 stage.

8) I agree that the authors show that, “In both mutants, the right ventricle was smaller than the left ventricle, and the myocardial thickness was reduced.” I however, disagree that these results, “suggest that the heart defects in both the Erbb3-/- and Erbb37A/7A mutants were caused by overall growth retardation.” If that were true, then you would expect an overall reduction in heart size, not in defects in only one ventricle. They may not have a good explanation, but no explanation is better than one that can’t be supported by the data.

Response: In our results, the right ventricle of both mutants was smaller than the left ventricle and had decreased myocardial thickness. Our results showing defects in cell proliferation and EndMT support this finding. In addition, although not quantified, Figure 2 shows that the heart size from E12.5 to E13.5 is progressively smaller than normal. These findings were the basis why we stated that heart defects in both Erbb3-/- and Erbb37A/7A mutants were caused by the overall growth retardation.

9) I also note that the change in the right ventricle size compared to the left ventricle is easy to see at E12.5, The authors show no embryo photos at that stage. Instead, the small embryo size is seen at E13.5 when the hearts in section also seem to be overall smaller than wild-type.

Response: Thank you for the query. In E12.5 embryos, there were no morphological differences between the normal and the two variants. Therefore, the heart sections of E12.5 and E13.5 shown in Figure 2 address the reviewer's concern.

10) Was the myocardial thickness of the left ventricle affected?

Response: We found decreased myocardial thickness in both Erbb3-/- and Erbb37A/7A, but the difference between the left and right myocardial thickness was not significant.

11) The authors state, “Overall, our results suggest that the YXXA mutation completely blocks the activation of ERBB3-dependent PI3K signaling than does the FXXM mutation of ERBB3.” This is not grammatically correct. Did they mean to say, “

PI3K signaling more so than does the FXXM mutation of ERBB3.”

Response: The constructive feedback is appreciated. We have modified the phrase on p.14, lines 351–353, as follows: “Overall, our results suggest that the YXXA mutation blocks the activation of ERBB3-dependent PI3K signaling more than the FXXM mutation of ERBB3.”

12) The authors also state, “Furthermore, our data suggest that ERBB3-dependent PI3K signaling is essential for AV cushion development and valve formation.” The AV cushions develop in ERBB3 null mice, right? Hence, ERRB3 can’t be essential for AV cushion development. In addition, I have seen no evidence of any effect on valve formation in this paper.

Response: Thank you for raising this important point. We agree and have removed the phrase on p.14, lines 353–354.

Attachment

Submitted filename: Response to Reviewers_20211005.docx

Click here for additional data file. (510.9KB, docx)

Decision Letter 2

Robert W Dettman

20 Oct 2021

ERBB3-dependent AKT and ERK pathways are essential for atrioventricular cushion development in mouse embryos

PONE-D-21-03408R2

Dear Dr. Kim,

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Kind regards,

Robert W Dettman, PhD

Academic Editor

PLOS ONE

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Thank you for your revised submission. I have reviewed your changes and found them to be acceptable. Thank you for your excellent submission.

Reviewers' comments:

Acceptance letter

Robert W Dettman

22 Oct 2021

PONE-D-21-03408R2

ERBB3-dependent AKT and ERK pathways are essential for atrioventricular cushion development in mouse embryos

Dear Dr. Kim:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Kind regards,

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on behalf of

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Academic Editor

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