Kimiko Fukuda

Early specification of intestinal epithelium in the chicken embryo: a study on the localization and regulation of CdxA expression.

CdxA, a chicken homeobox‐containing gene related to caudal in Drosophila, has been implicated in the regionalization of endoderm. It is reported here that, in the development of the chicken embryo, CdxA expression appears in the endoderm at day 1.5 of development as bilateral bands on either side of the splanchnopleure which later contribute to intestinal epithelium. The CdxA‐expressing area extends medially and caudally as formation of the gut tube progresses. It is also shown that the rostral limit of CdxA expression demarcates the boundary between stomach and duodenum after day 3 of development. CdxA is not expressed in digestive tract appendages which open into the intestine, such as pancreas, liver and allantois. Early restriction of CdxA expression in intestinal lineage suggests that the intestinal specification involving CdxA expression commences before the gut tube is formed. The expression of CdxA in epithelial‐mesenchymal tissue recombinants suggests that mesenchymal influence regulating CdxA expression plays an important role in confirming the boundary between the stomach and intestine. Chronological change in the spatial distribution of CdxA transcripts and the results of tissue recombination experiments, together with precise fate maps of early endoderm and splanchnic mesoderm, lead to a model of mechanisms by which intestinal specification is brought about.

The dual origin of the peripheral olfactory system: placode and neural crest

BackgroundThe olfactory epithelium (OE) has a unique capacity for continuous neurogenesis, extending axons to the olfactory bulb with the assistance of olfactory ensheathing cells (OECs). The OE and OECs have been believed to develop solely from the olfactory placode, while the neural crest (NC) cells have been believed to contribute only the underlying structural elements of the olfactory system. In order to further elucidate the role of NC cells in olfactory development, we examined the olfactory system in the transgenic mice Wnt1-Cre/Floxed-EGFP and P0-Cre/Floxed-EGFP, in which migrating NC cells and its descendents permanently express GFP, and conducted transposon-mediated cell lineage tracing studies in chick embryos.ResultsExamination of these transgenic mice revealed GFP-positive cells in the OE, demonstrating that NC-derived cells give rise to OE cells with morphologic and antigenic properties identical to placode-derived cells. OECs were also positive for GFP, confirming their NC origin. Cell lineage tracing studies performed in chick embryos confirmed the migration of NC cells into the OE. Furthermore, spheres cultured from the dissociated cells of the olfactory mucosa demonstrated self-renewal and trilineage differentiation capacities (neurons, glial cells, and myofibroblasts), demonstrating the presence of NC progenitors in the olfactory mucosa.ConclusionOur data demonstrates that the NC plays a larger role in the development of the olfactory system than previously believed, and suggests that NC-derived cells may in part be responsible for the remarkable capacity of the OE for neurogenesis and regeneration.

Endoderm development in vertebrates: fate mapping, induction and regional specification

The formation of the vertebrate body plan begins with the differentiation of cells into three germ layers: ectoderm, mesoderm and endoderm. Cells in the endoderm give rise to the epithelial lining of the digestive tract, associated glands and respiratory system. One of the fundamental problems in developmental biology is to elucidate how these three primary germ layers are established from the homologous population of cells in the early blastomere. To address this question, ectoderm and mesoderm development have been extensively analyzed, but study of endoderm development has only begun relatively recently. In this review, we focus on the ‘where’, ‘when’ and ‘how’ of endoderm development in four vertebrate model organisms: the zebrafish, Xenopus, chick and mouse. We discuss the classical fate mapping of the endoderm and the more recent progress in characterizing its induction, segregation and regional specification.

Application of efficient and specific gene transfer systems and organ culture techniques for the elucidation of mechanisms of epithelial- mesenchymal interaction in the developing gut

Epithelial–mesenchymal interactions are very important in the development of the vertebrate gut. In the avian embryonic stomach (proventriculus), expression of embryonic chick pepsinogen (ECPg) gene, which is specific to developing glandular cells in stomach epithelium, is regulated by mesenchymal influence. Molecular mechanisms of tissue‐specific transcriptional regulation of the ECPg gene and the molecular nature of the mesenchymal signals were analyzed using a combination of the classic organ culture system and gene transfer strategies. In the present review, three methods for the introduction of DNA into tissues are described: lipofection, electroporation and retroviral infection, and characteristics of each system are discussed.

