Expression analysis of chick Wnt and frizzled genes and selected inhibitors in early chick patterning.

Wnt signaling is an important component in patterning the early embryo and specifically the neural plate. Studies in Xenopus, mouse, and zebrafish have shown that signaling by members of the Wnt family of secreted signaling factors, their Frizzled receptors and several inhibitors (sFRP1, sFRP2, sFRP3/Frzb1, Crescent/Frzb2, Dkk1, and Cerberus) are involved. However, very little is known about the expression of genes in the Wnt signaling pathway during early anterior neural patterning in chick. We have performed an expression analysis at neural plate stages of several Wnts, Frizzled genes, and Wnt signaling pathway inhibitors using in situ hybridization. The gene expression patterns of these markers are extremely dynamic. We have identified two candidate molecules for anterior patterning of the neural plate, Wnt1 and Wnt8b, which are expressed in the rostral ectoderm at these stages. Further functional studies on the roles of these markers are underway.     

Xenopus Zic4: conservation and diversification of expression profiles and protein function among the Xenopus Zic family.

We compared the expression and function of Xenopus Zic4 with those of the other four Xenopus laevis Zic family members (Zic1, Zic2, Zic3, and Zic5). Zic4 expression was detected mainly in the neural plate border, dorsal neural tube, and somites, and was similar to that of Zic1, which is adjacent to Zic4 on the same chromosome. Injection of wild-type or mutant Zic4 RNA caused the induction of neural crest marker gene expression, hyperplastic neural tissue, and ectopic pigment cell formation, indicating that Zic4 can induce neural and neural crest tissue, as can other Xenopus Zic genes. Deletion analysis showed that the zinc-finger domain is critical for many Zic4 functions, but the C-terminal region is differently involved in induction of two neural crest markers, Slug and Sox10. The protein function as determined by the animal cap explant assay was similar to that of Zic5, but different from those of Zic1, Zic2, and Zic3, suggesting that Xenopus Zic genes can be divided into two groups based on function. These results indicate that the five Xenopus Zic genes cooperatively regulate both neural and neural crest development, despite significantly diverged expression profiles and functions.     

Dlx基因表达改变可影响颅神经嵴细胞迁移及第一鳃弓的发育？

背景：Dlx基因在颅神经嵴细胞中高表达,并调控颅神经嵴细胞的迁移及分化。目的：综述Dlx基因高表达对颅神经嵴细胞迁移及其分化的影响及机制。方法：由第一作者用计算机检索全国期刊全文数据库(CNKI),Medline数据库,检索词分别为“神经嵴细胞,颅神经嵴细胞迁移,Dlx,Dlx高表达,Fgf,成软骨,成骨”和“cranial neural crest cells, cranial neural crest cells’migration, Dlx, Dlx overexpression, Fgf, chodrogenesis, osteogenesis”。从颅神经嵴细胞的迁移,Dlx表达改变对颅神经嵴细胞迁移的影响,Dlx基因与细胞环境的相互作用3个方面进行总结。共检索到63篇文章,按纳入和排除标准对文献进行筛选,共纳入43篇文章。结果与结论：Dlx基因表达改变导致细胞间黏附因子表达的改变,高表达会使得绝大多数颅神经嵴细胞聚集,错误迁移,致使畸形的发生。并且Dlx基因表达增高,也将导致骨或软骨的异常生成,其原因可能在于相关细胞因子间的相互作用。     

Expression cloning in Xenopus identifies RNA-binding proteins as regulators of embryogenesis and Rbmx as necessary for neural and muscle development.

We have performed an expression cloning screen in Xenopus laevis with the aim of isolating novel gene activities from the neural plate. Of 8,064 clones screened, we isolated 61 clones that affected either neural plate patterning or tadpole morphology. Of these, 20 clones encoded RNA binding proteins, and the majority of these are heterogeneous nuclear ribonucleoproteins (hnRNPs) or SR-proteins, which are associated with alternative splicing. All of these genes are expressed in the nervous system, and in several cases specific to neural tissue. Injecting mRNA encoding these proteins results in neural plate mispatterning and abnormal muscle segmentation. To initiate characterization of these proteins, we selected Rbmx as a candidate for deeper analysis. Using morpholino mediated knockdown, we show that Rbmx is necessary for normal anterior neural plate patterning, neurogenesis, neural crest development, and muscle segmentation.     

A novel, activin-inducible, blastopore lip-specific gene of Xenopus laevis contains a fork head DNA-binding domain.

The organizer region, or dorsal blastopore lip, plays a central role in the initiation of gastrulation and the formation of the body axis during Xenopus development. A similar process can also be induced in ectodermal explants by activin or by injection of activin mRNA into embryos. We have searched early embryo-specific cDNA libraries for genes containing the fork head box sequence that encodes a DNA-binding domain similar to that of the Drosophila homeotic gene fork head and rat hepatocyte nuclear factor HFN3 beta. These genes were subsequently tested for expression in the organizer region of blastula/gastrula-stage embryos as well as inducibility by activin. Our effort resulted in the isolation of a gene, XFKH1, that is primarily expressed in the dorsal blastopore lip of early gastrulae and is inducible by activin. At later stages it is expressed in the notochord and neural floor plate. Because of its spatial and temporal expression pattern, as well as its inducibility by activin, this gene is a good candidate to have a regulatory function in the initial processes of axis formation in Xenopus laevis embryos.     

Pilot morpholino screen in Xenopus tropicalis identifies a novel gene involved in head development.

