External genitalia phenotypes of a Mab21l1-null mouse model for cerebellar, ocular, craniofacial, and genital (COFG) syndrome.

The cerebellar, ocular, craniofacial, and genital (COFG) syndrome is a human genetic disease that is caused by MAB21L1 mutations. A COFG mouse model with Mab21l1-null mutation causes severe microphthalmia and fontanelle dysosteogenesis, similar to the symptoms in human patients. One of the typical symptoms is scrotal agenesis in male infants, while male Mab21l1-null mice show hypoplastic preputial glands, a rodent-specific derivative of the cranial scrotal fold. However, it is still unclear where and how MAB21Ll acts in the external genitalia in both mice and humans. Here we show that, at the neonatal stage, MAB21L1 expression in the external genitalia was restricted to two mesenchymal cell populations-underneath the scrotal and labial skin and around the preputial and clitoral glands (PG/CG). Morphometric analyses of the Mab21l1-/- pups revealed a significant reduction in the external size of the scrotum, vulva, and CG, as well as PG. In the periglandular region around PG and CG, the periglandular mesenchymal cells showed a drastic reduction in both cell density and immunoreactive signals for several extracellular matrix proteins (e.g., collagen I, fibronectin, and proteoglycans), together with their reduced Ki67-positive cell proliferation index. In the Mab21l1-/- PG/CG, together with reduced vascularization, the glandular epithelia displayed atrophy with discontinuous basal lamina along the basal surface and defective glycogen accumulation in their cytoplasm. Under a 5-day organ culture of the isolated PG, the Mab21l1-/- explants showed poor outgrowth and retention of the glandular structure in vitro. However, the addition of exogenous Matrigel could partially rescue such tissue-autonomous phenotypes, showing glandular morphology similar to that of the wild-type explants. These findings suggest that MAB21L1+ mesenchymal cells play a crucial role in providing nutrient ECM support for glandular outgrowth and morphogenesis in the peripheral external genitalia.

SOX17-positive rete testis epithelium is required for Sertoli valve formation and normal spermiogenesis in the male mouse.

Seminiferous tubules (STs) in the mammalian testes are connected to the rete testis (RT) via a Sertoli valve (SV). Spermatozoa produced in the STs are released into the tubular luminal fluid and passively transported through the SV into the RT. However, the physiological functions of the RT and SV remain unclear. Here, we identified the expression of Sox17 in RT epithelia. The SV valve was disrupted before puberty in RT-specific Sox17 conditional knockout (Sox17-cKO) male mice. This induced a backflow of RT fluid into the STs, which caused aberrant detachment of immature spermatids. RT of Sox17-cKO mice had reduced expression levels of various growth factor genes, which presumably support SV formation. When transplanted next to the Sox17+ RT, Sertoli cells of Sox17-cKO mice reconstructed the SV and supported proper spermiogenesis in the STs. This study highlights the novel and unexpected modulatory roles of the RT in SV valve formation and spermatogenesis in mouse testes, as a downstream action of Sox17.

Anatomical and histological characteristics of the hepatobiliary system in adult Sox17 heterozygote mice.

Biliary atresia (BA) is a rare neonatal disease characterized by inflammation and obstruction of the extrahepatic bile ducts (EHBDs). The Sox17-haploinsufficient (Sox17+/- ) mouse is an animal model of BA that encompasses bile duct injury and subsequent BA-like inflammation by the neonatal stage. Most Sox17+/- neonates die soon after birth, but some Sox17+/- pups reach adulthood and have a normal life span, unlike human BA. However, the phenotype and BA-derived scars in the hepatobiliary organs of surviving Sox17+/- mice are unknown. Here, we examined the phenotypes of the hepatobiliary organs in post-weaning and young adult Sox17+/- mice. The results confirmed the significant reduction in liver weight, together with peripheral calcinosis and aberrant vasculature in the hepatic lobule, in surviving Sox17+/- mice as compared with their wild-type (WT) littermates. Such hepatic phenotypes may be sequelae of hepatobiliary damage at the fetal and neonatal stages, a notion supported by the slight, but significant, increases in the levels of serum markers of liver damage in adult Sox17+/- mice. The surviving Sox17+/- mice had a shorter gallbladder in which ectopic hepatic ducts were more frequent compared to WT mice. Also, the surviving Sox17+/- mice showed neither obstruction of the EHBDs nor atrophy or inflammation of hepatocytes or the intrahepatic ducts. These data suggest that some Sox17+/- pups with BA naturally escape lethality and recover from fetal hepatobiliary damages during the perinatal period, highlighting the usefulness of the in vivo model in understanding the hepatobiliary healing processes after surgical restoration of bile flow in human BA.

