Asymmetric Nodal expression in the mouse is governed by the combinatorial activities of two distinct regulatory elements

In all vertebrates, invariant left/right (L/R) positioning and organization of the internal viscera is controlled by a conserved pathway. Nodal, a member of the TGFbeta superfamily is a critical upstream component responsible for initiating L/R axis determination. Asymmetric Nodal expression in the node preceeds and foreshadows morphological L/R asymmetry. Here we address the mechanism of Nodal activation in the left LPM by studying the function of a novel enhancer element, the AIE. We show this element is exclusively active in cells of the left lateral plate mesoderm (LPM) and is not itself responding to Nodal asymmetry. To test the hypothesis that this element may initiate asymmetric Nodal expression in the LPM, we deleted it from the mouse germ line. Mice homozygous for the AIE deletion (Nodal(deltaaie/deltaaie)) show no defects. However, we find that the AIE contributes to regulating the level of asymmetric Nodal activity; analysis of transheterozygous embryos (Nodal(deltaaie/null)) shows reduced Nodal expression in the left LPM associated with a low penetrance of L/R defects. Our findings point to the existence of two independent pathways that control Nodal expression in the left LPM.     

csal1 is controlled by a combination of FGF and Wnt signals in developing limb buds

While some of the signaling molecules that govern establishment of the limb axis have been characterized, little is known about the downstream effector genes that interpret these signals. In Drosophila, the spalt gene is involved in cell fate determination and pattern formation in different tissues. We have cloned a chick homologue of Drosophila spalt, which we have termed csal1, and this study focuses on the regulation of csal1 expression in the limb bud. csal1 is expressed in limb buds from HH 17 to 26, in both the apical ectodermal ridge and the distal mesenchyme. Signals from the apical ridge are essential for csal1 expression, while the dorsal ectoderm is required for csal1 expression at a distance from the ridge. Our data indicate that both FGF and Wnt signals are required for the regulation of csal1 expression in the limb. Mutations in the human homologue of csal1, termed Hsal1/SALL1, result in a condition known as Townes-Brocks syndrome (TBS), which is characterized by preaxial polydactyly. The developmental expression of csal1 together with the digit phenotype in TBS patients suggests that csal1 may play a role in some aspects of distal patterning.     

Hydra pattern is controlled by two distinct but interacting morphogen sets

Summary Temporal course of regeneration of the hypostome and basal disc along the body length of the hydra is studied both in the presence and absence of the other determined centre. The regeneration times vary nonlinearly with distance from the original position indicating that the underlying processes are of non-linear nature. The presence of hypostome influences the regeneration of basal disc in an inhibitory manner throughout the body length, whereas, basal disc influences the regeneration of hypostome only in the lower portion of the body in a positive manner. A scheme in terms of the activators and inhibitors specific to hypostome and basal disc, is given. The implication of these results is that the two inhibitors are functionally distinct.     

Regulation of gene expression in preimplantation mouse embryos: effects of the zygotic clock and the first mitosis on promoter and enhancer activities

Previous studies have reported that promoters requiring enhancers for full activity in mammalian somatic cells also require enhancers when injected into mouse two-cell embryos, whereas the same promoters can be expressed just as efficiently in the absence of an enhancer when injected into arrested one-cell embryos. Experiments were designed to determine whether this phenomenon reflected normal developmental changes at the beginning of mammalian development, or simply differences in the physiological states of these cells under the experimental conditions employed. The activity of three different promoters that function in a wide variety of mammalian cells was measured both in embryos whose morphological development was arrested and in embryos that continued development in vitro. Expression of the injected gene was related to the onset of zygotic gene expression ("zygotic clock"), the phase of the cell proliferation cycle, the use of aphidicolin to arrest cell proliferation, and formation of two-cell embryos in vitro and in vivo. The results demonstrated that promoter activity was tightly linked to zygotic gene expression, while the need for enhancers to stimulate promoter activity depended only on formation of a two-cell embryo. These results further support the hypothesis that the first mitosis induces a general repression of promoters prior to initiation of zygotic gene expression that is relieved specifically by enhancers.     

经程序化冷冻的小鼠休眠胚胎的基因表达谱差异分析

目的探讨小鼠休眠胚胎经程序化冷冻后基因表达谱的变化及相关信号通路的改变趋势。方法采用Affymetrix基因芯片检测小鼠正常休眠胚胎和经程序化冷冻后的休眠胚胎的差异表达基因;采用GO分析和Pathway分析等生物信息学方法进一步了解相关信号通路的改变。结果经程序化冷冻后的小鼠休眠胚胎与正常休眠胚胎相比,存在228个差异表达基因,其中50个基因表达上调,178个基因表达下调。Pathway分析显示黏着斑通路、细胞外基质受体相互作用通路、肌动蛋白细胞骨架调节通路、细胞凋亡通路、细胞通讯通路、泛素介导的蛋白质水解通路、甘油磷脂代谢通路、小细胞肺癌通路、TGF-β信号通路、MAPK信号通路等基因差异表达变化趋势明显。结论小鼠休眠胚胎经程序化冷冻后会导致一系列基因调控变化,并可能影响多条信号通路的协同变化。     

