Sadao Yasugi

Region-Specific Expression of Chicken Sox2 in the Developing Gut and Lung Epithelium: Regulation by Epithelial-Mesenchymal Interactions

In situ analysis of the chicken cSox2 gene, a member of the transcription factor family containing an Sry‐like high‐mobility group (HMG) box, demonstrated localized expression in the embryonic endoderm. Transcripts of cSox2 appeared before commencement of morphogenesis and cytodifferentiation in the rostral gut epithelium from the pharynx to the stomach. The caudal limit of cSox2 expression coincided with that of the region competent for proventricular differentiation and to the rostral limit of the domain of CdxA, a homologue of Drosophila caudal. During morphogenesis, the level of transcripts of cSox2 decreased in epithelia invaginating into surrounding mesenchyme to form glandular or tubular structures, such as the primordia of the thyroid and lung, glandular epithelium of the proventriculus, and secondary bronchus of the lung. Tissue recombination experiments demonstrated that cSox2 expression is regulated by the underlying mesenchyme as well as morphogenesis and cytodifferentiation. The results suggest that cSox2 plays pivotal roles in generating morphologically and physiologically distinct types of epithelial cells in the gut. Dev. Dyn. 1998;213:464–475.

Spatially and temporally regulated expression of the LIM class homeobox gene Hrlim suggests multiple distinct functions in development of the ascidian, Halocynthia roretzi

Hrlim is a LIM class homeobox gene that was first isolated from the ascidian Halocynthia roretzi. To assess its roles in early development of the ascidian, spatial and temporal expression of Hrlim was examined by whole mount in situ hybridization. This revealed that transcription of Hrlim is activated at the 32-cell stage specifically in the endoderm lineage. Hrlim is also transiently expressed in all notochord precursor cells. Expression in the endoderm lineage continues through to the middle of gastrulation. After gastrulation, Hrlim is expressed in certain lineages that give rise to subsets of cells in the brain and spinal cord. Based on these observations, it is suggested that Hrlim plays multiple distinct roles in ascidian embryogenesis.

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.

Role of Epithelial‐Mesenchymal Interactions in Differentiation of Epithelium of Vertebrate Digestive Organs

The digestive tract of the vertebrates consists of endodermal epithelium and mesenchyme derived from splanchnic mesoderm. In birds and mammals, the initially flat endodermal sheet forms a tubular pocket by folding and fusion of left and right sheets at the midline. The endodermal tube becomes surrounded by mesodermal cells, which later differentiate into the mesenchymal part of the tract and finally into connective tissue and muscles. Regional differences of digestive tract are recognizable soon after the establishment of a tubular tract by the form and the position of digestive organ rudiments, which vary from one organ to another and from one developmental stage to another. Inside each organ, morphological and functional differentiation proceeds by the cooperation of the two components, the endodermal epithelium and the mesenchyme (28, 61). In general, the vertebrate digestive tract becomes divided anteroposteriorly into the esophagus, stomach, small intestine, cecum, large intestine and allantois. In birds, the stomach is further divided into the proventriculus (glandular stomach) and gizzard (muscular stomach). The liver and pancreas develop from the primordia formed in the small intestine. There have been extensive morphological studies on the formation and differentiations of these organs (4, 29, 58), and recently their molecular biological features have been also studied. These studies have been facilitated by the fact that each organ has its own characteristic morphology and function, which can be detected by rather simple methods such as histological, biochemical, immunocytochemical and in situ hybridization procedures. Special attention has been paid to

Susceptibility of epithelia to directive influences of mesenchymes during organogenesis: uncoupling of morphogenesis and cytodifferentiation

Morphogenesis and functional cytodifferentiation are two major events in organogenesis, and normally they take place inseparably either in vivo or in vitro conditions. In this article, we reviewed a series of our recent results on mesenchymal-epithelial interactions in organogenesis of digestive organs, urogenital organs and the skin of avian and mammalian embryos, giving special attention to the importance of the responses of epithelia to the directive influences of mesoderms and also to the uncoupling of morphogenesis and cytodifferentiation, which has often been observed during the course of these studies.

DIFFERENTIATION OF THE DIGESTIVE TRACT EPITHELIUM UNDER THE INFLUENCE OF THE HETEROLOGOUS MESENCHYME OF THE DIGESTIVE TRACT IN THE BIRD EMBRYOS

The endoderm of the oesophagus, proventriculus, gizzard or small intestine of the 5‐day‐old chick or quail embryo was cultivated in combination with homologous or heterologous mesenchyme on a WxxxOLFFyyy and HxxxAFFHNyyy medium for 7 to 21 days or on the chorio‐allantoic membrane (CAM) for 8 days. With homologous mesenchyme the epithelium always differentiated homotypically. In association with heterologous mesenchyme, the differentiation of the epithelium was both homotypical and heterotypical depending on the region of the digestive tract. The oesophagus and small intestine differentiate mainly homotypically both in culture and on CAM, but the gizzard and proventriculus show heterotypic differentiation particularly on CAM. Thus, the endoderm of the digestive tract of the 5‐day‐old chick or quail embryo, though rather “determined”, still reacts to the heterologous stimuli of the mesenchyme to some degree.