The diploid frog X. tropicalis has recently been adopted as a model genetic system, but loss-of-function screens in Xenopus have not yet been performed. We have undertaken a pilot functional knockdown screen in X. tropicalis for genes involved in nervous system development by injecting antisense morpholino (MO) oligos directed against X. tropicalis mRNAs. Twenty-six genes with primary expression in the nervous system were selected as targets based on an expression screen previously conducted in X. laevis. Reproducible phenotypes were observed for six and for four of these, a second MO gave a similar result. One of these genes encodes a novel protein with previously unknown function. Knocking down this gene, designated pinhead, results in severe microcephaly, whereas, overexpression results in macrocephaly. Together with the early embryonic expression in the anterior neural plate, these data indicate that pinhead is a novel gene involved in controlling head development.     

Expression and functional analysis of Tgif during mouse midline development.

The Tgif gene encodes a homeodomain protein that functions as a transforming growth factor beta (TGF-beta) repressor by binding to Smad2. Mutations in the TGIF gene are associated with human holoprosencephaly, a common birth defect caused by the failure of anterior ventral midline formation. However, Smad2-mediated TGF-beta signaling in the axial mesendoderm has been demonstrated to be essential for ventral midline formation, and loss of a Smad2 antagonist should in principle promote rather than inhibit ventral midline formation. This suggests a more complex mechanism for the function of TGIF in controlling ventral midline formation. To explore the role of TGIF in ventral forebrain formation and patterning, we investigated Tgif expression and function during mouse development by in situ hybridization and gene targeting. We found that Tgif is highly expressed in the anterior neural plate, consistent with the proposed neural differentiation model in which TGF-beta suppression is required for normal neural differentiation. This result suggests a possible role for Tgif in anterior neural differentiation and patterning. However, targeted disruption of the Tgif gene during mouse development does not cause any detectable defects in development and growth. Both histological examination and gene expression analysis showed that Tgif-/- embryos have a normal ventral specification in the central nervous system, including the forebrain region. One interpretation of these results is that the loss of TGIF function is compensated by other TGF-beta antagonists such as c-Ski and SnoN during vertebrate anterior neural development.     

Conserved cross-interactions in Drosophila and Xenopus between Ras/MAPK signaling and the dual-specificity phosphatase MKP3.

The extracellular signal-regulated kinase (ERK) is a key transducer of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) signaling pathways, and its function is required in multiple processes during animal development. The activity of ERK depends on the phosphorylation state of conserved threonine and tyrosine residues, and this state is regulated by different kinases and phosphatases. A family of phosphatases with specificity toward both threonine and tyrosine residues in ERK (dual-specificity phosphatases) play a conserved role in its dephosphorylation and consequent inactivation. Here, we characterize the function of the dual-specificity phosphatase MKP3 in Drosophila EGFR and Xenopus FGFR signaling. The function of MKP3 is required during Drosophila wing vein formation and Xenopus anteroposterior neural patterning. We find that the expression of the MKP3 gene is localized in places of high EGFR and FGFR signaling. Furthermore, this restricted expression depends on ERK function both in Drosophila and Xenopus, suggesting that MKP3 constitutes a conserved negative feedback loop on the activity of the Ras/ERK signaling pathway.     

XIdax, an inhibitor of the canonical Wnt pathway, is required for anterior neural structure formation in Xenopus.

Wnt signaling pathways are involved during various stages in the development of many species. In Xenopus, the accumulation of beta-catenin on the dorsal side of embryo is required for induction of the organizer, while the head structure formation requires inhibition of Wnt signaling. Here, we report a role for xIdax, a negative regulator of Wnt signaling. XIdax is expressed in neural tissues at the neurula stage, and in the restricted region of the tadpole brain. Ectopic expression of xIdax inhibits the target gene expression, suggesting that xIdax can inhibit canonical Wnt signaling. To examine the function of xIdax, a morpholino oligo for xIdax (xIdaxMO) was designed. An injection into an animal pole cell caused a loss of forebrain. The anterior neural marker expression is decreased in xIdaxMO-injected embryo, suggesting that xIdax is required for anterior neural development. Moreover, a negative regulator that acts downstream of xIdax rescued this defect. We propose that Idax functions are dependent on the canonical Wnt pathway and are crucial for the anterior neural development.     

功能性SDF-1受体在非洲爪蟾胚胎发育中的表达研究

本文目的在于探索骨髓基质细胞源因子-1(SDF-1)的功能性受体在非洲爪蟾胚胎发育过程中的表达状况,进而探讨SDF-1在胚胎发育中的作用。采用胚胎细胞分散培养及钙成像技术对不同发育时期及胚胎不同部位的分散培养细胞进行了检测,结果证明:在主要由神经板、神经嵴细胞和基板迁移而来的胚胎头部组织中,其功能性受体表达量远高于背部及尾部组织(P<0.001),并从胚胎14～21期呈上升趋势,在21～35期保持高表达率。结果提示,此功能性受体在胚胎期的表达密度与成体相应区域的器官结构复杂程度呈正相关,说明SDF-1及其受体在爪蟾的生长发育中起作用。     

Dlx5, the mouse homologue of the human-imprinted DLX5 gene,is biallelically expressed in the mouse brain

The mouse Dlx5 gene encodes a distal-less-related DNA-binding homeobox protein first expressed during early embryonic development in anterior regions of mouse embryo and is located on chromosome 6, which is the syntenic region to the human chromosome 7q21–q31 imprinting cluster. Recently, its human homologue, DLX5, was identified to be imprinted and maternally expressed, at least in normal human lymphoblasts and in brain tissues. In our study, we analyzed the imprinting status of mouse Dlx5 by RT-PCR, first in the F1 of a reciprocal cross between two different mouse strains, and second in heterozygous Dlx5 mutant mice. Both approaches revealed that mouse Dlx5 followed a biallelic pattern of expression in brain tissue and in testis. Our findings suggest that the Dlx5 gene escapes genomic imprinting, at least in mice of certain genetic backgrounds.