Koko Urase

Expression of RA175 mRNA, a new member of the immunoglobulin superfamily, in developing mouse brain

RA175, a new member of the immunoglobulin superfamily, is highly expressed during neuronal differentiation of P19 embryonal carcinoma cells. In situ hybridization showed that RA175 mRNA was detected in the developing nervous system, as well as the epithelium of the various non-neuronal tissues of mouse embryo. In contrast with the epithelia of the non-neuronal tissues, RA175 mRNA was not co-expressed with Sonic hedgehog mRNA and Patched mRNA during brain morphogenesis. RA175 mRNA was highly expressed in the anterior horn cells and the peripheral nervous system at embryonic day (E) 11.5 and in the central nervous system at E14.5–E18.5, but its expression decreased after birth and was undetectable in the adult mouse brain.

DETECTION OF ACTIVATED CASPASE-3 (CPP32) IN THE VERTEBRATE NERVOUS SYSTEM DURING DEVELOPMENT BY A CLEAVAGE SITE-DIRECTED ANTISERUM

We previously demonstrated that Caspase-3 is highly expressed in dorsal root ganglia and trigeminal ganglia of mouse embryos [T. Mukasa, K. Urase, Y.M. Momoi, I. Kimura, T. Momoi, Specific expression of CPP32 in sensory neurons of mouse embryos and activation of CPP32 in the apoptosis induced by a withdrawal of NGF, Biochem. Biophys. Res. Commun., 231 (1997) 770-774.]. Since, however, Caspases are processed into active form during apoptosis, it is difficult to examine the involvement of activated Caspases in naturally occurring cell death during development by immunohistochemical staining or in situ hybridization method. We prepared a cleavage site-directed antiserum against Caspase-3 (anti-p20/17). This antiserum reacted with fragment (p20/17) of Caspase-3, but not proCaspase-3 (p32), proCaspase-7 (p34) and its cleaved fragment (p24). We examined the relationship between the activation of Caspase-3 and the appearance of the naturally occurring apoptotic cells in the nervous system during development. In the trigeminal ganglia and dorsal root ganglia, the expression of Caspase-3 mRNA was maximal before the appearance of p20/17-positive cells and apoptotic cells. In the mouse brain, many p20/17-positive cells and apoptotic cells were observed in the neuroepithelium in the early developmental stages, but very few p20/17-positive cells were detected in postmitotic neurons in the cerebral cortex although Caspase-3 mRNA was expressed highly. Caspase-3 is activated mainly during apoptosis of neuroepithelial cells in the early developmental stages but not of mature neurons at postnatal stages.

Induction of bud formation of embryonic mouse tracheal epithelium by fibroblast growth factor plus transferrin in mesenchyme-free culture

Embryonic mouse tracheal epithelium, which branches in an epithelial–mesenchymal recombination culture with bronchial mesenchyme, was cultured under mesenchyme‐free conditions. When embedded in a basement‐membrane–like matrix and cultured in a serum‐free medium supplemented with fibroblast growth factor 1 (FGF1), the tracheal epithelium did not branch, whereas the bronchial epithelium underwent branching morphogenesis. When the medium was enriched with transferrin (Tf), bud formation was induced in the tracheal epithelium and some buds branched secondarily. FGF7 and FGF10, in cooperation with Tf, induced tracheal bud formation to the same extent as FGF1, although the optimum concentrations differed. A bromodeoxyuridine‐labeling study comparing cultures with and without Tf showed no Tf‐specific amplification of cell proliferation. A whole‐mount in situ hybridization study of the expression of Bmp4 and Shh genes in explants of mesenchyme‐free culture revealed that both genes were ubiquitously expressed and that expression did not correlate with bud formation. This expression pattern was different from the distally localized expression pattern observed in normal lung rudiments and in extratracheal buds induced by the recombined bronchial mesenchyme. These results suggest that both bronchial and tracheal bud formations were initiated without localized exposure of the epithelium to FGFs and were not accompanied by localized expression of Bmp4 and Shh in the epithelium.

