Use of Bmp1/Tll1 doubly homozygous null mice and proteomics to identify and validate in vivo substrates of bone morphogenetic protein 1/tolloid-like metalloproteinases

Bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD), two proteinases encoded by Bmp1, provide procollagen C-proteinase (pCP) activity that converts procollagens I to III into the major fibrous components of mammalian extracellular matrix (ECM). Yet, although Bmp1(-/-) mice have aberrant collagen fibrils, they have residual pCP activity, indicative of genetic redundancy. Mammals possess two additional proteinases structurally similar to BMP-1 and mTLD: the genetically distinct mammalian Tolloid-like 1 (mTLL-1) and mTLL-2. Mice lacking the mTLL-1 gene Tll1 are embryonic lethal but have pCP activity levels similar to those of the wild type, suggesting that mTLL-1 might not be an in vivo pCP. In vitro studies have shown BMP-1 and mTLL-1 capable of cleaving Chordin, an extracellular antagonist of BMP signaling, suggesting that these proteases might also serve to modulate BMP signaling and to coordinate the latter with ECM formation. However, in vivo evidence of roles for BMP-1 and mTLL-1 in BMP signaling in mammals is lacking. To remove functional redundancy obscuring the in vivo functions of BMP-1-related proteases in mammals, we here characterize Bmp1 Tll1 doubly null mouse embryos. Although these appear morphologically indistinguishable from Tll1(-/-) embryos, biochemical analysis of cells derived from doubly null embryos shows functional redundancy removed to an extent enabling us to demonstrate that (i) products of Bmp1 and Tll1 are responsible for in vivo cleavage of Chordin in mammals and (ii) mTLL-1 is an in vivo pCP that provides residual activity observed in Bmp1(-/-) embryos. Removal of functional redundancy also enabled use of Bmp1(-/-) Tll1(-/-) cells in a proteomics approach for identifying novel substrates of Bmp1 and Tll1 products.     

Multiple bone morphogenetic protein 1-related mammalian metalloproteinases process pro-lysyl oxidase at the correct physiological site and control lysyl oxidase activation in mouse embryo fibroblast cultures

Lysyl oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-lysyl oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-lysyl oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-lysyl oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in lysyl oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for lysyl oxidase enzyme activity and for accumulation of pro-lysyl oxidase and mature approximately 30-kDa lysyl oxidase. Wild type cells or cells singly null for Bmp1 or Tll1 all produced both pro-lysyl oxidase and processed lysyl oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-lysyl oxidase and had lysyl oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, and Tll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of lysyl oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-lysyl oxidase as the first known substrate for mTLL-2.     

Bone morphogenetic protein-1 processes probiglycan

Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays important roles in regulating the deposition of fibrous extracellular matrix in vertebrates, including provision of the procollagen C-proteinase activity that processes the major fibrillar collagens I-III. Biglycan, a small leucine-rich proteoglycan, is a nonfibrillar extracellular matrix component with functions that include the positive regulation of bone formation. Biglycan is synthesized as a precursor with an NH(2)-terminal propeptide that is cleaved to yield the mature form found in vertebrate tissues. Here, we show that BMP-1 cleaves probiglycan at a single site, removing the propeptide and producing a biglycan molecule with an NH(2) terminus identical to that of the mature form found in tissues. BMP-1-related proteases mammalian Tolloid and mammalian Tolloid-like 1 (mTLL-1) are shown to have low but detectable levels of probiglycan-cleaving activity. Comparison shows that wild type mouse embryo fibroblasts (MEFs) produce only fully processed biglycan, whereas MEFs derived from embryos homozygous null for the Bmp1 gene, which encodes both BMP-1 and mammalian Tolloid, produce predominantly unprocessed probiglycan, and MEFs homozygous null for both the Bmp1 gene and the mTLL-1 gene Tll1 produce only unprocessed probiglycan. Thus, all detectable probiglycan-processing activity in MEFs is accounted for by the products of these two genes.     

