Characterization of the Enigma family in zebrafish.

The three Enigma subfamily proteins, Enigma, Enigma homologue, and Cypher/ZASP belong to the PDZ and LIM encoding protein family, which is characterized by the presence of a PDZ- and one or more LIM domains. PDZ/LIM proteins play important biological roles, and all members have been shown to associate with the actin cytoskeleton. We describe here the splice form specific expression patterns for the three Enigma subfamily members during zebrafish embryogenesis. Whole-mount in situ hybridization revealed common and distinct expression patterns for the different PDZ or LIM domain encoding splice variants. We further studied the role of enigma in zebrafish development. Enigma knockdown appeared to be embryonic lethal shortly after the end of gastrulation and in few surviving embryos led to elongation defects and disorganized somites. In summary, we show here the temporal and spatial expression patterns of the three Enigma family members and their PDZ and LIM domain encoding splice forms during zebrafish embryogenesis. Our results suggest that enigma is important for the formation and organization of somites and might play an important role for actin cytoskeleton organization during development.     

Identification and expression of voltage‐gated calcium channel β subunits in Zebrafish

Voltage‐gated calcium channels (VGCC) play important roles in electrically excitable cells and embryonic development. The VGCC β subunits are essential for membrane localization of the channel and exert modulatory effects on channel functions. In mammals, the VGCC β subunit gene family contains four members. In zebrafish, there appear to be seven VGCC β subunits including the previously identified β1 subunit. cDNAs for six additional VGCC β subunit homologs were identified in zebrafish, their chromosomal locations determined and their expression patterns characterized during embryonic development. These six genes are primarily expressed in the nervous system with cacnb4a also expressed in the developing heart. Sequence homology, genomic synteny and expression patterns suggest that there are three pairs of duplicate genes for β2, β3, and β4 in zebrafish with distinct expression patterns during embryonic development. Developmental Dynamics 237:3842–3852, 2008. © 2008 Wiley‐Liss, Inc.     

一个在小鼠头部特异表达的新基因BSG1 cDNA的克隆及研究

目的 克隆小鼠头部发育过程中特异表达的新基因并对新基因可能的功能进行初步分析。方法 采用消减差异筛选的方法筛选小鼠头部特异表达的新基因，并用生物信息学的方法对克隆到的新基因进行序列分析和蛋白结构域的预测。同时，还采用了小鼠胚胎原位杂交、小鼠脑部切片的原位杂交以及鸡胚的原位杂交方法研究BS61基因的表达情况。结果 克隆到1个与小鼠头部发育相关的新基因BSG1(brain specific gene 1)，其Gen Bank的登录号为AY210402。该基因包含1个2133bp的完整阅读编码框，编码1个710aa的C2H2型锌指蛋白。它与人的KIAA0441基因在氨基酸水平上有82.8％的同源性。该基因定位在小鼠的第10号染色体上，含7个外显子，6个内含子。BSG1主要在小鼠胚胎、鸡胚的头部表达，并且BSG1在小鼠头部表达的部位局限在海马、齿状回和小脑。结论 BSG1是1个可能的转录因子，在脑部特异表达。对其研究有助于进一步揭示大脑发育的分子机理。     

Cellular nonmuscle myosins NMHC‐IIA and NMHC‐IIB and vertebrate heart looping

Flectin, a protein previously described to be expressed in a left‐dominant manner in the embryonic chick heart during looping, is a member of the nonmuscle myosin II (NMHC‐II) protein class. During looping, both NMHC‐IIA and NMHC‐IIB are expressed in the mouse heart on embryonic day 9.5. The patterns of localization of NMHC‐IIB, rather than NMHC‐IIA in the mouse looping heart and in neural crest cells, are equivalent to what we reported previously for flectin. Expression of full‐length human NMHC‐IIA and ‐IIB in 10 T1/2 cells demonstrated that flectin antibody recognizes both isoforms. Electron microscopy revealed that flectin antibody localizes in short cardiomyocyte cell processes extending from the basal layer of the cardiomyocytes into the cardiac jelly. Flectin antibody also recognizes stress fibrils in the cardiac jelly in the mouse and chick heart; while NMHC‐IIB antibody does not. Abnormally looping hearts of the Nodal^{Δ 600} homozygous mouse embryos show decreased NMHC‐IIB expression on both the mRNA and protein levels. These results document the characterization of flectin and extend the importance of NMHC‐II and the cytoskeletal actomyosin complex to the mammalian heart and cardiac looping. Developmental Dynamics 237:3577–3590, 2008. © 2008 Wiley‐Liss, Inc.     

Expression of GPR177 (Wntless/Evi/Sprinter), a highly conserved Wnt‐transport protein,in rat tissues,zebrafish embryos,and cultured human cells

GPR177 is an evolutionarily conserved transmembrane protein necessary for Wnt protein secretion. Little is currently known, however, regarding expression of GPR177, especially in vertebrate species. We have developed an antiserum against GPR177, and used it to examine expression of GPR177 in human tissue culture cells, adult mouse, and rat tissues, as well as developing zebrafish embryos. In rodents, GPR177 is expressed in virtually all tissue types and brain regions examined. In zebrafish, GPR177 polypeptides are expressed throughout embryogenesis, and are detectable as early as 1 hr post‐fertilization. In situ hybridization analysis reveals that gpr177 mRNA expression is prominent in embryonic zebrafish brain and ear. Structural studies suggest that GPR177 is modified by N‐linked sugars, and that the protein contains an even number of transmembrane segments. The relatively ubiquitous expression of GPR177 suggests that this protein may serve to regulate Wnt secretion in a variety of embryonic and adult tissue types. Developmental Dynamics 239:2426–2434, 2010. © 2010 Wiley‐Liss, Inc.     

