Lise Zakin

Inactivation of mouse Twisted gastrulation reveals its role in promoting Bmp4 activity during forebrain development.

Twisted gastrulation (Tsg) is a secreted protein that regulates Bmp signaling in the extracellular space through its direct interaction with Bmp/Dpp and Chordin (Chd)/Short gastrulation (Sog). The ternary complex of Tsg/Chd/Bmp is cleaved by the metalloprotease Tolloid (Tld)/Xolloid (Xld). Studies in Drosophila, Xenopus and zebrafish suggest that Tsg can act both as an anti-Bmp and as a pro-Bmp. We have analyzed Tsg loss-of-function in the mouse. Tsg homozygous mutants are viable but of smaller size and display mild vertebral abnormalities and osteoporosis. We provide evidence that Tsg interacts genetically with Bmp4. When only one copy of Bmp4 is present, a requirement of Tsg for embryonic development is revealed. Tsg-/-;Bmp4+/- compound mutants die at birth and display holoprosencephaly, first branchial arch and eye defects. The results show that Tsg functions to promote Bmp4 signaling during mouse head development.

Sirenomelia in Bmp7 and Tsg compound mutant mice: requirement for Bmp signaling in the development of ventral posterior mesoderm

Sirenomelia or mermaid-like phenotype is one of the principal human congenital malformations that can be traced back to the stage of gastrulation. Sirenomelia is characterized by the fusion of the two hindlimbs into a single one. In the mouse, sirens have been observed in crosses between specific strains and as the consequence of mutations that increase retinoic acid levels. We report that the loss of bone morphogenetic protein 7 (Bmp7) in combination with a half dose or complete loss of twisted gastrulation (Tsg) causes sirenomelia in the mouse. Tsg is a Bmp- and chordin-binding protein that has multiple effects on Bmp metabolism in the extracellular space; Bmp7 is one of many Bmps and is shown here to bind to Tsg. In Xenopus, co-injection of Tsg and Bmp7 morpholino oligonucleotides (MO) has a synergistic effect, greatly inhibiting formation of ventral mesoderm and ventral fin tissue. In the mouse, molecular marker studies indicate that the sirenomelia phenotype is associated with a defect in the formation of ventroposterior mesoderm. These experiments demonstrate that dorsoventral patterning of the mouse posterior mesoderm is regulated by Bmp signaling, as is the case in other vertebrates. Sirens result from a fusion of the hindlimb buds caused by a defect in the formation of ventral mesoderm.

Development of the vertebral morphogenetic field in the mouse: interactions between Crossveinless-2 and Twisted gastrulation

Crossveinless-2 (Cv2), Twisted Gastrulation (Tsg) and Chordin (Chd) are components of an extracellular biochemical pathway that regulates Bone Morphogenetic Protein (BMP) activity during dorso-ventral patterning of Drosophila and Xenopus embryos, the formation of the fly wing, and mouse skeletogenesis. Because the nature of their genetic interactions remained untested in the mouse, we generated a null allele for Cv2 which was crossed to Tsg and Chd mutants to obtain Cv2; Tsg and Cv2; Chd compound mutants. We found that Cv2 is essential for skeletogenesis as its mutation caused the loss of multiple bone structures and posterior homeotic transformation of the last thoracic vertebra. During early vertebral development, Smad1 phosphorylation in the intervertebral region was decreased in the Cv2 mutant, even though CV2 protein is normally located in the future vertebral bodies. Because Cv2 mutation affects BMP signaling at a distance, this suggested that CV2 is involved in the localization of the BMP morphogenetic signal. Cv2 and Chd mutations did not interact significantly. However, mutation of Tsg was epistatic to all CV2 phenotypes. We propose a model in which CV2 and Tsg participate in the generation of a BMP signaling morphogenetic field during vertebral formation in which CV2 serves to concentrate diffusible Tsg/BMP4 complexes in the vertebral body cartilage.