Gizzard epithelium of chick embryos can express embryonic pepsinogen antigen, a marker protein of proventriculus

SummaryThe avian stomach is composed of two distinct organs, the proventriculus and the gizzard. Pepsinogen expression in the proventricular and gizzard epithelia of chick embryos was investigated immunohistochemically with anti-embryonic chick pepsinogen (anti-ECPg) antiserum. In normal development, the ECPg antigen was expressed only in the glandular epithelial cells of the embryonic proventriculus from the 8th day of incubation onwards. However, both proventricular and gizzard epithelia of 6-day embryos expressed the ECPg antigen when recombined and cultured with the proventricular mesenchyme. Chronological studies revealed that the ECPg antigen was first detected in a few epithelial cells at 3 days of cultivation. The percentage of ECPg-positive cells among the total epithelial cells in each recombinant increased with the length of the culture period and all the glandular epithelial cells were positive at 9 days. During this process, the percentage of ECPg-positive cells in each cultured recombinant was similar in proventricular and gizzard epithelia. Moreover, both epithelia could express the ECPg antigen when recombined and cultured with the oesophageal or small-intestine mesenchyme for 9 days, though the percentage of ECPg-positive cells in each cultured recombinant was much lower than that in the cultured recombinant with the proventricular mesenchyme. These results indicate that the gizzard epithelium of 6-day chick embryos possesses a similar potential for pepsinogen expression as the proventricular epithelium of the same age.

Correlation between Musashi-1 and c-hairy-1 expression and cell proliferation activity in the developing intestine and stomach of both chicken and mouse

Musashi‐1 (Msi‐1) is an RNA‐binding protein that plays key roles in the maintenance of neural stem cell states and in their differentiation into neural cells. Msi‐1 has also been proposed as a candidate marker gene of mammalian intestinal stem cells and their immediate lineages. In this study, we examined Msi‐1 expression in the small intestine and the stomach of both chicken and mouse during embryonic, fetal and postnatal development. In addition, we analyzed the expression of c‐hairy‐1, a chicken homologue of mouse Hes1, and assessed the proliferative activity of the cells expressing both of these factors. Significantly, during the development of these digestive organs in both species Msi‐1 expression showed dynamic changes, suggesting that it is important for digestive organ development, particularly for epithelial differentiation. Based on our observations of the expression patterns of Msi‐1 and c‐hairy‐1 in the adult small intestine, we speculate that Msi‐1 is also a stem cell marker of the chicken small intestinal epithelium.

Sonic hedgehog expression in developing chicken digestive organs is regulated by epithelial–mesenchymal interactions

Sonic hedgehog (Shh) gene encodes a secreted protein that acts as an important mediator of cell–cell interactions. A detailed analysis of Shh expression in the digestive organs of the chicken embryo was carried out. Shh expression in the endoderm begins at stage 7, when the formation of the foregut commences, and is found as narrow bands in the midgut. Shh expression around the anterior intestinal portal at stage 15 is restricted to the columnar endoderm lined by the thick splanchnic mesoderm, suggesting that the existence of thick splanchnic mesoderm might be necessary for Shh expression in the columnar endoderm. After the gut is closed, Shh expression is found universally in digestive epithelia, including the cecal epithelium. However, its expression ceases in the epithelium of the proventricular glands, the ductus choledochus and ductus pancreaticus that protrude from the main digestive duct. When the gizzard epithelium differentiated into glands under the influence of the proventricular mesenchyme, the glandular epithelium lost the ability to express Shh. These findings suggest that Shh expression in the epithelium may be regulated by surrounding mesenchyme throughout organogenesis of the digestive organs and is closely involved in epithelial–mesenchymal interactions in developing digestive organs.

Phospholipase C-δ1 is an essential molecule downstream of Foxn1, the gene responsible for the nude mutation, in normal hair development

Nude mice exhibit athymia and hairless‐ness by a loss‐of‐function mutation in the transcription factor Foxnl gene. Although the immunological functions of Foxn1 have been studied intensively, there have been relatively few studies of its functions in skin. Foxn1 regulates expression of hair keratins, which is essential for normal hair structure; however, how Foxn1 regulates hair keratin expression and hair formation is largely unknown. In the present study, we found that mice lacking phospholipase C (PLC)‐δ1, a key molecule in the phosphoinositide signaling pathway, and nude mice show similar hair abnormalities, such as lack of cuticle and bending. We also found that expression of hair keratins was remarkably decreased in skin of PLC‐δ1 knockout mice. Furthermore, expression of PLC‐δ1 was induced in Foxn1‐transfected U2OS cells. In addition, we showed that PLC‐δ1 expression was remarkably decreased in skin of nude mice. In skin and keratinocytes of nude mice as well as PLC‐δ1 KO mice, activation of PLC downstream effectors, such as PKC and nuclear factor of activated T cells, was impaired. These results indicate that PLC‐δ1 is an essential molecule downstream of Foxn1 in normal hair formation, and strongly suggest that hairless‐ness in nude mice is caused by insufficient expression of PLC‐δ1.—Nakamura, Y., Ichinohe, M., Hirata, M., Matsuura, H., Fujiwara, T., Igarashi, T., Nakahara, M., Yamaguchi, H., Yasugi, S., Takenawa, T., Fukami, K. Phospholipase C‐δ1 is an essential molecule downstream of Foxnl, the gene responsible for the nude mutation, in normal hair development. FASEB J. 22, 841–849 (2008)