Activation of Tolloid‐like 1 gene expression by the cardiac specific homeobox gene Nkx2–5

Mammalian Tolloid‐like 1 (Tll‐1) is a pleiotropic metalloprotease that is expressed by a small subset of cells within the precardiac mesoderm and is necessary for proper heart development. Following heart tube formation Tll‐1 is expressed by the endocardium and regions of myocardium overlying the region of the muscular interventricular septum. Mutations in Tll‐1 lead to embryonic lethality due to cardiac defects. We demonstrate that the Tll‐1 promoter contains Nkx2–5 binding sites and that the Tll‐1 promoter is activated by and directly binds Nkx2–5. Tll‐1 expression is ablated by a dominant negative Nkx2–5 or by mutation of the Nkx2–5 binding sites within the Tll‐1 promoter. In vivo, Tll‐1 expression is decreased in the hearts of Nkx2–5 knockout embryos when compared with hemizygous and wild‐type embryos. These results show that Nkx2–5 is a direct activator of Tll‐1 expression and provide insight into the mechanism of the defects found in both the Tll‐1 and Nkx2–5 knockout mice.     

Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis.

Vertebrate bone morphogenetic protein 1 (BMP-1) and Drosophila Tolloid (TLD) are prototypes of a family of metalloproteases with important roles in various developmental events. BMP-1 affects morphogenesis, at least partly, via biosynthetic processing of fibrillar collagens, while TLD affects dorsal-ventral patterning by releasing TGFbeta-like ligands from latent complexes with the secreted protein Short Gastrulation (SOG). Here, in a screen for additional mammalian members of this family of developmental proteases, we identify novel family member mammalian Tolloid-like 2 (mTLL-2) and compare enzymatic activities and expression domains of all four known mammalian BMP-1/TLD-like proteases [BMP-1, mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mTLL-2]. Despite high sequence similarities, distinct differences are shown in ability to process fibrillar collagen precursors and to cleave Chordin, the vertebrate orthologue of SOG. As previously demonstrated for BMP-1 and mTLD, mTLL-1 is shown to specifically process procollagen C-propeptides at the physiologically relevant site, while mTLL-2 is shown to lack this activity. BMP-1 and mTLL-1 are shown to cleave Chordin, at sites similar to procollagen C-propeptide cleavage sites, and to counteract dorsalizing effects of Chordin upon overexpression in Xenopus embryos. Proteases mTLD and mTLL-2 do not cleave Chordin. Differences in enzymatic activities and expression domains of the four proteases suggest BMP-1 as the major Chordin antagonist in early mammalian embryogenesis and in pre- and postnatal skeletogenesis.     

Mammalian tolloid metalloproteinase,and not matrix metalloprotease 2 or membrane type 1 metalloprotease,processes laminin-5 in keratinocytes and skin

Laminin-5, a major adhesive ligand for epithelial cells, undergoes processing of its gamma2 and alpha3 chains. This study investigated the mechanism of laminin-5 processing by keratinocytes. BI-1 (BMP-1 isoenzyme inhibitor-1), a selective inhibitor of a small group of astacin-like metalloproteinases, which includes bone morphogenetic protein 1 (BMP-1), mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mammalian Tolloid-like 2 (mTLL-2), inhibited the processing of laminin-5 gamma2 and alpha3 chains in keratinocyte cultures in a dose-dependent manner. In a proteinase survey, all BMP-1 isoenzymes processed human laminin-5 gamma2 and alpha3 chains to 105- and 165-kDa fragments, respectively. In contrast, MT1-MMP and MMP-2 did not cleave the gamma2 chain of human laminin-5 but processed the rat laminin gamma2 chain to an 80-kDa fragment. An immunoblot and quantitative PCR survey of the BMP-1 isoenzymes revealed expression of mTLD in primary keratinocyte cultures but little or no expression of BMP-1, mTLL-1, or mTLL-2. mTLD was shown to cleave the gamma2 chain at the same site as the previously identified BMP-1 cleavage site. In addition, mTLD/BMP-1 null mice were shown to have deficient laminin-5 processing. Together, these data identify laminin-5 as a substrate for mTLD, suggesting a role for laminin-5 processing by mTLD in the skin.     