Chordin forms a self-organizing morphogen gradient in the extracellular space between ectoderm and mesoderm in the Xenopus embryo

Significance Cell differentiation in the embryo is regulated by diffusible substances called “morphogens,” but these have never been directly visualized as endogenous components of the extracellular space. Chordin is an antagonist of the bone morphogenetic protein (BMP) pathway copiously secreted by a dorsal region of the Xenopus embryo called “Spemann’s organizer” that has potent tissue-inducing activity. We report that Chordin protein forms a dorsal-to-ventral gradient in the embryo. This gradient is located in a narrow space containing extracellular matrix (ECM) that separates the ectoderm from the endomesoderm, which seems to serve as a highway for the diffusion of Chordin–BMP complexes over very long distances (2 mm) in the embryo. All vertebrate embryos have a similar ECM between ectoderm and mesoderm during gastrulation. The vertebrate body plan follows stereotypical dorsal–ventral (D-V) tissue differentiation controlled by bone morphogenetic proteins (BMPs) and secreted BMP antagonists, such as Chordin. The three germ layers—ectoderm, mesoderm, and endoderm—are affected coordinately by the Chordin–BMP morphogen system. However, extracellular morphogen gradients of endogenous proteins have not been directly visualized in vertebrate embryos to date. In this study, we improved immunolocalization methods in Xenopus embryos and analyzed the distribution of endogenous Chordin using a specific antibody. Chordin protein secreted by the dorsal Spemann organizer was found to diffuse along a narrow region that separates the ectoderm from the anterior endoderm and mesoderm. This Fibronectin-rich extracellular matrix is called “Brachet’s cleft” in the Xenopus gastrula and is present in all vertebrate embryos. Chordin protein formed a smooth gradient that encircled the embryo, reaching the ventral-most Brachet cleft. Depletion with morpholino oligos showed that this extracellular gradient was regulated by the Chordin protease Tolloid and its inhibitor Sizzled. The Chordin gradient, as well as the BMP signaling gradient, was self-regulating and, importantly, was able to rescale in dorsal half-embryos. Transplantation of Spemann organizer tissue showed that Chordin diffused over long distances along this signaling highway between the ectoderm and mesoderm. Chordin protein must reach very high concentrations in this narrow region. We suggest that as ectoderm and mesoderm undergo morphogenetic movements during gastrulation, cells in both germ layers read their positional information coordinately from a single morphogen gradient located in Brachet’s cleft.

Systems control of BMP morphogen flow in vertebrate embryos

Embryonic morphogenetic programs coordinate cell behavior to ensure robust pattern formation. Having identified components of those programs by molecular genetics, developmental biology is now borrowing concepts and tools from systems biology to decode their regulatory logic. Dorsal-ventral (D-V) patterning of the frog gastrula by Bone Morphogenetic Proteins (BMPs) is one of the best studied examples of a self-regulating embryonic patterning system. Embryological analyses and mathematical modeling are revealing that the BMP activity gradient is maintained by a directed flow of BMP ligands towards the ventral side. Pattern robustness is ensured through feedback control of the levels of extracellular BMP pathway modulators that adjust the flow to the dimensions of the embryonic field.

Foxh1 recruits Gsc to negatively regulate Mixl1 expression during early mouse development

Mixl1 is a member of the Mix/Bix family of paired‐like homeodomain proteins and is required for proper axial mesendoderm morphogenesis and endoderm formation during mouse development. Mix/Bix proteins are transcription factors that function in Nodal‐like signaling pathways and are themselves regulated by Nodal. Here, we show that Foxh1 forms a DNA‐binding complex with Smads to regulate transforming growth factor β (TGFβ)/Nodal‐dependent Mixl1 gene expression. Whereas Foxh1 is commonly described as a transcriptional activator, we observed that Foxh1‐null embryos exhibit expanded and enhanced Mixl1 expression during gastrulation, indicating that Foxh1 negatively regulates expression of Mixl1 during early mouse embryogenesis. We demonstrate that Foxh1 associates with the homeodomain‐containing protein Goosecoid (Gsc), which in turn recruits histone deacetylases to repress Mixl1 gene expression. Ectopic expression of Gsc in embryoid bodies represses endogenous Mixl1 expression and this effect is dependent on Foxh1. As Gsc is itself induced in a Foxh1‐dependent manner, we propose that Foxh1 initiates positive and negative transcriptional circuits to refine cell fate decisions during gastrulation.

