HNF3beta and Lim1 interact in the visceral endoderm to regulate primitive streak formation and anterior-posterior polarity in the mouse embryo.

Recent embryological and genetic experiments have suggested that the anterior visceral endoderm and the anterior primitive streak of the early mouse gastrula function as head- and trunk-organising centers, respectively. Here, we report that HNF3beta and Lim1 are coexpressed in both organising centers suggesting synergistic roles of these genes in regulating organiser functions and hence axis development in the mouse embryo. To investigate this possibility, we generated compound HNF3beta and Lim1 mutant embryos. An enlarged primitive streak and a lack of axis formation were observed in HNF3beta (-)(/)(-);Lim1(-)(/)(-), but not in single homozygous mutant embryos. Chimera experiments indicate that the primary defect in these double homozygous mutants is due to loss of activity of HNF3beta and Lim1 in the visceral endoderm. Altogether, these data provide evidence that these genes function synergistically to regulate organiser activity of the anterior visceral endoderm. Moreover, HNF3beta (-)(/)(-);Lim1(-)(/)(-) mutant embryos also exhibit defects in mesoderm patterning that are likely due to lack of specification of anterior primitive streak cells.     

Microautophagy in the visceral endoderm is essential for mouse early development

During early embryogenesis, before the conceptus forms the placenta, maternal nutrients as well as signaling molecules must reach the embryo proper through a tightly sealed epithelial tissue, the visceral endoderm (VE). The VE serves as a signaling center for embryogenesis, where exocytic and endocytic processes integrate signal production, perception and termination. However, the endocytic process in this important tissue has not been well characterized. We show that endocytic delivery to the lysosomes occurs via RAB7-dependent microautophagy. This process is essential for early mammalian development.     

Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks

The anterior visceral endoderm plays a pivotal role in establishing anterior-posterior polarity of the mouse embryo, but the molecular nature of the signals required remains to be determined. Here, we demonstrate that Cerberus-like(-/-);Lefty1(-/-) compound mutants can develop a primitive streak ectopically in the embryo. This defect is not rescued in chimeras containing wild-type embryonic, and Cerberus-like(-/-);Lefty1(-/-) extraembryonic, cells but is rescued in Cerberus-like(-/-); Lefty1(-/-) embryos after removal of one copy of the Nodal gene. Our findings provide support for a model whereby Cerberus-like and Lefty1 in the anterior visceral endoderm restrict primitive streak formation to the posterior end of mouse embryos by antagonizing Nodal signaling. Both antagonists are also required for proper patterning of the primitive streak.     

Cell lineage analysis of the primitive and visceral endoderm of mouse embryos cultured in vitro

Cell lineages of the primitive endoderm and the visceral endoderm of mouse embryos were examined by culturing whole embryos in vitro. The primitive endoderm and visceral endoderm cells could be labelled by incubation of embryos in a medium containing horse radish peroxidase (HRP). HRP localization was chased throughout the culture period. The results show that the visceral endoderm derives from the primitive endoderm, and the visceral endoderm forms only the extra-embryonic endoderm (yolk sac endoderm) of the conceptus. The definitive endoderm which is probably derived from the head process, newly appears on the ventral surface of the embryo.     

Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst

The first two lineages to differentiate from a pluripotent cell population during mammalian development are the extraembryonic trophectoderm (TE) and the primitive endoderm (PrE). Whereas the mechanisms of TE specification have been extensively studied, segregation of PrE and the pluripotent epiblast (EPI) has received comparatively little attention. A current model of PrE specification suggests PrE precursors exhibit an apparently random distribution within the inner cell mass of the early blastocyst and then segregate to their final position lining the cavity by the late blastocyst. We have identified platelet-derived growth factor receptor alpha (Pdgfralpha) as an early-expressed protein that is also a marker of the later PrE lineage. By combining live imaging of embryos expressing a histone H2B-GFP fusion protein reporter under the control of Pdgfra regulatory elements with the analysis of lineage-specific markers, we investigated the events leading to PrE and EPI lineage segregation in the mouse, and correlated our findings using an embryo staging system based on total cell number. Before blastocyst formation, lineage-specific factors are expressed in an overlapping manner. Subsequently, a gradual progression towards a mutually exclusive expression of PrE- and EPI-specific markers occurs. Finally, cell sorting is achieved by a variety of cell behaviours and by selective apoptosis.     