Role of Hand1/eHAND in the dorso-ventral patterning and interventricular septum formation in the embryonic heart

Molecular mechanisms for the dorso-ventral patterning and interventricular septum formation in the embryonic heart are unknown. To investigate a role of Hand1/eHAND in cardiac chamber formation, we generated Hand1/eHAND knock-in mice where Hand1/eHAND cDNA was placed under the control of the MLC2V promoter. In Hand1/eHAND knock-in mice, the outer curvature of the right and left ventricles expanded more markedly. Moreover, there was no interventricular groove or septum formation, although molecularly, Hand1/eHAND knock-in hearts had two ventricles. However, the morphology of the inner curvature of the ventricles, the atrioventricular canal, and the outflow tract was not affected by Hand1/eHAND expression. Furthermore, expression of Hand1/eHAND in the whole ventricles altered the expression patterns of Chisel, ANF, and Hand2/dHAND but did not affect Tbx5 expression. In contrast, the interventricular septum formed normally in transgenic embryos overexpressing Hand1/eHAND in the right ventricle but not in the boundary region. These results suggested that Hand1/eHAND is involved in expansion of the ventricular walls and that absence of Hand1/eHAND expression in the boundary region between the right and left ventricles may be critical in the proper formation of the interventricular groove and septum. Furthermore, Hand1/eHAND is not a master regulatory gene that specifies the left ventricle myocyte lineage but may control the dorso-ventral patterning in concert with additional genes.     

Identification of the minimal domain structure of bone morphogenetic protein-1 (BMP-1) for chordinase activity: chordinase activity is not enhanced by procollagen C-proteinase enhancer-1 (PCPE-1)

Bone morphogenetic protein 1 (BMP-1), which is a tolloid member of the astacin-like family of zinc metalloproteinases, is a highly effective procollagen C-proteinase (PCP) and chordinase. On the other hand, mammalian tolloid like-2 (mTLL-2) does not cleave chordin or procollagen; procollagen is cleaved by mTLL-2 in the presence of high levels of procollagen C-proteinase enhancer-1 (PCPE-1), for reasons that are unknown. We used these differences in activity between BMP-1 and mTLL-2 to narrow in on the domains in BMP-1 that specify PCP and chordinase activity. Using a domain swap approach, we showed that: 1) the metalloproteinase and CUB2 domains of BMP-1 are absolutely required for PCP activity; swaps with either of the corresponding domains in BMP-1 and mTLL-2 did not result in procollagen cleavage and 2) the proteinase domain of mTLL-2 can cleave chordin if coupled to the CUB1 domain of BMP-1. Therefore, the minimal structure for chordinase activity comprises a metalloproteinase domain (either from BMP-1 or from mTLL-2) and the CUB1 domain of BMP-1 (the CUB1 domain of mTLL-2 cannot substitute for the CUB1 domain of BMP-1). We showed that the minimal procollagen C-proteinase (BMP-1 lacking the EGF and CUB3 domain) was enhanced by PCPE-1 but not as well as BMP-1 retaining the CUB3 domain. Further studies showed that PCPE-1 had no effect on the ability of BMP-1 to cleave chordin. The data support a previously suggested mechanism of PCPE-1 whereby PCPE-1 interacts with procollagen, but in addition, the CUB3 domain of BMP-1 appears to augment the interaction.     

Bone morphogenetic protein-1 processes insulin-like growth factor-binding protein 3