Gallbladder wall abnormality in biliary atresia of mouse Sox17+/- neonates and human infants.

Biliary atresia (BA) is characterized by the inflammation and obstruction of the extrahepatic bile ducts (EHBDs) in newborn infants. SOX17 is a master regulator of fetal EHBD formation. In mouse Sox17+/- BA models, SOX17 reduction causes cell-autonomous epithelial shedding together with the ectopic appearance of SOX9-positive cystic duct-like epithelia in the gallbladder walls, resulting in BA-like symptoms during the perinatal period. However, the similarities with human BA gallbladders are still unclear. In the present study, we conducted phenotypic analysis of Sox17+/- BA neonate mice, in order to compare with the gallbladder wall phenotype of human BA infants. The most characteristic phenotype of the Sox17+/- BA gallbladders is the ectopic appearance of SOX9-positive peribiliary glands (PBGs), so-called pseudopyloric glands (PPGs). Next, we examined SOX17/SOX9 expression profiles of human gallbladders in 13 BA infants. Among them, five BA cases showed a loss or drastic reduction of SOX17-positive signals throughout the whole region of gallbladder epithelia (SOX17-low group). Even in the remaining eight gallbladders (SOX17-high group), the epithelial cells near the decidual sites were frequently reduced in the SOX17-positive signal intensity. Most interestingly, the most characteristic phenotype of human BA gallbladders is the increased density of PBG/PPG-like glands in the gallbladder body, especially near the epithelial decidual site, indicating that PBG/PPG formation is a common phenotype between human BA and mouse Sox17+/- BA gallbladders. These findings provide the first evidence of the potential contribution of SOX17 reduction and PBG/PPG formation to the early pathogenesis of human BA gallbladders.This article has an associated First Person interview with the joint first authors of the paper.

Endocardium differentiation through Sox17 expression in endocardium precursor cells regulates heart development in mice.

The endocardium is the endothelial component of the vertebrate heart and plays a key role in heart development. Where, when, and how the endocardium segregates during embryogenesis have remained largely unknown, however. We now show that Nkx2-5+ cardiac progenitor cells (CPCs) that express the Sry-type HMG box gene Sox17 from embryonic day (E) 7.5 to E8.5 specifically differentiate into the endocardium in mouse embryos. Although Sox17 is not essential or sufficient for endocardium fate, it can bias the fate of CPCs toward the endocardium. On the other hand, Sox17 expression in the endocardium is required for heart development. Deletion of Sox17 specifically in the mesoderm markedly impaired endocardium development with regard to cell proliferation and behavior. The proliferation of cardiomyocytes, ventricular trabeculation, and myocardium thickening were also impaired in a non-cell-autonomous manner in the Sox17 mutant, likely as a consequence of down-regulation of NOTCH signaling. An unknown signal, regulated by Sox17 and required for nurturing of the myocardium, is responsible for the reduction in NOTCH-related genes in the mutant embryos. Our results thus provide insight into differentiation of the endocardium and its role in heart development.

Sox17 is essential for proper formation of the marginal zone of extraembryonic endoderm adjacent to a developing mouse placental disk.

