The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice

Recent work has identified LDL receptor-related family members, Lrp5 and Lrp6, as co-receptors for the transduction of Wnt signals. Our analysis of mice carrying mutations in both Lrp5 and Lrp6 demonstrates that the functions of these genes are redundant and are essential for gastrulation. Lrp5;Lrp6 double homozygous mutants fail to establish a primitive streak, although the anterior visceral endoderm and anterior epiblast fates are specified. Thus, Lrp5 and Lrp6 are required for posterior patterning of the epiblast, consistent with a role in transducing Wnt signals in the early embryo. Interestingly, Lrp5(+/-);Lrp6(-/-) embryos die shortly after gastrulation and exhibit an accumulation of cells at the primitive streak and a selective loss of paraxial mesoderm. A similar phenotype is observed in Fgf8 and Fgfr1 mutant embryos and provides genetic evidence in support of a molecular link between the Fgf and Wnt signaling pathways in patterning nascent mesoderm. Lrp5(+/-);Lrp6(-/-) embryos also display an expansion of anterior primitive streak derivatives and anterior neurectoderm that correlates with increased Nodal expression in these embryos. The effect of reducing, but not eliminating, Wnt signaling in Lrp5(+/-);Lrp6(-/-) mutant embryos provides important insight into the interplay between Wnt, Fgf and Nodal signals in patterning the early mouse embryo.     

ALK7, a receptor for nodal, is dispensable for embryogenesis and left-right patterning in the mouse

Mesendoderm formation and left-right patterning during vertebrate development depend upon selected members of the transforming growth factor beta superfamily, particularly Nodal and Nodal-related ligands. Two type I serine/threonine kinase receptors have been identified for Nodal, ALK4 and ALK7. Mouse embryos lacking ALK4 fail to produce mesendoderm and die shortly after gastrulation, resembling the phenotype of Nodal knockout mice. Whether ALK4 contributes to left-right patterning is still unknown. Here we report the generation and initial characterization of mice lacking ALK7. Homozygous mutant mice were born at the expected frequency and remained viable and fertile. Viability at weaning was not different from that of the wild type in ALK7(-/-); Nodal(+/-) and ALK7(-/-); ALK4(+/-) compound mutants. ALK7 and ALK4 were highly expressed in interdigital regions of the developing limb bud. However, ALK7 mutant mice displayed no skeletal abnormalities or limb malformations. None of the left-right patterning abnormalities and organogenesis defects identified in mice carrying mutations in Nodal or in genes encoding ActRIIA and ActRIIB coreceptors, including heart malformations, pulmonary isomerism, right-sided gut, and spleen hypoplasia, were observed in mice lacking ALK7. Finally, the histological organization of the cerebellum, cortex, and hippocampus, all sites of significant ALK7 expression in the rodent brain, appeared normal in ALK7 mutant mice. We conclude that ALK7 is not an essential mediator of Nodal signaling during mesendoderm formation and left-right patterning in the mouse but may instead mediate other activities of Nodal and related ligands in the development or function of particular tissues and organs.     

Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation.

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

Cripto is required for mesoderm and endoderm cell allocation during mouse gastrulation

During mouse gastrulation, cells in the primitive streak undergo epithelial–mesenchymal transformation and the resulting mesenchymal cells migrate out laterally to form mesoderm and definitive endoderm across the entire embryonic cylinder. The mechanisms underlying mesoderm and endoderm specification, migration, and allocation are poorly understood. In this study, we focused on the function of mouse Cripto, a member of the EGF-CFC gene family that is highly expressed in the primitive streak and migrating mesoderm cells on embryonic day 6.5. Conditional inactivation of Cripto during gastrulation leads to varied defects in mesoderm and endoderm development. Mutant embryos display accumulation of mesenchymal cells around the shortened primitive streak indicating a functional requirement of Cripto during the formation of mesoderm layer in gastrulation. In addition, some mutant embryos showed poor formation and abnormal allocation of definitive endoderm cells on embryonic day 7.5. Consistently, many mutant embryos that survived to embryonic day 8.5 displayed defects in ventral closure of the gut endoderm causing cardia bifida. Detailed analyses revealed that both the Fgf8–Fgfr1 pathway and p38 MAP kinase activation are partially affected by the loss of Cripto function. These results demonstrate a critical role for Cripto during mouse gastrulation, especially in mesoderm and endoderm formation and allocation.     