The mouse homeobox gene Not is required for caudal notochord development and affected by the truncate mutation

The floating head (flh) gene in zebrafish encodes a homeodomain protein, which is essential for notochord formation along the entire body axis. flh orthologs, termed Not genes, have been isolated from chick and Xenopus, but no mammalian ortholog has yet been identified. Truncate (tc) is an autosomal recessive mutation in mouse that specifically disrupts the development of the caudal notochord. Here, we demonstrate that truncate arose by a mutation in the mouse Not gene. The truncate allele (Nottc) contains a point mutation in the homeobox of Not that changes a conserved Phenylalanine residue in helix 1 to a Cysteine (F20C), and significantly destabilizes the homeodomain. Reversion of F20C in one allele of homozygous tc embryonic stem (ES) cells is sufficient to restore normal notochord formation in completely ES cell-derived embryos. We have generated a targeted mutation of Not by replacing most of the Not coding sequence, including the homeobox with the eGFP gene. The phenotype of NoteGFP/eGFP, NoteGFP/tc, and Nottc/tc embryos is very similar but slightly more severe in NoteGFP/eGFP than in Nottc/tc embryos. This confirms allelism of truncate and Not, and indicates that tc is not a complete null allele. Not expression is abolished in Foxa2 and T mutant embryos, suggesting that Not acts downstream of both genes during notochord development. This is in contrast to zebrafish embryos, in which flh interacts with ntl (zebrafish T) in a regulatory loop and is essential for development of the entire notochord, and suggests that different genetic control circuits act in different vertebrate species during notochord formation.     

Expression of strawberry notch family genes during zebrafish embryogenesis

Our previous study suggested a possible role for Sbno1, a mouse homologue of strawberry notch gene during brain development. In this report, we cloned the zebrafish homologues of sbno, and examined their expression pattern during embryogenesis by whole‐mount in situ hybridization. Zebrafish have three sbno genes: one Sbno1 homologue and two Sbno2 homologues, sbno2a and sbno2b. We observed that the expression of sbno1 and sbno2a was initially ubiquitous and gradually became predominant in the central nervous system as development progressed. The expression of sbno2b was observed in non‐neural tissues in contrast to the other two genes. sbno1 and sbno2a exhibited higher expression in distinct regions within the nervous system of pharyngula‐stage embryos, suggesting possible differing roles for sbno1 and sbno2a during later stages of embryogenesis. Together, the observed gene expression patterns suggest an important role of sbno‐family genes during development of the vertebrate central nervous system. Developmental Dynamics 239:1789–1796, 2010. © 2010 Wiley‐Liss, Inc.     

Enforced expression of the transcription factor HOXD3 under the control of the Wnt1 regulatory element modulates cell adhesion properties in the developing mouse neural tube

HOX genes expressed in a specific spatial and temporal manner play a crucial role in determining the body plan during the early development of vertebrates. In adult tissues, many HOX genes participate in normal hematopoiesis and carcinogenesis. We previously found that overexpression of the homeobox gene HOXD3 alters expression levels of cell adhesion molecules in human cancer cell lines. Here, we have investigated whether HOXD3 expression is related to the cell adhesion processes during mouse development focusing on dorsal midline cells or roof-plate cells of the neural tube and neural crest cells. We created transgenic mouse embryos, in which HOXD3 is expressed in the dorsal midline under the control of the Wnt1 regulatory element, and analyzed these embryos at embryonic day 10.5-13.5. In HOXD3-expressing transgenic embryos, although neural crest-derived structures in the trunk region appeared to be normal, striking abnormalities were found in the neural tube. In transgenic embryos expressing the lacZ gene under the control of the Wnt1 regulatory element, expression of lacZ was restricted to roof-plate cells within the neural tube. By contrast, in HOXD3-expressing transgenic embryos, expression of HOXD3 was not only located in the dorsal neural tube, but also had spread inside the ventricular zone in more ventral regions of the neural tube. These findings show that the HOXD3 transgene is expressed more broadly than the Wnt1 gene is normally expressed. Expression of both Wnt1 and Msx1, marker genes in the roof plate, was further extended ventrally in HOXD3-expressing embryos than in normal embryos, suggesting that expression of the HOXD3 transgene expands the roof plate ventrally within the neural tube. In the ventricular zone of HOXD3-expressing embryos at embryonic day 10.5, we observed an increase in the number of mitotic cells and failure of interkinetic nuclear migration of progenitor cells. Furthermore, in HOXD3-expressing embryos at embryonic day 12.5, the ventricular zone, in which progenitor cells became more loosely connected to each other, was composed of a large number of cells that did not express N-cadherin. Our results indicate that expression of HOXD3 is closely associated with modulation of cell-adhesive properties during embryonic development.