In mouse conceptus, two yolk-sac membranes, the parietal endoderm (PE) and visceral endoderm (VE), are involved in protecting and nourishing early-somite-stage embryos prior to the establishment of placental circulation. Both PE and VE membranes are tightly anchored to the marginal edge of the developing placental disk, in which the extraembryonic endoderm (marginal zone endoderm: ME) shows the typical flat epithelial morphology intermediate between those of PE and VE in vivo. However, the molecular characteristics and functions of the ME in mouse placentation remain unclear. Here, we show that SOX17, not SOX7, is continuously expressed in the ME cells, whereas both SOX17 and SOX7 are coexpressed in PE cells, by at least 10.5 days postconception. The Sox17-null conceptus, but not the Sox7-null one, showed the ectopic appearance of squamous VE-like epithelial cells in the presumptive ME region, together with reduced cell density and aberrant morphology of PE cells. Such aberrant ME formation in the Sox17-null extraembryonic endoderm was not rescued by the chimeric embryo replaced with the wild-type gut endoderm by the injection of wild-type ES cells into the Sox17-null blastocyst, suggesting the cell autonomous defects in the extraembryonic endoderm of Sox17-null concepti. These findings provide direct evidence of the crucial roles of SOX17 in proper formation and maintenance of the ME region, highlighting a novel entry point to understand the in vivo VE-to-PE transition in the marginal edge of developing placenta.

Anatomy and development of the extrahepatic biliary system in mouse and rat: a perspective on the evolutionary loss of the gallbladder.

The gallbladder is the hepatobiliary organ for storing and secreting bile fluid, and is a synapomorphy of extant vertebrates. However, this organ has been frequently lost in several lineages of birds and mammals, including rodents. Although it is known as the traditional problem, the differences in development between animals with and without gallbladders are not well understood. To address this research gap, we compared the anatomy and development of the hepatobiliary systems in mice (gallbladder is present) and rats (gallbladder is absent). Anatomically, almost all parts of the hepatobiliary system of rats are topographically the same as those of mice, but rats have lost the gallbladder and cystic duct completely. During morphogenesis, the gallbladder-cystic duct domain (Gb-Cd domain) and its primordium, the biliary bud, do not develop in the rat. In the early stages, SOX17, a master regulator of gallbladder formation, is positive in the murine biliary bud epithelium, as seen in other vertebrates with a gallbladder, but there is no SOX17-positive domain in the rat hepatobiliary primordia. These findings suggest that the evolutionary loss of the Gb-Cd domain should be translated simply as the absence of a biliary bud at an early stage, which may correlate with alterations in regulatory genes, such as Sox17, in the rat. A SOX17-positive biliary bud is clearly definable as a developmental module that may be involved in the frequent loss of gallbladder in mammals.

Embryonic cholecystitis and defective gallbladder contraction in the Sox17-haploinsufficient mouse model of biliary atresia.

The gallbladder excretes cytotoxic bile acids into the duodenum through the cystic duct and common bile duct system. Sox17 haploinsufficiency causes biliary atresia-like phenotypes and hepatitis in late organogenesis mouse embryos, but the molecular and cellular mechanisms underlying this remain unclear. In this study, transcriptomic analyses revealed the early onset of cholecystitis in Sox17+/- embryos, together with the appearance of ectopic cystic duct-like epithelia in their gallbladders. The embryonic hepatitis showed positive correlations with the severity of cholecystitis in individual Sox17+/- embryos. Embryonic hepatitis could be induced by conditional deletion of Sox17 in the primordial gallbladder epithelia but not in fetal liver hepatoblasts. The Sox17+/- gallbladder also showed a drastic reduction in sonic hedgehog expression, leading to aberrant smooth muscle formation and defective contraction of the fetal gallbladder. The defective gallbladder contraction positively correlated with the severity of embryonic hepatitis in Sox17+/- embryos, suggesting a potential contribution of embryonic cholecystitis and fetal gallbladder contraction in the early pathogenesis of congenital biliary atresia.

Osteocrin, a peptide secreted from the heart and other tissues, contributes to cranial osteogenesis and chondrogenesis in zebrafish.

