Expression and regulation of ANTXR1 in the chick embryo.

Anthrax Toxin Receptor 1 (ANTXR1; also known as Tumor Endothelial Marker 8, TEM8) is one of several genes that was recently found to be up-regulated in tumor-associated endothelial cells. In vitro, the protein can link extracellular matrix components with the actin cytoskeleton to promote cell adhesion and cell spreading. Both, ANTXR1 and the closely related ANTXR2 can bind anthrax toxin and interact with lipoprotein receptor-related protein 5 and 6, which also work as coreceptors in the WNT signaling pathway. Here, we report the cloning of chick ANTXR1 from a suppression subtractive hybridization screen for fibroblast growth factor (FGF) -inducible genes in chicken embryonic facial mesenchyme. We show that chicken ANTXR1 is dynamically expressed throughout embryogenesis, starting from Hamburger and Hamilton stage 10. Furthermore, we demonstrate that FGF signaling is sufficient, but not necessary, to induce ANTXR1 expression in chicken facial mesenchyme.

Expression and regulation of HTRA1 during chick and early mouse development.

HTRA1, a member of the high temperature requirement factor A family, is a secreted serine protease that can bind to and inactivate members of the transforming growth factor-beta (TGFbeta) family, modulate insulin-like growth factor signaling and stimulate long range fibroblast growth factor (FGF) signaling in Xenopus. In vertebrates, so far homologues from mouse, human, and Xenopus have been cloned and studied. Here we report the cloning of the chicken HTRA1 homologue from a screen for FGF8 inducible genes in chick facial mesenchyme. We characterize its expression pattern from gastrulation (Hamburger and Hamilton stage 4) to day 4 of development, and in forming inner organs and limbs. We show that chick HTRA1 has a dynamic expression pattern that differs significantly from the expression of its mouse homolog. We, furthermore, demonstrate that FGF signaling is necessary and sufficient for HTRA1 expression in chick facial and forelimb mesenchyme, but is not required for HTRA1 expression in HH11 embryos.

Expression of ASK1 during chick and early mouse development.

Apoptosis signal-regulating kinase 1 (ASK1) is an important regulator of stress-induced cell death. ASK1 is activated by oxidative stress, TNF and endoplasmatic reticulum stress and activates the JNK- and p38-dependent intracellular death pathways. A number of studies have suggested that ASK1 may also have other roles in addition to its pro-apoptotic activity. Expression of ASK1 during early embryonic development has so far not been analyzed. We have identified and cloned chick ASK1 in a screen for FGF8 inducible genes in chick facial mesenchyme. Here we report the expression of chick ASK1 from the gastrulation stage (HH4) to day 4 of development, its expression in the developing inner organs and limbs, and we compare its expression to the expression of Ask1 during mouse development. Furthermore, we provide evidence that FGF signaling is required for ASK1 expression in chick nasal mesenchyme. In contrast, expression in the mouse nasal region was restricted to the epithelium and was independent of FGF signaling. Our analysis demonstrates that ASK1 has a spatially restricted and temporally dynamic expression pattern in both chick and mouse embryos, which includes conserved as well as species-specific expression domains.

Separase: a universal trigger for sister chromatid disjunction but not chromosome cycle progression.

Separase is a protease whose liberation from its inhibitory chaperone Securin triggers sister chromatid disjunction at anaphase onset in yeast by cleaving cohesin's kleisin subunit. We have created conditional knockout alleles of the mouse Separase and Securin genes. Deletion of both copies of Separase but not Securin causes embryonic lethality. Loss of Securin reduces Separase activity because deletion of just one copy of the Separase gene is lethal to embryos lacking Securin. In embryonic fibroblasts, Separase depletion blocks sister chromatid separation but does not prevent other aspects of mitosis, cytokinesis, or chromosome replication. Thus, fibroblasts lacking Separase become highly polyploid. Hepatocytes stimulated to proliferate in vivo by hepatectomy also become unusually large and polyploid in the absence of Separase but are able to regenerate functional livers. Separase depletion in bone marrow causes aplasia and the presumed death of hematopoietic cells other than erythrocytes. Destruction of sister chromatid cohesion by Separase may be a universal feature of mitosis in eukaryotic cells.

Mammalian inscuteable regulates spindle orientation and cell fate in the developing retina.

During mammalian neurogenesis, progenitor cells can divide with the mitotic spindle oriented parallel or perpendicular to the surface of the neuroepithelium. Perpendicular divisions are more likely to be asymmetric and generate one progenitor and one neuronal precursor. Whether the orientation of the mitotic spindle actually determines their asymmetric outcome is unclear. Here, we characterize a mammalian homolog of Inscuteable (mInsc), a key regulator of spindle orientation in Drosophila. mInsc is expressed temporally and spatially in a manner that suggests a role in orienting the mitotic spindle in the developing nervous system. Using retroviral RNAi in rat retinal explants, we show that downregulation of mInsc inhibits vertical divisions. This results in enhanced proliferation, consistent with a higher frequency of symmetric divisions generating two proliferating cells. Our results suggest that the orientation of neural progenitor divisions is important for cell fate specification in the retina and determines their symmetric or asymmetric outcome.

FGF signaling regulates expression of Tbx2, Erm, Pea3, and Pax3 in the early nasal region.

Fgf8 is required for normal development of the nasal region. Here, we have used a candidate approach to identify genes that are induced in chick nasal mesenchyme in response to FGF signaling. Using an explant culture system, we show that expression of the transcription factors Tbx2, Erm, Pea3, and Pax3, but not Pax7, in nasal mesenchyme is regulated by ectodermal signals in a stage-dependent manner. Using beads soaked in recombinant FGF protein and an FGF receptor antagonist, we furthermore demonstrate that FGF signaling is necessary and sufficient for expression of Tbx2, Erm, Pea3, and Pax3, but has no effect on Pax7 expression. We also show that, within the nasal mesenchyme, competence to respond to FGF signaling is initially widespread and uniform but becomes restricted to regions normally exposed to FGF at later stages of development, coincident with changes in FGF receptor expression. Finally, we provide evidence that FGF8 also regulates Erm and Pea3 expression in the nasal placodes. Together, these results identify Tbx2, Erm, Pea3, and Pax3 as downstream targets of FGF signaling in the facial area and suggest that these genes may mediate some of the effects of FGF8 during development of the nasal region.