Immunolocalization of fibroblast growth factor receptors 1 and 2 in mouse palate development.

Recent evidence has implicated mutations of fibroblast growth factor receptors (FGF-R) in the pathogenesis of craniosynostotic syndromes. Cleft palate can be a component of such syndromes. The expression of FGF-R1 and FGF-R2 has been delineated in normally developing cranium, where they seem to regulate cellular differentiation and proliferation, respectively. The specific role of fibroblast growth factor signaling in mammalian palate development is unclear. The authors investigated the patterns of expression of FGF-R1 and FGF-R2 throughout mouse palatal development in the embryo. Time-dated CD-1 mouse heads (n = 135) were harvested at embryonic ages 12.5, 13.5, 14.5, 15.5, and 16.5 days (term gestation = 19.5 days), fixed in paraformaldehyde, embedded in paraffin, and sectioned. In addition, paired palatal shelves (n = 30) were isolated by means of microdissection from embryonic day--13.5 embryos, grown on Millipore filters in serum-free medium in vitro for 24, 48, 72, or 96 hours and processed for histological analysis. Immunohistochemical analysis for FGF-R1 and FGF-R2 was performed on the in vivo and in vitro specimens. FGF-R1 and FGF-R2 were found to be specifically expressed in the epithelium of the developing palatal shelves from the time of their outgrowth from the maxillary processes through completion of fusion in vivo and in vitro. Expression of both receptors was particularly strong during the phases of medial epithelial-medial epithelial contact between the individual shelves, through the formation of the medial epithelial seam, to the ultimate dissolution of the seam. Such a pattern of expression seems to implicate fibroblast growth factor signaling in the regulation of the critical phase of fusion of the bilateral shelves. The expression of both FGF-R1 and FGF-R2 in the lateral palatal mesenchyme, where such secondary structures as tooth primordia and bone begin to appear, also suggests a role for fibroblast growth factor signaling in the induction of ongoing differentiation and maturation of the palate after fusion. These data suggest that fibroblast growth factor signaling may play a role in the epithelial-mesenchymal interactions that dictate fusion and maturation of the developing palate. Furthermore, the data are consistent with the correlation of cleft palate formation with aberrant fibroblast growth factor signaling.

Transforming growth factor-beta 1 in the developing mouse pancreas: a potential regulator of exocrine differentiation.

Transforming growth factor-beta 1 (TGF-beta 1) is known to regulate cell growth, differentiation, and function in developing mammalian systems. Altering TGF-beta 1 expression in the developing pancreas has been shown to affect both exocrine and endocrine development, suggesting that it is an important regulator of pancreatic organogenesis. We proposed to examine the ontogeny of TGF-beta 1 mRNA expression in the developing pancreas, as well as characterize the patterns of relative TGF-beta 1 gene expression and activity. We performed in situ hybridization for TGF-beta 1 on pancreas specimens obtained from CD-1 mice on gestational days 12.5 (E12.5), 15.5 (E15.5), and 18.5 (18.5). We also isolated mRNA from the pancreas on each of these days and performed a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to assess relative TGF-beta 1 expression as a function of gestational age. Finally, we performed a TGF-beta 1 ELISA with media conditioned by embryonic pancreas from gestational days 15.5 and 18.5. By in situ hybridization, TGF-beta 1 mRNA is expressed exclusively in the E12.5 pancreatic epithelium, sparing the surrounding mesenchyme. As pancreatic organogenesis progresses, TGF-beta 1 mRNA expression localizes predominantly to the developing acini. TGF-beta 1 gene expression appears modest through E15.5 but is upregulated near the end of gestation, at E18.5. TGF-beta 1 activity, by ELISA, is also upregulated at E18.5. TGF-beta 1 may thus be a modulator of pancreatic organogenesis. Modest TGF-beta 1 expression through E15.5 may be permissive for exocrine lineage selection. TGF-beta 1 expression may then become critical for terminal acinar differentiation. Upregulated TGF-beta 1 expression at the end of gestation may be important for islet formation, and it may be necessary to inhibit continued proliferation and differentiation of pluripotent cells within the pancreatic ductal epithelium.

Basement membrane exposure defines a critical window of competence for pancreatic duct differentiation from undifferentiated pancreatic precursor cells.

We previously showed that the undifferentiated pancreatic epithelium can differentiate into islets, ducts, or acini depending on its milieu and that laminin is necessary for pancreatic duct formation. Therefore we wanted to study the plasticity of laminin-induced duct differentiation the better to understand mechanisms of pancreatic duct lineage selection induced by basement membrane. Mouse embryonic pancreases were dissected at gestational day 11 (E11.5), and epithelium was isolated from its surrounding mesenchyme. Some epithelia were cultured in a collagen gel devoid of laminin. These epithelia were "rescued" at days 1-7 of culture by transferring them to a laminin-rich matrix (Matrigel) for 7 additional days. Other epithelia were instead first cultured in Matrigel, and then placed into collagen. Immunohistochemistry was performed for insulin, amylase, and carbonic anhydrase II. Pancreatic epithelia rescued from collagen into laminin during days 1-4 after harvest were still able to form ducts, whereas epithelia deprived of laminin for longer than this 4-day window were not. Pancreatic epithelia exposed to laminin for as little as 1 day, and then placed into collagen, still retained the ability to make ducts. Thus there is a clear cut-off in the development of the pancreatic epithelium at E11.5, after which laminin appears necessary to induce duct formation. We believe that such "windows of competence" in embryonic development imply that developmental programs in the embryo allow some flexibility.

Ontogeny of activin B and follistatin in developing embryonic mouse pancreas: implications for lineage selection.

Activin, a member of the transforming growth factor-beta superfamily, has been shown to be a critical regulator in exocrine and endocrine pancreas formation. The purpose of our study was to describe the ontogeny of activin B and its inhibitor, follistatin, in developing pancreas and to elucidate potential mechanisms for exocrine and endocrine lineage selection. Mouse embryonic pancreata were dissected at various ages (day 10 [E10.5] to birth [E18.5]), sectioned, and immunostained for activin B (one of two existing isomers, A and B), follistatin, insulin, and glucagon. In addition, reverse transcriptase-polymerase chain reaction was employed to determine the messenger RNA expression of follistatin in isolated pancreatic epithelia and mesenchyme of various ages. Activin B was first detected at E12.5 in epithelial cells coexpressing glucagon. At E16.5 these coexpressors appeared as clusters in close proximity to early ducts. By E18.5 activin B was localized to forming islets where cells coexpressed glucagon and were arranged in the mantle formation characteristic of mature alpha cells. Follistatin was found to be ubiquitous in pancreatic mesenchyme at early ages by immunohistochemical analysis, disappearing sometime after E12.5. Follistatin reappeared in E18.5 islets and remains expressed in adult islets. Follistatin messenger RNA was first detected in epithelium at E11.5, preceding its protein expression in islets later in gestation. We propose that mesenchyme-derived follistatin inhibits epithelium-derived activin at early embryonic ages allowing for unopposed exocrine differentiation and relative suppression of endocrine differentiation. At later ages the decrease in the amount of mesenchyme relative to epithelium and the subsequent drop in follistatin levels liberates epithelial activin to allow differentiation of endocrine cells to form mature islets by the time of birth.