Cloning and expression of a novel cysteine-rich secreted protein family member expressed in thyroid and pancreatic mesoderm within the chicken embryo.

We have isolated a new chicken gene that is a member of the cysteine-rich secreted protein family (CRISP). The CRISP family is composed of over 70 members that are found in many phyla of organisms, including: vertebrates, plants, fungi, yeast, and insects. Here we describe the cloning of a novel member of this family, SugarCrisp, and its expression pattern throughout chicken embryogenesis. We also describe its utility as a marker of thyroid and pancreatic mesoderm in the developing chicken embryo and its expression within the human and mouse in glandular tissue.

The Evi1 proto-oncogene is required at midgestation for neural, heart, and paraxial mesenchyme development.

The ecotropic viral integration site-1 (Evi1) locus was initially identified as a common site of retroviral integration in myeloid tumors of the AKXD-23 recombinant inbred mouse strain. The full-length Evi1 transcript encodes a putative transcription factor, containing ten zinc finger motifs found within two domains of the protein. To determine the biological function of the Evi1 proto-oncogene, the full-length, but not an alternately spliced, transcript was disrupted using targeted mutagenesis in embryonic stem cells. Evi1 homozygous mutant embryos die at approximately 10.5 days post coitum. Mutants were distinguished at 10.5 days post coitum by widespread hypocellularity, hemorrhaging, and disruption in the development of paraxial mesenchyme. In addition, defects in the heart, somites, and cranial ganglia were detected and the peripheral nervous system failed to develop. These results correlated with whole-mount in situ hybridization analyses of embryos which showed expression of the Evi1 proto-oncogene in embryonic mesoderm and neural crest-derived cells associated with the peripheral nervous system. These data suggest that Evi1 has important roles in general cell proliferation, vascularization, and cell-specific developmental signaling, at midgestation.

Mechanism of infertility in male guinea pigs immunized with sperm PH-20.

PH-20, a testis-specific protein first expressed in haploid germ cells, is present on the posterior head plasma membrane and inner acrosomal membrane of mature guinea pig sperm. PH-20 is bifunctional, having a hyaluronidase activity that allows sperm to penetrate the cumulus layer and a separate activity required for binding of acrosome-reacted sperm to the zona pellucida. The immunization of male guinea pigs with PH-20 reproducibly results in infertility with a duration of 6-12 mo or longer. In this study, we analyzed the immunopathology in the reproductive tract of PH-20-immunized males to probe the mechanism(s) responsible for the induced infertility and found two separate effects. Remarkably, in almost all infertile, PH-20-immunized males, the caudae epididymides were empty (contained no sperm) or contained only abnormal sperm. The complete loss of normal sperm in the epididymis apparently results in infertility. A second effect was the induction of experimental autoimmune orchitis (EAO), representing the first report of EAO induced by a purified testis/sperm molecule of known functions. PH-20-induced EAO differed from EAO induced by crude testis antigens in two respects: 1) an absence of epididymitis with abscess and granuloma and 2) the presence of antibody on germ cells within seminiferous tubules and inside the cauda epididymidis. The former suggests that crude testis antigens other than PH-20 are responsible for epididymitis, and the latter suggests a possible role of antibody in EAO pathogenesis and infertility induction. Return to fertility, after 6-12 mo, was accompanied by regression of EAO and reappearance of spermatozoa in the caudae epididymides.

Autonomous endodermal determination in Xenopus: regulation of expression of the pancreatic gene XlHbox 8.

In neural plate stage Xenopus embryos, XlHbox 8 expression marks anterior endodermal cells fated to develop into pancreas/duodenum, and expression continues in adult pancreas in exocrine duct, acinar, and islet cells. Here, XlHbox 8 is used as a marker in experiments addressing the mechanisms of early endodermal patterning, particularly with respect to the role of specific polypeptide growth factors. When mesoderm-free vegetal explants (VEs) from early blastula stage embryos are cultured in isolation, XlHbox 8 expression develops autonomously in the dorsal region, strongly suggesting that endodermal region-specific determination occurs before MBT. Data from microinjection experiments using RNA encoding the activin and FGF dominant negative receptors and growth factor treatments of isolated VEs suggest that activin positively regulates XlHbox 8 expression, whereas bFGF is a potent negative regulator. Moreover, bFGF induces mesodermal marker expression in VEs. This suggests that the early endodermal determination state is plastic and that elevated levels of bFGF may convert vegetal (endodermal) cells into mesoderm. We propose a model for XlHbox 8 regulation in which an early signal from the Nieuwkoop center (whose eventual fate is endoderm) predisposes dorsovegetal cells for autonomous XlHbox 8 expression, in an area of high local activin (or activin-like) ligand concentration, and low relative concentrations of bFGF.

XIHbox 8, an endoderm-specific Xenopus homeodomain protein, is closely related to a mammalian insulin gene transcription factor.

The cis-acting sequences that mediate insulin gene expression exclusively in pancreatic islet beta-cells are localized within the 5'-flanking region between nucleotides -340 and -91. We have identified an evolutionarily conserved, A+T-rich element at -201/-196 basepairs in the rat insulin II gene that is essential for efficient expression in beta-cells. Affinity-purified antibody to the XIHbox 8 protein super-shifted the major beta-cell-activator factor complex binding to the -201/-196 element. XIHbox 8 is a Xenopus endoderm-specific homeodomain protein whose expression is restricted to the nucleus of endodermal cells of the duodenum and developing pancreas. Antibody to XIHbox 8 specifically interacts with a 47-kilodalton protein present in this DNA complex. Immunohistochemical studies revealed XIHbox 8-like proteins within the nucleus of almost all mouse islet beta-cells and a subset of islet alpha- and beta-cells. These results are consistent with the proposal that an XIHbox 8-related homeoprotein of 47 kilodalton is required for expression of the mammalian insulin gene in beta-cells. Experiments conducted with antiserum raised to somatostatin transcription factor-1 (STF-1), a recently isolated mammalian XIHbox 8-related homeoprotein, indicate that the STF-1 protein is the mammalian homolog of Xenopus XIHbox 8.