Otx2 is required for visceral endoderm movement and for the restriction of posterior signals in the epiblast of the mouse embryo

Genetic and embryological experiments have demonstrated an essential role for the visceral endoderm in the formation of the forebrain; however, the precise molecular and cellular mechanisms of this requirement are poorly understood. We have performed lineage tracing in combination with molecular marker studies to follow morphogenetic movements and cell fates before and during gastrulation in embryos mutant for the homeobox gene Otx2. Our results show, first, that Otx2 is not required for proliferation of the visceral endoderm, but is essential for anteriorly directed morphogenetic movement. Second, molecules that are normally expressed in the anterior visceral endoderm, such as Lefty1 and Mdkk1, are not expressed in Otx2 mutants. These secreted proteins have been reported to antagonise, respectively, the activities of Nodal and Wnt signals, which have a role in regulating primitive streak formation. The visceral endoderm defects of the Otx2 mutants are associated with abnormal expression of primitive streak markers in the epiblast, suggesting that anterior epiblast cells acquire primitive streak characteristics. Taken together, our data support a model whereby Otx2 functions in the anterior visceral endoderm to influence the ability of the adjacent epiblast cells to differentiate into anterior neurectoderm, indirectly, by preventing them from coming under the influence of posterior signals that regulate primitive streak formation.     

Dickkopf1 is required for embryonic head induction and limb morphogenesis in the mouse.

Dickkopf1 (Dkk1) is a secreted protein that acts as a Wnt inhibitor and, together with BMP inhibitors, is able to induce the formation of ectopic heads in Xenopus. Here, we show that Dkk1 null mutant embryos lack head structures anterior of the midbrain. Analysis of chimeric embryos implicates the requirement of Dkk1 in anterior axial mesendoderm but not in anterior visceral endoderm for head induction. In addition, mutant embryos show duplications and fusions of limb digits. Characterization of the limb phenotype strongly suggests a role for Dkk1 both in cell proliferation and in programmed cell death. Our data provide direct genetic evidence for the requirement of secreted Wnt antagonists during embryonic patterning and implicate Dkk1 as an essential inducer during anterior specification as well as a regulator during distal limb patterning.     

Multi-cellular rosettes in the mouse visceral endoderm facilitate the ordered migration of anterior visceral endoderm cells

The visceral endoderm (VE) is a simple epithelium that forms the outer layer of the egg-cylinder stage mouse embryo. The anterior visceral endoderm (AVE), a specialised subset of VE cells, is responsible for specifying anterior pattern. AVE cells show a stereotypic migratory behaviour within the VE, which is responsible for correctly orientating the anterior-posterior axis. The epithelial integrity of the VE is maintained during the course of AVE migration, which takes place by intercalation of AVE and other VE cells. Though a continuous epithelial sheet, the VE is characterised by two regions of dramatically different behaviour, one showing robust cell movement and intercalation (in which the AVE migrates) and one that is static, with relatively little cell movement and mixing. Little is known about the cellular rearrangements that accommodate and influence the sustained directional movement of subsets of cells (such as the AVE) within epithelia like the VE. This study uses an interdisciplinary approach to further our understanding of cell movement in epithelia. Using both wild-type embryos as well as mutants in which AVE migration is abnormal or arrested, we show that AVE migration is specifically linked to changes in cell packing in the VE and an increase in multi-cellular rosette arrangements (five or more cells meeting at a point). To probe the role of rosettes during AVE migration, we develop a mathematical model of cell movement in the VE. To do this, we use a vertex-based model, implemented on an ellipsoidal surface to represent a realistic geometry for the mouse egg-cylinder. The potential for rosette formation is included, along with various junctional rearrangements. Simulations suggest that while rosettes are not essential for AVE migration, they are crucial for the orderliness of this migration observed in embryos. Our simulations are similar to results from transgenic embryos in which Planar Cell Polarity (PCP) signalling is disrupted. Such embryos have significantly reduced rosette numbers, altered epithelial packing, and show abnormalities in AVE migration. Our results show that the formation of multi-cellular rosettes in the mouse VE is dependent on normal PCP signalling. Taken together, our model and experimental observations suggest that rosettes in the VE epithelium do not form passively in response to AVE migration. Instead, they are a PCP-dependent arrangement of cells that acts to buffer the disequilibrium in cell packing generated in the VE by AVE migration, enabling AVE cells to migrate in an orderly manner.     

Distinct roles for visceral endoderm during embryonic mouse development.

The murine visceral endoderm is an extraembryonic cell layer that appears prior to gastrulation and performs critical functions during embryogenesis. The traditional role ascribed to the visceral endoderm entails nutrient uptake and transport. Besides synthesizing a number of specialized proteins that facilitate uptake, digestion, and secretion of nutrients, the extraembryonic visceral endoderm coordinates blood cell differentiation and vessel formation in the adjoining mesoderm, thereby facilitating efficient exchange of nutrients and gases between the mother and embryo. Recent studies suggest that in addition to this nutrient exchange function the visceral endoderm overlying the egg cylinder stage embryo plays an active role in guiding early development. Cells in the anterior visceral endoderm function as an early organizer. Prior to formation of the primitive streak, these cells express specific gene products that specify the fate of underlying embryonic tissues. In this review we highlight recent investigations demonstrating this dual role for visceral endoderm as a provider of both nutrients and developmental cues for the early embryo.     

