scholarly journals Localization of CRABP-I and CRABP-II mRNA in the early mouse embryo by whole-mount in situ hybridization: Implications for teratogenesis and neural development

1994 ◽  
Vol 199 (4) ◽  
pp. 280-291 ◽  
Author(s):  
Sally Lyn ◽  
Vincent Giguère
Endocrinology ◽  
2001 ◽  
Vol 142 (7) ◽  
pp. 3223-3230 ◽  
Author(s):  
Masayo Yamagata ◽  
Akihito Kimoto ◽  
Toshimi Michigami ◽  
Masahiro Nakayama ◽  
Keiichi Ozono

Abstract In this study we examined the expression of 25-hydroxyvitamin D-1α-hydroxylase (1α-hydroxylase) and 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) by RT-PCR and whole mount in situ hybridization using organ culture of kidney taken from mouse embryo. First, the kidneys of mouse embryo at 11.5–17.5 days gestation were cultured in the presence or absence of forskolin and 1,25-dihydroxyvitamin D3[ 1α,25-(OH)2D3]. Forskolin and 1α,25-(OH)2D3 induced the expression of 1α-hydroxylase and 24-hydroxylase, respectively, in a dose- and time-dependent manner. In the absence of stimulants, the expression of 1α-hydroxylase and 24-hydroxylase was detected from days 13.5–17.5 gestation. The expression of vitamin D receptor and megalin was detected from days 13.5 and 11.5, respectively. Next, signals for the expression of either 1α-hydroxylase or 24-hydroxylase were detected by whole mount in situ hybridization in kidney explants taken from embryo at 15.5 days gestation after the appropriate stimulation. However, the localization of signals differed between the two enzymes; 1α-hydroxylase messenger RNA was expressed in the inner area of the kidney explants, whereas 24-hydroxylase messenger RNA was expressed in the surface area. The expression of both hydroxylases was restricted to the epithelium of developing renal tubules. The pattern of megalin expression was similar to that of 1α-hydroxylase expression. To confirm the difference in distribution of 1α-hydroxylase and 24-hydroxylase transcripts, the explants were hybridized with probes for both 1α-hydroxylase and 24-hydroxylase using double labeling techniques after simultaneous stimulation with forskolin and 1α,25-(OH)2D3, resulting in the detection at different locations of positive signals for the two enzymes. These results suggest that the expression of 1α-hydroxylase is induced in a distinct epithelium of renal tubules from that of 24-hydroxylase even at the early stage of kidney development before glomerulogenesis.


Author(s):  
A.E. Sutherland ◽  
P.G. Calarco ◽  
C.H. Damsky

Cell-extracellular matrix (ECM) interactions mediated by the integrin family of receptors are critical for morphogenesis and may also play a regulatory role in differentiation during early development. We have examined the onset of expression of individual integrin subunit proteins in the early mouse embryo, and their roles in early morphogenetic events. As detected by immunoprecipitation, the α6, αV, β1, and β3 subunits are detected as early as the 4-cell stage, α5 at the hatched blastocyst stage and αl and α3 following blastocyst attachment. We tested the role of these integrins in the attachment and migratory activity of two cell populations of the early mouse embryo: the trophoblast giant cells, which invade the uterine stroma and ultimately contribute to the chorio-allantoic placenta, and the parietal endoderm, which migrates over the inner surface of the trophoblast and ultimately forms Reichert's membrane and the parietal yolk sac. Experiments were done in serum-free medium on substrates coated with laminin (Ln) and fibronectin (Fn). Trophoblast outgrowth occurs on Ln and its E8 fragment (long arm), but not on the E1’ fragment (cross region) (Figs. 1, 2 ). This outgrowth is inhibited by anti-E8, anti-Ln, and by the anti-β1 family antiserum anti-ECMR, but not by anti-αV or the function-perturbing GoH3 antibody that recognizes the α6/β1 integrin, a major Ln (E8) receptor. This suggests that trophoblast outgrowth on Ln or E8 is mediated by a different β1 integrin such as α3/β1. Early stages of trophoblast outgrowth (up to 48 hours) on Fn are inhibited by anti-Fn and by function-perturbing anti-αV antibodies, whereas at later times outgrowth becomes insensitive to anti-αV but remains sensitive to the anti-β1 family antiserum anti-ECMr, indicating that trophoblast cells modulate their interaction with Fn during outgrowth. Trophoblast outgrowth on vitronectin (Vn) is sensitive to anti-αV antibodies throughout the 5-day period examined.


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