Gastrulation in the mouse: the role of the homeobox gene goosecoid.

Mouse goosecoid is a homeobox gene expressed briefly during early gastrulation. Its mRNA accumulates as a patch on the side of the epiblast at the site where the primitive streak is first formed. goosecoid-expressing cells are then found at the anterior end of the developing primitive streak, and finally in the anteriormost mesoderm at the tip of the early mouse gastrula, a region that gives rise to the head process. Treatment of early mouse embryos with activin results in goosecoid mRNA accumulation in the entire epiblast, suggesting that a localized signal induces goosecoid expression during development. Transplantation experiments indicate that the tip of the murine early gastrula is the equivalent of the organizer of the amphibian gastrula.     

Wnt3 signaling in the epiblast is required for proper orientation of the anteroposterior axis

The establishment of anteroposterior (AP) polarity in the early mouse epiblast is crucial for the initiation of gastrulation and the subsequent formation of the embryonic (head to tail) axis. The localization of anterior and posterior determining genes to the appropriate region of the embryo is a dynamic process that underlies this early polarity. Several studies indicate that morphological and molecular markers which define the early AP axis are first aligned along the short axis of the elliptical egg cylinder. Subsequently, just prior to the time of primitive streak formation, a conformational change in the embryo realigns these markers with the long axis. We demonstrate that embryos lacking the signaling factor Wnt3 exhibit defects in this axial realignment. In addition, chimeric analyses and conditional removal of Wnt3 activity reveal that Wnt3 expression in the epiblast is required for induction of the primitive streak and mesoderm whereas activity in the posterior visceral endoderm is dispensable.     

Crystal structure of activin receptor type IIB kinase domain from human at 2.0 Angstrom resolution

Activin receptor type IIB (ActRIIB), a type II TGF-beta serine/threonine kinase receptor, is integral to the activin and myostatin signaling pathway. Ligands such as activin and myostatin bind to activin type II receptors (ActRIIA, ActRIIB), and the GS domains of type I receptors are phosphorylated by type II receptors. Myostatin, a negative regulator of skeletal muscle growth, is regarded as a potential therapeutic target and binds to ActRIIB effectively, and to a lesser extent, to ActRIIA. The high-resolution structure of human ActRIIB kinase domain in complex with adenine establishes the conserved bilobal architecture consistent with all other catalytic kinase domains. The crystal structure reveals that the adenine has a considerably different orientation from that of the adenine moiety of ATP observed in other kinase structures due to the lack of an interaction by ribose-phosphate moiety and the presence of tautomers with two different protonation states at the N9 nitrogen. Although the Lys217-Glu230 salt bridge is absent, the unphosphorylated activation loop of ActRIIB adopts a conformation similar to that of the fully active form. Unlike the type I TGF-beta receptor, where a partially conserved Ser280 is a gatekeeper residue, the AcRIIB structure possesses Thr265 with a back pocket supported by Phe247. Taken together, these structural features provide a molecular basis for understanding the coupled activity and recognition specificity for human ActRIIB kinase domain and for the rational design of selective inhibitors.     

Distinct and cooperative roles of mammalian Vg1 homologs GDF1 and GDF3 during early embryonic development

Vg1, a member of the TGF-β superfamily of ligands, has been implicated in the induction of mesoderm, formation of primitive streak, and left-right patterning in Xenopus and chick embryos. In mice, GDF1 and GDF3 - two TGF-β superfamily ligands that share high sequence identity with Vg1 - have been shown to independently mimic distinct aspects of Vg1's functions. However, the extent to which the developmental processes controlled by GDF1 and GDF3 and the underlying signaling mechanisms are evolutionarily conserved remains unclear. Here we show that phylogenetic and genomic analyses indicate that Gdf1 is the true Vg1 ortholog in mammals. In addition, and similar to GDF1, we find that GDF3 signaling can be mediated by the type I receptor ALK4, type II receptors ActRIIA and ActRIIB, and the co-receptor Cripto to activate Smad-dependent reporter genes. When expressed in heterologous cells, the native forms of either GDF1 or GDF3 were incapable of inducing downstream signaling. This could be circumvented by using chimeric constructs carrying heterologous prodomains, or by co-expression with the Furin pro-protein convertase, indicating poor processing of the native GDF1 and GDF3 precursors. Unexpectedly, co-expression with Nodal - another TGF-β superfamily ligand involved in mesoderm formation - could also expose the activities of native GDF1 and GDF3, suggesting a potentially novel mode of cooperation between these ligands. Functional complementarity between GDF1 and GDF3 during embryonic development was investigated by analyzing genetic interactions between their corresponding genes. This analysis showed that Gdf1^−/−;Gdf3^−/− compound mutants are more severely affected than either Gdf1^−/− or Gdf3^−/− single mutants, with defects in the formation of anterior visceral endoderm and mesoderm that recapitulate Vg1 loss of function, suggesting that GDF1 and GDF3 together represent the functional mammalian homologs of Vg1.     

FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression

Background  

FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.     

Differential requirements for Smad4 in TGFbeta-dependent patterning of the early mouse embryo

Genetic and biochemical data have identified Smad4 as a key intracellular effector of the transforming growth factor beta (TGFbeta superfamily of secreted ligands. In mouse, Smad4-null embryos do not gastrulate, a phenotype consistent with loss of other TGFbeta-related signaling components. Chimeric analysis reveals a primary requirement for Smad4 in the extra-embryonic lineages; however, within the embryo proper, characterization of the specific roles of Smad4 during gastrulation and lineage specification remains limited. We have employed a Smad4 conditional allele to specifically inactivate the Smad4 gene in the early mouse epiblast. Loss of Smad4 in this tissue results in a profound failure to pattern derivatives of the anterior primitive streak, such as prechordal plate, node, notochord and definitive endoderm. In contrast to these focal defects, many well-characterized TGFbeta- and Bmp-regulated processes involved in mesoderm formation and patterning are surprisingly unaffected. Mutant embryos form abundant extra-embryonic mesoderm, including allantois, a rudimentary heart and middle primitive streak derivatives such as somites and lateral plate mesoderm. Thus, loss of Smad4 in the epiblast results not in global developmental abnormalities but instead in restricted patterning defects. These results suggest that Smad4 potentiates a subset of TGFbeta-related signals during early embryonic development, but is dispensable for others.     

BMP signaling in the epiblast is required for proper recruitment of the prospective paraxial mesoderm and development of the somites

Bmpr1a encodes the BMP type IA receptor for bone morphogenetic proteins (BMPs), including 2 and 4. Here, we use mosaic inactivation of Bmpr1a in the epiblast of the mouse embryo (Bmpr-MORE embryos) to assess functions of this gene in mesoderm development. Unlike Bmpr1a-null embryos, which fail to gastrulate, Bmpr-MORE embryos initiate gastrulation, but the recruitment of prospective paraxial mesoderm cells to the primitive streak is delayed. This delay causes a more proximal distribution of cells with paraxial mesoderm character within the primitive streak, resulting in a lateral expansion of somitic mesoderm to form multiple columns. Inhibition of FGF signaling restores the normal timing of recruitment of prospective paraxial mesoderm and partially rescues the development of somites. This suggests that BMP and FGF signaling function antagonistically during paraxial mesoderm development.     

The orphan steroid receptor Nur77 family member Nor-1 is essential for early mouse embryogenesis

Nur77 and its family members, Nor-1 and Nurr1, are orphan steroid receptors implicated in a wide variety of biological processes, including apoptosis and dopamine neuron agenesis. Expression of these family members can be detected at low levels in many tissues but they are expressed at very high levels when cells are stimulated by outside signals, including serum, nerve growth factor, and receptor engagement. Introduction of a dominant negative Nur77 protein that blocks the activities of all family members led to inhibition of apoptosis in T cells. Nur77-deficient mice, however, exhibit no phenotype, and a line of Nor-1 mutant mice was reported to exhibit a mild ear development phenotype but no other gross abnormalities. Here, we report the generation of Nor-1-deficient mice with a block in early embryonic development. Nor-1 is expressed early during embryogenesis, and its loss leads to embryonic lethality around embryonic day 8.5 of gestation. The mutant embryos fail to complete gastrulation and display distinct morphological abnormalities, including a decrease in overall size, developmental delay and an accumulation of mesoderm in the primitive streak during gastrulation. Abnormal expression of a number of early developmental markers and defects in growth or distribution of emerging mesoderm cells were also detected. These data suggest that Nor-1 plays a crucial role in gastrulation.