The heart is an endocrine organ, as cardiomyocytes (CMs) secrete natriuretic peptide (NP) hormones. Since the discovery of NPs, no other peptide hormones that affect remote organs have been identified from the heart. We identified osteocrin (Ostn) as an osteogenesis/chondrogenesis regulatory hormone secreted from CMs in zebrafish. ostn mutant larvae exhibit impaired membranous and chondral bone formation. The impaired bones were recovered by CM-specific overexpression of OSTN. We analyzed the parasphenoid (ps) as a representative of membranous bones. In the shortened ps of ostn morphants, nuclear Yap1/Wwtr1-dependent transcription was increased, suggesting that Ostn might induce the nuclear export of Yap1/Wwtr1 in osteoblasts. Although OSTN is proposed to bind to NPR3 (clearance receptor for NPs) to enhance the binding of NPs to NPR1 or NPR2, OSTN enhanced C-type NP (CNP)-dependent nuclear export of YAP1/WWTR1 of cultured mouse osteoblasts stimulated with saturable CNP. OSTN might therefore activate unidentified receptors that augment protein kinase G signaling mediated by a CNP-NPR2 signaling axis. These data demonstrate that Ostn secreted from the heart contributes to bone formation as an endocrine hormone.

Yap/Taz transcriptional activity in endothelial cells promotes intramembranous ossification via the BMP pathway.

Osteogenesis is categorized into two groups based on developmental histology, intramembranous and endochondral ossification. The role of blood vessels during endochondral ossification is well known, while their role in intramembranous ossification, especially the intertissue pathway, is poorly understood. Here, we demonstrate endothelial Yap/Taz is a novel regulator of intramembranous ossification in zebrafish. Appropriate blood flow is required for Yap/Taz transcriptional activation in endothelial cells and intramembranous ossification. Additionally, Yap/Taz transcriptional activity in endothelial cells specifically promotes intramembranous ossification. BMP expression by Yap/Taz transactivation in endothelial cells is also identified as a bridging factor between blood vessels and intramembranous ossification. Furthermore, the expression of Runx2 in pre-osteoblast cells is a downstream target of Yap/Taz transcriptional activity in endothelial cells. Our results provide novel insight into the relationship between blood flow and ossification by demonstrating intertissue regulation.

Fate mapping of gallbladder progenitors in posteroventral foregut endoderm of mouse early somite-stage embryos.

In early embryogenesis, the posteroventral foregut endoderm gives rise to the budding endodermal organs including the liver, ventral pancreas and gallbladder during early somitogenesis. Despite the detailed fate maps of the liver and pancreatic progenitors in the mouse foregut endoderm, the exact location of the gallbladder progenitors remains unclear. In this study, we performed a DiI fate-mapping analysis using whole-embryo cultures of mouse early somite-stage embryos. Here, we show that the majority of gallbladder progenitors in 9-11-somite-stage embryos are located in the lateral-most domain of the foregut endoderm at the first intersomite junction level along the anteroposterior axis. This definition of their location highlights a novel entry point to understanding of the molecular mechanisms of initial specification of the gallbladder.

Sox17-mediated maintenance of fetal intra-aortic hematopoietic cell clusters.

During mouse development, definitive hematopoiesis is first detected around embryonic day 10.5 (E10.5) in the aorta-gonad-mesonephros (AGM) region, which exhibits intra-aortic cell clusters. These clusters are known to contain hematopoietic stem cells (HSCs). On the other hand, it is not clear how the cells in such clusters maintain their HSC phenotype and how they are triggered to differentiate. Here we found that an endodermal transcription factor marker, Sox17, and other F-group (SoxF) proteins, Sox7 and Sox18, were expressed in E10.5 intra-aortic cell clusters. Forced expression of any of these SoxF proteins, particularly Sox17, in E10.5 AGM CD45(low) c-Kit(high) cells, which are the major component of intra-aortic clusters, led to consistent formation of cell clusters in vitro during several passages of cocultures with stromal cells. Cluster-forming cells with constitutive Sox17 expression retained long-term bone marrow reconstitution activity in vivo. Notably, shutdown of exogenously introduced Sox17 gene expression resulted in immediate hematopoietic differentiation. These results indicate that SoxF proteins, especially Sox17, contribute to the maintenance of cell clusters containing HSCs in the midgestation AGM region. Furthermore, SoxF proteins play a pivotal role in controlling the HSC fate decision between indefinite self-renewal and differentiation during fetal hematopoiesis.