Induction and Inhibition of Epithelial Differentiation by the Mixed Cell Aggregates of the Mesenchymes from the Chicken Embryonic Digestive Tract

Epithelial‐mesenchymal interaction plays an important role in the differentiation of digestive tract. However, the factors of these mesenchymes involved in induction of the epithelial differentiation of each organs are still unknown. In the present study, we made reconstituted mesenchymal cell aggregates by mixing proventricular mesenchymal cells with other mesenchymal cells, recombined the reconstituted mesenchyme with gizzard epithelium, and observed the differentiation of the gizzard epithelium in the explants with special attention to the appearance of embryonic chicken pepsinogen, one of the molecular marker of the proventricular epithelial cells, in the gizzard epithelium. The results showed that the proventricular mesenchymal cells induce gland formation and pepsinogen in the gizzard epithelium and that the esophageal and gizzard mesenchymal cells have the inhibitory influence on the differentiation of epithelia toward proventricular epithelium. The cells from small‐intestinal, lung and dorsal dermal mesenchyme have no such effect. Based on the results obtained so far, a hypothesis was presented to explain the mechanism regulating the differentiation of the epithelium in the digestive tract in the chicken embryo.

Foregut endoderm is specified early in avian development through signal(s) emanating from Hensen’s node or its derivatives

In this study, the initial specification of foregut endoderm in the chick embryo was analyzed. A fate map constructed for the area pellucida endoderm at definitive streak-stage showed centrally-located presumptive cells of foregut-derived organs around Hensens node. Intracoelomic cultivation of the area pellucida endoderm at this stage combined with somatic mesoderm resulted in the differentiation predominantly into intestinal epithelium, suggesting that this endoderm may not yet be regionally specified. In vitro cultivation of this endoderm for 1-1.5 day combined with Hensens node or its derivatives but not with other embryonic structures/tissues elicited endodermal expression of cSox2 but not of cHoxb9, which is characteristic of specified foregut endoderm. When the anteriormost or posteriormost part of the area pellucida endoderm at this stage, whose fate is extraembryonic, was combined with Hensens node or its derivatives for 1 day, then enwrapped with somatic mesoderm and cultivated for a long period intracoelomically, differentiation of various foregut organ epithelia was observed. Such epithelia never appeared in the endoderm associated with other embryonic structures/tissues and cultured similarly. Thus, Hensens node and its derivatives that lie centrally in the presumptive endodermal area of the foregut are likely to play an important role in the initial specification of the foregut. Chordin-expressing COS cells or noggin-producing CHO cells transplanted into the anteriormost area pellucida of the definitve streak-stage embryo could induce endodermal expression of cSox2 but not of cHoxb9, suggesting that chordin and noggin that emanate from Hensens node and its derivatives, may be involved in this process.

Spatial and temporal expression of RA70/Scap2 in the developing neural tube.

Src kinase-associated phosphoprotein 2 (Ra70/scap2), which was originally isolated as a retinoic acid (RA)-induced gene, associates with molecules that modulate integrin-survival signals. Although RA is essential for vertebrate organogenesis in the posterior region, little is known about the biological role of RA70/Scap2 during development. In the present study, we demonstrate that Ra70/scap2 mRNA is temporally expressed during the RA-induced neuronal differentiation of P19 embryonic carcinoma cells. Homozygous knockout mice in which the Ra70/scap2 gene was replaced with LacZ exhibited embryonic lethality, while heterozygous mice displayed preferential expression of LacZ in posterior neural tissues, including the neural tube and hindbrain during development (E7.5-11.5), but not the forebrain. Ra70/scap2 was expressed in the ependymal layer and ventricular zone in the neural tube, where neuroepithelial cells and neuroblasts with proliferation capacity are localized, respectively. Thus, RA70/Scap2 may be necessary for RA-induced neuronal differentiation from the posterior neuroectoderm.

The analysis of naturally occurring neuronal cell death by cleavage site-directed antiserum against Caspases

Some granule neurons naturally undergo apoptosis in the external granular layer of the postnatally developing rat cerebellum. We previously established an organotypic slice culture system as an experimental model for studying mechanisms of this apoptosis, in which deprivation of insulin or IGF-I analog induces apoptosis of external granular layer neurons. In the present study, we examined involvement of caspase-3 in this apoptosis using the slice culture system and an antibody specific for the active form of caspase-3. AC-DEVD-CHO, a peptide inhibitor of caspase-3-like proteases, partially prevented this apoptosis. Double staining by in situ nick end labeling and immunohistochemistry against the active caspase-3 revealed that the active caspase-3 is present in some of the apoptotic granule neurons. A similar staining pattern was also observed in the postnatal cerebellum in vivo. Thus, while caspase-3 was shown to be involved in apoptosis of some external granular layer neurons, there might exist a mechanism of apoptosis independent of caspase-3. Alternatively, activation of caspase-3 might peak before granule neurons become positive to the nick end labeling.