Modulation of two distinct galactosyltransferase activities in populations of mouse peritoneal macrophages

We have examined two galactosyltransferase activities in membrane preparations obtained from resident macrophages, from resident macrophages maintained in culture for 24 hr, and from thioglycollate (TG)-elicited macrophages. Transfer of galactose from uridine diphosphate (UDP)-galactose to N-acetylglucosamine is 2.6 times higher in membranes prepared from TG macrophages (107 +/- 5.5 nmol/hr/mg) than in membranes prepared from resident macrophages (41 +/- 2.0 nmol/hr/mg). Membranes obtained from resident macrophages cultured for 24 hr exhibit a 2.5 times higher activity (102 +/- 4.4 nmol/hr/mg) than membranes from resident cells plated for 4 hr. Transferase activity in membranes derived from TG macrophages is not significantly affected by overnight culture. The transferase reaction product, isolated on Bio-Gel P-4 and analyzed by galactosidase treatments, was identified as galactosyl-beta 1, 4-N-acetylglucosamine. The enzyme, therefore, is UDP-galactose:2-acetamido-2-deoxy-D-glucose 4 beta-galactosyltransferase. This is supported by the fact that this galactosyltransferase activity is specifically inhibited by high concentrations of N-acetylglucosamine (200 mM). We have also examined the transfer of galactose to N-acetyllactosamine. Membranes from TG-elicited macrophages contain a UDP-galactose:galactosyl-beta 1, 4-N-acetylglucosamine 3 alpha-galactosyltransferase which synthesizes the trisaccharide, galactosyl-alpha 1, 3-galactosyl-beta 1,4-N-acetylglucosamine. This product was identified by gel filtration chromatography, high performance liquid chromatography, and galactosidase digestions. This alpha-galactosyltransferase activity was not detected in membranes prepared from resident macrophages. These results indicate that glycosyltransferase activities are modulated in populations of mouse macrophages, and that these changes correlate with changes in cell surface lactosaminoglycans reported previously.     

Temporal pattern of posterior positional identity in mouse limb buds

This study describes the temporal pattern of posterior positional identity in mouse limb bud cells. To do this wedges of tissue from the posterior edge of mouse limb buds at various stages (limb stages: Wanek et al., 1989b. J. Exp. Zool. 249, 41-49) were grafted to the anterior edge of a host chick embryo wing bud. Grafts of mouse posterior cells are able to induce the formation of supernumerary digits every time when they are taken from buds from stage 3 through stage 6. At stage 7, the frequency declines and by stage 8 the chick cells no longer respond. The results indicate a change in tissue properties at stage 7, which progresses by stage 8 to the point at which posterior positional identity is no longer detectable by this assay. These temporal changes in this aspect of limb pattern formation can be used as an additional criterion to guide the identification of genes involved in the specification of posterior positional identity.     

Mesenchyme with fgf-10 expression is responsible for regenerative capacity in Xenopus limb buds

A young tadpole of an anuran amphibian can completely regenerate an amputated limb, and it exhibits an ontogenetic decline in the ability to regenerate its limbs. However, whether mesenchymal or epidermal tissue is responsible for this decrease of the capacity remains unclear. Moreover, little is known about the molecular interactions between these two tissues during regeneration. The results of this study showed that fgf-10 expression in the limb mesenchymal cells clearly corresponds to the regenerative capacity and that fgf-10 and fgf-8 are synergistically reexpressed in regenerating blastemas. However, neither fgf-10 nor fgf-8 is reexpressed after amputation of a nonregenerative limb. Nevertheless, nonregenerative epidermal tissue can reexpress fgf-8 under the influence of regenerative mesenchyme, as was demonstrated by experiments using a recombinant limb composed of regenerative limb mesenchyme and nonregenerative limb epidermis. Taken together, our data demonstrate that the regenerative capacity depends on mesenchymal tissue and suggest that fgf-10 is likely to be involved in this capacity.     

hMAM启动子／增强子调控表达载体构建和调控作用

目的构建人乳腺珠蛋白（human mammaglobin,hMAM）启动子／增强子调控报告基因表达载体,探讨hMAM启动子／增强子序列在乳腺癌细胞中的特异性调控作用。方法应用PCR技术,从基因组DNA中扩增出hMAM启动子／增强子DNA序列,构建于PGL3报告基因上游,分别转染体外培养的乳腺癌细胞MDA—MB-415、T47D及胃癌细胞7901,分析启动子和增强子序列对乳腺癌细胞的基因表达调控作用。结果酶切图谱分析、DNA序列测定表明成功构建hMAM启动子／增强子调控的表达载体;荧光素酶报告基因检测结果分析表明,hMAM启动子／增强子能够调控报告基因的表达。结论hMAM启动子／增强子,在MDA—MB-415乳腺癌细胞具有调控基因表达的作用;