Versatile roles for sonic hedgehog in gut development

Sonic hedgehog (Shh) is a gene encoding a protein that can be secreted and act as a morphogen. The protein exerts versatile and important effects on the surrounding cells by binding a specific receptor, named patched. So far Shh has been shown to be involved in the morphogenesis and cytodifferentiation of many organ systems, such as notochord, floor plate, limb, pancreas, and pituitary gland, to mention only a few examples. Shh is also involved in the determination of left-right asymmetry, at least in the chicken embryo. Here we present evidence that Shh is one of the key genes whose activity is pivotal for the normal morphogenesis and differentiation of digestive organs. Epithelial Shh regulates the formation of stomach glands and stratification of the mesenchyme into connective tissue and smooth muscle. It exerts its effect often through the induction of bone morphogenetic protein (BMP) genes in the mesenchyme. Thus, Shh is a key player in the epithelial-mesenchymal interactions in the development of the gut.

Notch signaling functions as a binary switch for the determination of glandular and luminal fates of endodermal epithelium during chicken stomach development.

During development of the chicken proventriculus (glandular stomach), gut endoderm differentiates into glandular and luminal epithelium. We found that Delta1-expressing cells, undifferentiated cells and Notch-activated cells colocalize within the endodermal epithelium during early gland formation. Inhibition of Notch signaling using Numb or dominant-negative form of Su(H) resulted in a luminal differentiation, while forced activation of Notch signaling promoted the specification of immature glandular cells, but prevented the subsequent differentiation and the invagination of the glands. These results suggest that Delta1-mediated Notch signaling among endodermal cells functions as a binary switch for determination of glandular and luminal fates, and regulates patterned differentiation of glands in the chicken proventriculus.

The molecular mechanisms of stomach development in vertebrates

The tissue interactions between endodermal epithelium and mesenchyme originated from splanchnic mesoderm are essential during the formation of digestive tract. In this review, we introduce a series of works to elucidate the molecular mechanisms of the epithelial–mesenchymal interaction of stomach development in mainly the chicken embryo. We also describe some molecular studies in mouse stomach development.

The mesenchymal factors regulating epithelial morphogenesis and differentiation of the chicken stomach.

Abstract It is now well established that epithelial-mesenchymal interactions are essential for the formation of many organs in the development of the animals. Chicken digestive organs provide a valuable model system for analysis of the mechanisms underlying the epithelial-mesenchymal interactions. Here we will present our recent data indicating that the mesenchymal factors necessary for the epithelial differentiation in the chicken stomach are composed of several components such as growth factors and extracellular matrices. The possible involvement of bone morphogenetic protein-2 will be discussed.

Analysis of mesenchymal influence on the pepsinogen gene expression in the epithelium of chicken embryonic digestive tract

We performed tissue recombination experiments to discover the mesenchymal influences on differentiation of epithelia in chicken digestive organs. Epithelia and mesenchymes were taken from the lung, esophagus, proventriculus, gizzard, small intestine and large intestine of 6-day chicken embryos and recombined in various associations and cultivated in vitro for 6 days. Rather unexpectedly, embryonic chicken pepsinogen (ECPg) gene, a marker of the proventricular epithelium, was induced in the gizzard epithelium, which does not express ECPg in normal development, by the proventricular and lung mesenchymes. In the second half of this study, we investigated the mode of action of mesenchymal cells on ECPg expression in gizzard epithelial cells more precisely using the cell aggregate culture system, in which gizzard epithelial cells were mixed with proventricular, gizzard or lung mesenchymal cells. We found that supporting action of lung mesenchymal cells on ECPg expression was even stronger than that of proventricular mesenchymal cells, and suggest that the action of lung mesenchyme may be due partly to the enhancement of epithelial cell proliferation. According to the results of this study, together with many facts obtained so far, we will discuss a new model for restricted expression of ECPg in the proventricular epithelium in normal development.

Regional specification of the endoderm in the early chick embryo.

In the avian embryo, the endoderm, which forms a simple flat‐sheet structure after gastrulation, is regionally specified in a gradual manner along the antero‐posterior and dorso‐ventral axes, and eventually differentiates into specific organs with defined morphologies and gene expression profiles. In our study, we carried out transplantation experiments using early chick embryos to elucidate the timing of fate establishment in the endoderm. We showed that at stage 5, posteriorly grafted presumptive foregut endoderm expressed CdxA, a posterior endoderm marker, but not cSox2, an anterior endoderm marker. Conversely, anteriorly grafted presumptive mid‐hindgut endoderm expressed cSox2 but not CdxA. At stage 8, posteriorly grafted presumptive foregut endoderm also expressed CdxA and not cSox2, but anteriorly grafted presumptive mid‐hindgut endoderm showed no changes in its posterior‐specific gene expression pattern. At stage 10, both posteriorly grafted foregut endoderm and anteriorly grafted mid‐hindgut endoderm maintain their original gene expression patterns. These results suggest that the regional specification of the endoderm occurs between stages 8 and 10 in the foregut, and between stages 5 and 8 in the mid‐hindgut.