The bone morphogenetic protein-1 (BMP1)-like metalloproteinases play key roles in extracellular matrix formation, by converting precursors into mature functional proteins involved in forming the extracellular matrix. The BMP1-like proteinases also play roles in activating growth factors, such as BMP2/4, myostatin, growth differentiation factor 11, and transforming growth factor β1, by cleaving extracellular antagonists. The extracellular insulin-like growth factor-binding proteins (IGFBPs) are involved in regulating the effects of insulin-like growth factors (IGFs) on growth, development, and metabolism. Of the six IGFBPs, IGFBP3 has the greatest interaction with the large pool of circulating IGFs. It is also produced locally in tissues and is itself regulated by proteolytic processing. Here, we show that BMP1 cleaves human and mouse IGFBP3 at a single conserved site, resulting in markedly reduced ability of cleaved IGFBP3 to bind IGF-I or to block IGF-I-induced cell signaling. In contrast, such cleavage is shown to result in enhanced IGF-I-independent ability of cleaved IGFBP3 to block FGF-induced proliferation and to induce Smad phosphorylation. Consistent with in vivo roles for such cleavage, it is shown that, whereas wild type mouse embryo fibroblasts (MEFs) produce cleaved IGFBP3, MEFs doubly null for the Bmp1 gene and for the Tll1 gene, which encodes the related metalloproteinase mammalian Tolloid-like 1 (mTLL1), produce only unprocessed IGFBP3, thus demonstrating endogenous BMP1-related proteinases to be responsible for IGFBP3-processing activity in MEFs. Similarly, in zebrafish embryos, overexpression of Bmp1a is shown to reverse an Igfbp3-induced phenotype, consistent with the ability of BMP1-like proteinases to cleave IGFBP3 in vivo.     

Proteinases of the bone morphogenetic protein-1 family convert procollagen VII to mature anchoring fibril collagen

Collagen VII is the major structural component of the anchoring fibrils at the dermal-epidermal junction in the skin. It is secreted by keratinocytes as a precursor, procollagen VII, and processed into mature collagen during polymerization of the anchoring fibrils. We show that bone morphogenetic protein-1 (BMP-1), which exhibits procollagen C-proteinase activity, cleaves the C-terminal propeptide from human procollagen VII. The cleavage occurs at the BMP-1 consensus cleavage site SYAA/DTAG within the NC-2 domain. Mammalian tolloid-like (mTLL)-1 and -2, two other proteases of the astacin enzyme family, were able to process procollagen VII at the same site in vitro. Immunohistochemical and genetic evidence supported the involvement of these enzymes in cleaving type VII procollagen in vivo. Both BMP-1 and mTLL-1 are expressed in the skin and in cultured cutaneous cells. A naturally occurring deletion in the human COL7A1 gene, 8523del14, which is associated with dystrophic epidermolysis bullosa and eliminates the BMP-1 consensus sequence, abolished processing of procollagen VII, and in mutant skin procollagen VII accumulated at the dermal-epidermal junction. On the other hand, deficiency of BMP-1 in the skin of knockout mouse embryos did not prevent processing of procollagen VII to mature collagen, suggesting that mTLL-1 and/or mTLL-2 can substitute for BMP-1 in the processing of procollagen VII in situ.     

BMP is an important regulator of proepicardial identity in the chick embryo

The proepicardium (PE) is a transient structure formed by pericardial coelomic mesothelium at the venous pole of the embryonic heart and gives rise to several cell types of the mature heart. In order to study PE development in chick embryos, we have analyzed the expression pattern of the marker genes Tbx18, Wt1, and Cfc. During PE induction, the three marker genes displayed a left-right asymmetric expression pattern. In each case, expression on the right side was stronger than on the left side. The left-right asymmetric gene expression observed here is in accord with the asymmetric formation of the proepicardium in the chick embryo. While initially the marker genes were expressed in the primitive sinus horn, subsequently, expression became confined to the PE mesothelium. In order to search for signaling factors involved in PE development, we studied Bmp2 and Bmp4 expression. Bmp2 was bilaterally expressed in the sinus venosus. In contrast, Bmp4 expression was initially expressed unilaterally in the right sinus horn and subsequently in the PE. In order to assess its functional role, BMP signaling was experimentally modulated by supplying exogenous BMP2 and by inhibiting endogenous BMP signaling through the addition of Noggin. Both supplying BMP and blocking BMP signaling resulted in a loss of PE marker gene expression. Surprisingly, both experimental situations lead to cardiac myocyte formation in the PE cultures. Careful titration experiments with exogenously added BMP2 or Noggin revealed that PE-specific marker gene expression depends on a low level of BMP signaling. Implantation of BMP2-secreting cells or beads filled with Noggin protein into the right sinus horn of HH stage 11 embryos resulted in downregulation of Tbx18 expression, corresponding to the results of the explant assay. Thus, a distinct level of BMP signaling is required for PE formation in the chick embryo.     