Identification and expression of the mammalian homologue of Bicaudal-C

Translational activation and repression play an important role in the spatial-temporal regulation of gene expression in embryonic development. Bicaudal-C is an RNA-binding molecule believed to function at this post-transcriptional level. Loss-of-function mutants in Drosophila affect anterior-posterior patterning because of ectopic and premature translation of the posterior determinant oskar. The Xenopus homologue of Bicaudal-C is one of the few molecules that, when microinjected ectopically, results in endoderm formation in the absence of mesoderm induction. Here we report the sequence and expression pattern of the murine and human homologues of Bicaudal-C. The human gene is located on chromosome 10q21.2. Expression analysis in mouse using in situ hybridization detects expression of Bicaudal-C not only in domains detected in Xenopus, but also in previously unreported regions. As in Xenopus, mouse Bicaudal-C mRNA is found in the growing oocyte, Hensens node, and the developing kidney. Additionally, at later stages it is strongly expressed in the developing gut endoderm, in areas of cartilage formation, in pleuro-peritoneal membrane derivatives, in lung mesenchyme, and in the stroma of the ovary. We conclude that mouse Bicaudal-C is a maternally provided gene product that is tightly regulated during mammalian cell differentiation.

Crossveinless-2 is required for the relocalization of Chordin protein within the vertebral field in mouse embryos.

Bone morphogenetic proteins (BMPs), as well as the BMP-binding molecules Chordin (Chd), Crossveinless-2 (CV2) and Twisted Gastrulation (Tsg), are essential for axial skeletal development in the mouse embryo. We previously reported a strong genetic interaction between CV2 and Tsg and proposed a role for this interaction in the shaping of the BMP morphogenetic field during vertebral development. In the present study we investigated the roles of CV2 and Chd in the formation of the vertebral morphogenetic field. We performed immunostainings for CV2 and Chd protein on wild-type, CV2(-/-) or Chd(-/-) mouse embryo sections at the stage of onset of the vertebral phenotypes. By comparing mRNA and protein localizations we found that CV2 does not diffuse away from its place of synthesis, the vertebral body. The most interesting finding of this study was that Chd synthesized in the intervertebral disc accumulates in the vertebral body. This relocalization does not take place in CV2(-/-) mutants. Instead, Chd was found to accumulate at its site of synthesis in CV2(-/-) embryos. These results indicate a CV2-dependent flow of Chd protein from the intervertebral disc to the vertebral body. Smad1/5/8 phosphorylation was decreased in CV2(-/-)vertebral bodies. This impaired BMP signaling may result from the decreased levels of Chd/BMP complexes diffusing from the intervertebral region. The data indicate a role for CV2 and Chd in the establishment of the vertebral morphogenetic field through the long-range relocalization of Chd/BMP complexes. The results may have general implications for the formation of embryonic organ-forming morphogenetic fields.

09-P030 The role of Bicaudal-C in kidney development

ter identify mechanisms of islet formation in the zebrafish, with true homology to those observed in mammals, we have temporally and spatially characterized zebrafish secondary islet formation. As is the case in the mouse, we show that Notch inhibition leads to precocious differentiation of endocrine tissues. Furthermore, we have used transgenic fish expressing fluorescent markers under the control of a Notch-responsive element to observe the precursors of these induced endocrine cells. These pancreatic Notch-responsive cells represent a novel population of progenitors that are associated with larval pancreatic ductal epithelium, suggesting functional homology between secondary islet formation in zebrafish and the secondary transition in mammals. We also show that Notch-responsive cells persist in the adult pancreas and possess the classical characteristics of centroacinar cells, a cell type believed to be multipotent progenitor cells in adult mammalian pancreas.