The pitx2 homeobox protein is required early for endoderm formation and nodal signaling.

Nodal and Nodal-related factors play fundamental roles in a number of developmental processes, including mesoderm and endoderm formation, patterning of the anterior neural plate, and determination of bilateral asymmetry in vertebrates. pitx2, a paired-like homeobox gene, has been proposed to act downstream of Nodal in the gene cascade providing left-right cues to the developing organs. Here, we report that pitx2 is required early in the Nodal signaling pathway for specification of the endodermal and mesodermal germ layers. We found that pitx2 is expressed very early during Xenopus and zebrafish development and in many regions where Nodal signaling is required, including the presumptive mesoderm and endoderm at the blastula and gastrula stages and the prechordal mesoderm at later stages. In Xenopus embryos, overexpression of pitx2 caused ectopic expression of goosecoid and sox-17 and interfered with mesoderm formation. Overexpression of pitx2 in Xenopus animal cap explants partially mimics the effects of Nodal overexpression, suggesting that pitx2 is a mediator of Nodal signaling during specification of the endoderm and prechordal plate, but not during mesoderm induction. We further demonstrate that pitx2 is induced by Nodal signaling in Xenopus animal caps and that the early expression of zebrafish pitx2 is absent when the Nodal signaling pathway is inactive. Inhibition of pitx2 function using a chimeric EnR-pitx2 blocked specification of the mesoderm and endoderm and caused severe embryonic defects resembling those seen when Nodal signaling is inhibited. Following inhibition of pitx2 function, the fate of ventral vegetal blastomeres was shifted from an endodermal to a more mesodermal fate, an effect that was reversed by wild-type pitx2. Finally, we show that inhibition of pitx2 function interferes with the response of cells to Nodal signaling. Our results provide direct evidence that pitx2 function is required for normal specification of the endodermal and mesodermal germ layers.     

Tiki1 is required for head formation via Wnt cleavage-oxidation and inactivation

Secreted Wnt morphogens are signaling molecules essential for embryogenesis, pathogenesis, and regeneration and require distinct modifications for secretion, gradient formation, and activity. Whether Wnt proteins can be posttranslationally inactivated during development and homeostasis is unknown. Here we identify, through functional cDNA screening, a transmembrane protein Tiki1 that is expressed specifically in the dorsal Spemann-Mangold Organizer and is required for anterior development during Xenopus embryogenesis. Tiki1 antagonizes Wnt function in embryos and human cells via a TIKI homology domain that is conserved from bacteria to mammals and acts likely as a protease to cleave eight amino-terminal residues of a Wnt protein, resulting in oxidized Wnt oligomers that exhibit normal secretion but minimized receptor-binding capability. Our findings identify a Wnt-specific protease that controls head formation, reveal a mechanism for morphogen inactivation through proteolysis-induced oxidation-oligomerization, and suggest a role of the?Wnt amino terminus in evasion of oxidizing inactivation. TIKI proteins may represent potential therapeutic targets.     

Lim 1 is required for nephric duct extension and ureteric bud morphogenesis

The nephric duct plays a central role in orchestrating the development of the mammalian urogenital system. Lim 1 is a homeobox gene required for head and urogenital development in the mouse but most Lim 1-deficient embryos die by embryonic day 10. To determine the role of Lim 1 in the development of the nephric duct, we conditionally removed Lim 1 in the nephric epithelium just after the nephric duct begins to form using a floxed allele of Lim 1 and Pax2-cre transgenic mice. We report that Lim 1 conditional knockout mice have renal hypoplasia and hydronephrosis. Developmental studies revealed that the caudal portion of the nephric duct did not reach the urogenital sinus at embryonic day 10.5, formation of the ureteric bud was delayed, the ureteric bud was smaller and branching of the ureteric bud reduced. We also found that the nephric duct was generally not maintained and extension of the Müllerian duct inhibited. Molecular analysis indicated that Pax2 was expressed normally but the expression of Wnt9b and E-cadherin in the nephric duct was markedly altered. These results suggest that Lim 1 influences nephric duct extension and ureteric bud outgrowth by regulating and or maintaining the differentiation of the nephric epithelium.     