Bone morphogenetic protein-1/Tolloid-like proteinases process dentin matrix protein-1

Bone morphogenetic protein-1 (BMP-1)/Tolloid-like metalloproteinases play key roles in formation of mammalian extracellular matrix (ECM), through the biosynthetic conversion of precursor proteins into their mature functional forms. These proteinases probably play a further role in formation of bone through activation of transforming growth factor beta-like BMPs. Dentin matrix protein-1 (DMP1), deposited into the ECM during assembly and involved in initiating mineralization of bones and teeth, is thought to undergo proteolysis in vivo to generate functional cleavage fragments found in extracts of mineralized tissues. Here, we have generated recombinant DMP1 and demonstrate that it is cleaved, to varying extents, by all four mammalian BMP-1/Tolloid-like proteinases, to generate fragments similar in size to those previously isolated from bone. Consistent with possible roles for the BMP-1/Tolloid-like proteinases in the physiological processing of DMP1, NH2-terminal sequences of products generated by BMP-1 cleavage of DMP1 match those predicted from processing at the predicted DMP1 site that shows greatest cross-species conservation of sequences. Moreover, fibroblasts derived from mouse embryos homozygous null for genes encoding three of the four mammalian BMP-1/Tolloid-like proteinases appear to be deficient in processing of DMP1. Thus, a further role for BMP-1-Tolloid-like proteinases in formation of mineralized tissues is indicated, via proteolytic processing of DMP1.     

DSCR1 gene expression is dependent on NFATc1 during cardiac valve formation and colocalizes with anomalous organ development in trisomy 16 mice

The Down syndrome critical region 1 (DSCR1) gene is present in the region of human chromosome 21 and the syntenic region of mouse chromosome 16, trisomy of which is associated with congenital heart defects observed in Down syndrome. DSCR1 encodes a regulatory protein in the calcineurin/NFAT signal transduction pathway. During valvuloseptal development in the heart, DSCR1 is expressed in the endocardium of the developing atrioventricular and semilunar valves, the muscular interventricular septum, and the ventricular myocardium. Human DSCR1 contains an NFAT-rich calcineurin-responsive element adjacent to exon 4. Transgenic mice generated with a homologous regulatory region of the mouse DSCR1 gene linked to lacZ (DSCR1(e4)/lacZ) show gene activation in the endocardium of the developing valves and aorticopulmonary septum of the heart, recapitulating a specific subdomain of endogenous DSCR1 cardiac expression. DSCR1(e4)/lacZ expression in the developing valve endocardium colocalizes with NFATc1 and, endocardial DSCR1(e4)/lacZ, is notably reduced or absent in NFATc1(-/-) embryos. Furthermore, expression of the endogenous DSCR1(e4) isoform is decreased in the outflow tract of NFATc1(-/-) hearts, and the DSCR1(e4) intragenic element is trans-activated by NFATc1 in cell culture. In trisomy 16 (Ts16) mice, expression of endogenous DSCR1 and DSCR1(e4)/lacZ colocalizes with anomalous valvuloseptal development, and transgenic Ts16 hearts have increased beta-galactosidase activity. DSCR1 and DSCR1(e4)/lacZ also are expressed in other organ systems affected by trisomy 16 in mice or trisomy 21 in humans including the brain, eye, ear, face, and limbs. Together, these results show that DSCR1(e4) expression in the developing valve endocardium is dependent on NFATc1 and support a role for DSCR1 in normal cardiac valvuloseptal formation as well as the abnormal development of several organ systems affected in individuals with Down syndrome.     

hch-1, a gene required for normal hatching and normal migration of a neuroblast in C. elegans, encodes a protein related to TOLLOID and BMP-1.

