Coordinated Expression of Abd-B Subfamily Genes of the HoxA Cluster in the Developing Digestive Tract of Chick Embryo

AbstractTo elucidate the molecular mechanism for determining the positional specificity and morphogenesis of the chicken digestive tract, we analyzed expression patterns of Hox genes during development of the digestive tract, focusing on the Abd-B subfamily genes of the HoxA cluster, Hoxa-9, -10, -11 , and -13. Region-specific expression of these genes was found in the visceral mesoderm. In early development, before commencement of hindgut closure, they were expressed in a Russian doll pattern with the more 5′ located gene on the cluster showing the more posterior restricted expression domain. At middle stage, when hindgut closure had occurred and morphological subdivision of the hindgut was gradually becoming distinct, the expression domains of each gene became mutually exclusive and restricted to the subdivisions; i.e., Hoxa-9 in the posterior part of the small intestine and the ceca, Hoxa-10 in the ceca, Hoxa-11 in the ceca, the large intestine, and the cloaca, and Hoxa-13 in the cloaca. At later stages, when the bilateral branches of the ceca had formed, the overlapping expression domains of Hoxa-10 and -11 were strongly correlated with the budding processes of the ceca. These observations suggest the possibility that Hox genes are responsible for determination of the position-specific differentiation of the visceral mesoderm of the digestive system and regulate the budding processes of the ceca

A Secreted BMP Antagonist, Cer1, Fine Tunes the Spatial Organization of the Ureteric Bud Tree during Mouse Kidney Development

The epithelial ureteric bud is critical for mammalian kidney development as it generates the ureter and the collecting duct system that induces nephrogenesis in dicrete locations in the kidney mesenchyme during its emergence. We show that a secreted Bmp antagonist Cerberus homologue (Cer1) fine tunes the organization of the ureteric tree during organogenesis in the mouse embryo. Both enhanced ureteric expression of Cer1 and Cer1 knock out enlarge kidney size, and these changes are associated with an altered three-dimensional structure of the ureteric tree as revealed by optical projection tomography. Enhanced Cer1 expression changes the ureteric bud branching programme so that more trifid and lateral branches rather than bifid ones develop, as seen in time-lapse organ culture. These changes may be the reasons for the modified spatial arrangement of the ureteric tree in the kidneys of Cer1+ embryos. Cer1 gain of function is associated with moderately elevated expression of Gdnf and Wnt11, which is also induced in the case of Cer1 deficiency, where Bmp4 expression is reduced, indicating the dependence of Bmp expression on Cer1. Cer1 binds at least Bmp2/4 and antagonizes Bmp signalling in cell culture. In line with this, supplementation of Bmp4 restored the ureteric bud tip number, which was reduced by Cer1+ to bring it closer to the normal, consistent with models suggesting that Bmp signalling inhibits ureteric bud development. Genetic reduction of Wnt11 inhibited the Cer1-stimulated kidney development, but Cer1 did not influence Wnt11 signalling in cell culture, although it did inhibit the Wnt3a-induced canonical Top Flash reporter to some extent. We conclude that Cer1 fine tunes the spatial organization of the ureteric tree by coordinating the activities of the growth-promoting ureteric bud signals Gndf and Wnt11 via Bmp-mediated antagonism and to some degree via the canonical Wnt signalling involved in branching

Unfolding intermediate of a multidomain protein, calmodulin, in urea as revealed by small-angle X-ray scattering

AbstractThe denaturation of calmodulin (CaM) induced by urea has been studied by small-angle X-ray scattering, which is a direct way to evaluate the shape changes in a protein molecule. In the absence of Ca2+, the radii of gyration (Rg) of CaM are 20.8±0.3 Å in the native state and about 34±1.0 Å in the unfolded state. The transition curve derived from Kratky plots indicates a bimodal transition via a stable unfolding intermediate around 2.5 M urea. In the presence of Ca2+ and in the presence of both Ca2+ and a target peptide, the Rg values are 21.5±0.3 and 18.1±0.3 Å in the native state and 26.7±0.4 and 24.9±0.4 Å at 9 M urea, respectively. The results indicate that a stable unfolding intermediate still persists in 9 M urea. The present results suggest that the shape of unfolding intermediates is an asymmetric dumbbell-like structure, one in the folded and one in the unfolded state

Multi-modal effects of BMP signaling on Nodal expression in the lateral plate mesoderm during left–right axis formation in the chick embryo

AbstractDuring development of left–right asymmetry in the vertebrate embryo, Nodal plays a central role for determination of left-handedness. Bone morphogenetic protein (BMP) signaling has an important role for regulation of Nodal expression, although there is controversy over whether BMP signaling has a positive or negative effect on Nodal expression in the chick embryo. As BMP is a morphogen, we speculated that different concentrations might induce different responses in the cells of the lateral plate mesoderm (LPM). To test this hypothesis, we analyzed the effects of various concentrations of BMP4 and NOGGIN on Nodal expression in the LPM. We found that the effect on Nodal expression varied in a complex fashion with the concentration of BMP. In agreement with previous reports, we found that a high level of BMP signaling induced Nodal expression in the LPM, whereas a low level inhibited expression. However, a high intermediate level of BMP signaling was found to suppress Nodal expression in the left LPM, whereas a low intermediate level induced Nodal expression in the right LPM. Thus, the high and the low intermediate levels of BMP signaling up-regulated Nodal expression, but the high intermediate and low levels of BMP signaling down-regulated Nodal expression. Next, we sought to identify the mechanisms of this complex regulation of Nodal expression by BMP signaling. At the low intermediate level of BMP signaling, regulation depended on a NODAL positive-feedback loop suggesting the possibility of crosstalk between BMP and NODAL signaling. Overexpression of a constitutively active BMP receptor, a constitutively active ACTIVIN/NODAL receptor and SMAD4 indicated that SMAD1 and SMAD2 competed for binding to SMAD4 in the cells of the LPM. Nodal regulation by the high and low levels of BMP signaling was dependent on Cfc up-regulation or down-regulation, respectively. We propose a model for the variable effects of BMP signaling on Nodal expression in which different levels of BMP signaling regulate Nodal expression by a balance between BMP-pSMAD1/4 signaling and NODAL-pSMAD2/4 signaling