Sox17 haploinsufficiency results in perinatal biliary atresia and hepatitis in C57BL/6 background mice.

Congenital biliary atresia is an incurable disease of newborn infants, of unknown genetic causes, that results in congenital deformation of the gallbladder and biliary duct system. Here, we show that during mouse organogenesis, insufficient SOX17 expression in the gallbladder and bile duct epithelia results in congenital biliary atresia and subsequent acute 'embryonic hepatitis', leading to perinatal death in ~95% of the Sox17 heterozygote neonates in C57BL/6 (B6) background mice. During gallbladder and bile duct development, Sox17 was expressed at the distal edge of the gallbladder primordium. In the Sox17(+/-) B6 embryos, gallbladder epithelia were hypoplastic, and some were detached from the luminal wall, leading to bile duct stenosis or atresia. The shredding of the gallbladder epithelia is probably caused by cell-autonomous defects in proliferation and maintenance of the Sox17(+/-) gallbladder/bile duct epithelia. Our results suggest that Sox17 plays a dosage-dependent function in the morphogenesis and maturation of gallbladder and bile duct epithelia during the late-organogenic stages, highlighting a novel entry point to the understanding of the etiology and pathogenesis of human congenital biliary atresia.

Expression and function of mouse Sox17 gene in the specification of gallbladder/bile-duct progenitors during early foregut morphogenesis.

In early-organogenesis-stage mouse embryos, the posteroventral foregut endoderm adjacent to the heart tube gives rise to liver, ventral pancreas and gallbladder. Hepatic and pancreatic primordia become specified in the posterior segment of the ventral foregut endoderm at early somite stages. The mechanisms for demarcating gallbladder and bile duct primordium, however, are poorly understood. Here, we demonstrate that the gallbladder and bile duct progenitors are specified in the paired lateral endoderm domains outside the heart field at almost the same timing as hepatic and pancreatic induction. In the anterior definitive endoderm, Sox17 reactivation occurs in a certain population within the most lateral domains posterolateral to the anterior intestinal portal (AIP) lip on both the left and right sides. During foregut formation, the paired Sox17-positive domains expand ventromedially to merge in the midline of the AIP lip and become localized between the liver and pancreatic primordia. In Sox17-null embryos, these lateral domains are missing, resulting in a complete loss of the gallbladder/bile-duct structure. Chimera analyses revealed that Sox17-null endoderm cells in the posteroventral foregut do not display any gallbladder/bile-duct molecular characters. Our findings show that Sox17 functions cell-autonomously to specify gallbladder/bile-duct in the mouse embryo.

Evidence for crucial role of hindgut expansion in directing proper migration of primordial germ cells in mouse early embryogenesis.

During mouse gastrulation, primordial germ cells (PGCs) become clustered at the base of the allantois and move caudally into the hindgut endoderm before entering the genital ridges. The precise roles of endoderm tissues in PGC migration, however, remain unclear. By using Sox17 mutants with a specific endoderm deficiency, we provide direct evidence for the crucial role of hindgut expansion in directing proper PGC migration. In Sox17-null embryos, PGCs normally colonize in the allantois and then a small front-row population of PGCs moves properly into the most posterior gut endoderm. Defective hindgut expansion, however, causes the failure of further lateral PGC movement, resulting in the immobilization of PGCs in the hindgut entrance at the later stages. In contrast, the majority of the remaining PGCs moves into the visceral endoderm layer, but relocate outside of the embryonic gut domain. This leads to a scattering of PGCs in the extraembryonic yolk sac endoderm. This aberrant migration of Sox17-null PGCs can be rescued by the supply of wildtype hindgut cells in chimeric embryos. Therefore, these data indicate that hindgut morphogenic movement is crucial for directing PGC movement toward the embryonic gut side, but not for their relocation from the mesoderm into the endoderm.