Proteins of the tolloid/bone morphogenetic protein (BMP)-1 family play important roles in the differentiation of cell fates. Among those proteins are BMP-1, which plays a role in cartilage and bone formation in mammals, the TOLLOID protein, which is required for the establishment of the dorsoventral axis of Drosophila embryos and BP10/SpAN, which are thought to act in the morphogenesis of sea urchins. These proteins have some properties in common. First, they contain the astacin metalloprotease domain, the CUB domain and the epidermal growth factor-like domain. Second, they are expressed in embryos at stages expected for their role in cell differentiation. Third, at least BMP-1 and TOLLOID are thought to interact with proteins of the transforming growth factor-beta family. We report that the hch-1 gene of the nematode Caenorhabditis elegans encodes a tolloid/BMP-1 family protein. The protein has the characteristic domains common to the tolloid/ BMP-1 family. Like other members of the family, it is expressed in embryos. However, the phenotype of hch-1 mutants shows that it is required for normal hatching and normal migration of a post-embryonic neuroblast. Furthermore, in spite of its expression in embryogenesis, it is not required for the viability of embryos. These results show new functions of the tolloid/BMP-1 family proteins and give insight into their evolution.     

Developmental consequences of abnormal folate transport during murine heart morphogenesis

BACKGROUND: Folic acid is essential for the synthesis of nucleotides and methyl transfer reactions. Folic acid-binding protein one (Folbp1) is the primary mediator of folic acid transport into murine cells. Folbp1 knockout mouse embryos die in utero with multiple malformations, including severe congenital heart defects (CHDs). Although maternal folate supplementation is believed to prevent human conotruncal heart defects, its precise role during cardiac morphogenesis remains unclear. In this study, we examined the role of folic acid on the phenotypic expression of heart defects in Folbp1 mice, mindful of the importance of neural crest cells to the formation of the conotruncus. METHODS: To determine if the Folbp1 gene participates in the commitment and differentiation of the cardiomyocytes, relative levels of dead and proliferating precursor cells in the heart were examined by flow cytometry, Western blot, and immunohistostaining. RESULTS: Our studies revealed that impaired folic acid transport results in extensive apoptosis-mediated cell death, which concentrated in the interventricular septum and truncus arteriosus, thus being anatomically restricted to the two regions of congenital heart defects. Together with a reduced proliferative capacity of the cardiomyocytes, the limited size of the available precursor cell pool may contribute to the observed cardiac defects. Notably, there is a substantial reduction in Pax-3 expression in the region of the presumptive migrating cardiac neural crest, suggesting that this cell population may be the most severely affected by the massive cell death. CONCLUSIONS: Our findings demonstrate for the first time a prominent role of the Folbp1 gene in mediating susceptibility to heart defects.     

Hesr1 and Hesr2 regulate atrioventricular boundary formation in the developing heart through the repression of Tbx2

The establishment of chamber specificity is an essential requirement for cardiac morphogenesis and function. Hesr1 (Hey1) and Hesr2 (Hey2) are specifically expressed in the atrium and ventricle, respectively, implicating these genes in chamber specification. In our current study, we show that the forced expression of Hesr1 or Hesr2 in the entire cardiac lineage of the mouse results in the reduction or loss of the atrioventricular (AV) canal. In the Hesr1-misexpressing heart, the boundaries of the AV canal are poorly defined, and the expression levels of specific markers of the AV myocardium, Bmp2 and Tbx2, are either very weak or undetectable. More potent effects were observed in Hesr2-misexpressing embryos, in which the AV canal appears to be absent entirely. These data suggest that Hesr1 and Hesr2 may prevent cells from expressing the AV canal-specific genes that lead to the precise formation of the AV boundary. Our findings suggest that Tbx2 expression might be directly suppressed by Hesr1 and Hesr2. Furthermore, we find that the expression of Hesr1 and Hesr2 is independent of Notch2 signaling. Taken together, our data demonstrate that Hesr1 and Hesr2 play crucial roles in AV boundary formation through the suppression of Tbx2.