Mash1 is required for glomus cell formation in the mouse carotid body

The carotid body consists of chemoreceptive glomus cells, sustentacular cells and nerve endings. The murine carotid body, located at the carotid bifurcation, is always joined to the superior cervical ganglion of the sympathetic trunk. Glomus cells and sympathetic neurons are immunoreactive for the TuJ1, PGP9.5, tyrosine hydroxylase (TH) and neuropeptide Y (NPY) markers. Glomus cells are also immunoreactive for serotonin (5-HT). A targeted mutation of Mash1, a mouse homolog of the Drosophila achaete-scute complex, results in the elimination of sympathetic ganglia. In Mash1 null mutant mice, the carotid body primordium forms normally in the wall of the third arch artery at embryonic day (E) 13.0 and continues to develop, although the superior cervical ganglion is completely absent. However, no cells in the mutant carotid body display the TuJ1, PGP 9.5, TH, NPY and 5-HT markers throughout development. The absence of glomus cells was also confirmed by electron microscopy. The carotid body of newborn null mutants is composed of mesenchymal-like cells and nerve fibers. Many cells immunoreactive for the S-100 protein, a sustentacular cell marker, appear in the mutant carotid body during fetal development. The Mash1 gene is thus required for the genesis of glomus cells but not for sustentacular cells.     

Netrin 1 is required for semicircular canal formation in the mouse inner ear

The morphogenetic development of the mammalian inner ear is a complex multistep process, the molecular and cellular details of which are only beginning to be unraveled. We show here that mouse netrin 1, known to be involved in axon guidance and cell migration in the central nervous system, also plays a critical morphogenetic role during semicircular canal formation. netrin 1 is expressed at high levels in the otic epithelium, in cells that will come together to form a fusion plate, a prerequisite for the formation of semicircular canals. In netrin 1 mutant mice, fusion plate formation is severely affected resulting in a reduced anterior semicircular canal and the complete lack of the posterior and lateral canals. Our results suggest that netrin 1 facilitates semicircular canal formation through two different mechanisms: (1) it participates in the detachment of the fusion plate epithelia from the basement membrane, and (2) it stimulates proliferation of the periotic mesenchymal cells which then push the epithelial cell walls together to form the fusion plate.     

Laminin alpha 5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung

Laminin alpha/beta/gamma heterotrimers are the major noncollagenous components of all basement membranes. To date, five alpha, three beta, and three gamma chains have been identified. Laminin alpha 5 is expressed early in lung development and colocalizes with laminin alpha1. While laminin alpha1 expression in the lung is restricted to the embryonic period, laminin alpha 5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin alpha 5 gene Lama5 causes embryonic lethality at E14-E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama5(-/-) mice up to E16.5 was examined. We observed normal lung branching morphogenesis and vasculogenesis, but incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin alpha 4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlated with absence of laminin alpha 5, which was found to be the only laminin alpha chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked.     

Mouse amnionless, which is required for primitive streak assembly, mediates cell-surface localization and endocytic function of cubilin on visceral endoderm and kidney proximal tubules

Impaired primitive streak assembly in the mouse amnionless (amn) mutant results in the absence of non-axial trunk mesoderm, a derivative of the middle region of the primitive streak. In addition, the epiblast of amn mutants fails to increase significantly in size after E7.0, indicating that middle primitive streak assembly is mechanistically tied to the growth of the embryo during gastrulation. Amn, a novel transmembrane protein, is expressed exclusively in an extra-embryonic tissue, visceral endoderm (VE), during the early post-implantation stages. We show that Amn is also expressed in kidney proximal tubules (KPT) and intestinal epithelium, which, like the VE, are polarized epithelia specialized for resorption and secretion. To explore whether Amn participates in the development or function of KPT and intestinal epithelia and to gain insight into the function of Amn during gastrulation, we constructed Amn(-/-) ES cell<-->+/+ blastocyst chimeras. While chimeras form anatomically normal kidneys and intestine, they exhibit variable, selective proteinuria, a sign of KPT malfunction. In humans, AMN has been genetically connected to Cubilin (CUBN), a multi-ligand scavenger receptor expressed by KPT, intestine and yolk sac. Loss of CUBN, the intestinal intrinsic factor (IF)-vitamin B12 receptor, results in hereditary megaloblastic anemia (MGA1), owing to vitamin B12 malabsorption. The recent report of MGA1 families with mutations in AMN suggests that AMN functions in the same pathway as CUBN. We demonstrate that Cubn is not properly localized to the cell surface in Amn(-/-) tissues in the embryo and adult mouse, and that adult chimeras exhibit selective proteinuria of Cubn ligands. This study demonstrates that Amn is an essential component of the Cubn receptor complex in vivo and suggests that Amn/Cubn is required for endocytosis/transcytosis of one or more ligands in the VE during gastrulation to coordinate growth and patterning of the embryo. Furthermore, as AMN is apparently not required for gastrulation in humans, the developmental requirements for Amn/Cubn function may not be evolutionarily conserved, possibly reflecting differences between species in the role and organization of extra-embryonic tissues.