Expression of Regulatory Homeobox Genes during Retina Regeneration in Adult Newts

We studied molecular-genetic mechanisms of retina regeneration in amphibians and, specifically, expression of the homeobox genes Pax6, Prox1, and Six3 in normal development and during retina regeneration in the newt. Based on the structural analysis of genes in closely related amphibian species, primers were constructed that flank certain regions of these genes. PCR fragments of calculated length were obtained. The relationship of PCR products to the above genes was confirmed by sequencing. A comparative PCR analysis of expression of Pax6, Prox1, and Six3 was carried out in the native and regenerating newt retina, which allowed estimation of the level of expression. cDNA libraries obtained from the native and regenerating retina were used as templates. The libraries were preliminary standardized according to glyceraldehydes-3-phosphate dehydrogenase, an enzyme of general cell metabolism. The genes we studied were expressed in both native and regenerating retina. The level of Pax6 and Prox1 expression increased during regeneration, while that of Six3 decreased. The decrease in the level of Six3 expression could be due to antagonistic interrelations of Prox1 and Six3. The changed level of Prox1 and Six3 expression is a new fact and requires further studies. The interactions between these and other regulatory genes and localization of their expression in the cells of native and regenerating retina will be studied using in situ hybridization and immunohistochemistry.     

Pax6-dependence of Six3, Eya1 and Dach1 expression during lens and nasal placode induction

The Drosophila eyeless gene plays a central role in fly eye development and controls a subordinate regulatory network consisting of the so, eya and dac genes. All three genes have highly conserved mammalian homologs, suggesting possible conservation of this eye forming regulatory network. sine oculis (so) belongs to the so/Six gene family, and Six3 is prominently expressed in the developing mammalian eye. Eya1 and Dach1 are mammalian homologs of eya and dac, respectively, and although neither Eya1 nor Dach1 knockout mice express prenatal eye defects, possibilities exist for postnatal ocular phenotypes or for functional redundancy between related family members. To examine whether expression relationships analogous to those between ey, so, eya and dac exist in early mammalian oculogenesis, we investigated Pax6, Six3, Eya1 and Dach1 protein expression in murine lens and nasal placode development. Six3 expression in the pre-placode lens ectoderm is initially Pax6-independent, but subsequently both its expression and nuclear localization become Pax6-dependent. Six3, Dach1 and Eya1 nasal expression in pre-placode ectoderm are also initially Pax6-independent, but thereafter become Pax6-dependent. Pax6, Six3, Dach1 and Eya1 are all co-expressed in the developing ciliary marginal zone, a source of retinal stem cells in some vertebrates. An in vitro protein-protein interaction is detected between Six3 and Eya1. Collectively, these findings suggest that the Pax-Eya-Six-Dach network is at best only partly conserved during lens and nasal placode development. However, the findings do not rule out the possibility that such a regulatory network acts at later stages of oculogenesis.     

Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo

In mammals, Six5, Six4 and Six1 genes are co-expressed during mouse myogenesis. Six4 and Six5 single knockout (KO) mice have no developmental defects, while Six1 KO mice die at birth and show multiple organ developmental defects. We have generated Six1Six4 double KO mice and show an aggravation of the phenotype previously reported for the single Six1 KO. Six1Six4 double KO mice are characterized by severe craniofacial and rib defects, and general muscle hypoplasia. At the limb bud level, Six1 and Six4 homeogenes control early steps of myogenic cell delamination and migration from the somite through the control of Pax3 gene expression. Impaired in their migratory pathway, cells of the somitic ventrolateral dermomyotome are rerouted, lose their identity and die by apoptosis. At the interlimb level, epaxial Met expression is abolished, while it is preserved in Pax3-deficient embryos. Within the myotome, absence of Six1 and Six4 impairs the expression of the myogenic regulatory factors myogenin and Myod1, and Mrf4 expression becomes undetectable. Myf5 expression is correctly initiated but becomes restricted to the caudal region of each somite. Early syndetomal expression of scleraxis is reduced in the Six1Six4 embryo, while the myotomal expression of Fgfr4 and Fgf8 but not Fgf4 and Fgf6 is maintained. These results highlight the different roles played by Six proteins during skeletal myogenesis.     

金鱼Prox1基因cDNA的克隆及其在眼睛发育中的表达分析

脊椎动物的Prox1基因,与果蝇的转录因子prospero同源。为了探讨Prox1基因在金鱼眼睛发生过程中的表达图式,我们从金鱼眼睛SMART库中克隆了Prox1cDNA。它全长共2851bp,编码739个氨基酸。组织分布研究表明,Prox1主要分布于眼、脑、心、肝、脾和肾中。整体原位杂交显示,Prox1mRNA首先是在晶体期的晶体原基中有转录,心跳期则在未成熟晶体的细胞中和视网膜的幼芽区可以检测到。晶体纤维形成后,它主要定位于视纤维层和内网织细胞层。免疫组化显示,心跳期Prox1蛋白的定位与mRNA相同,晶体纤维形成以后,Prox1蛋白主要定位在晶体上皮细胞内侧的晶体纤维上一个环状区域,与Prox1mRNA的定位不同。这说明,Prox1基因在晶体发生过程中有重要作用,且在晶体的不同发育时期起的作用可能有所不同。另外,Prox1在晶体发育过程中有一个从内向外的变化过程。     

牡丹开花相关SWEET家族基因生物信息学与表达模式分析

为了揭示SWEET家族基因在牡丹开花过程中的作用,该研究以‘洛阳红’牡丹花瓣转录组数据库为基础,利用生物信息学方法对SWEET家族基因进行鉴定和分析。结果表明:(1)实验共得到10个具有完整开放阅读框的牡丹SWEET基因。(2)牡丹SWEET家族成员蛋白等电点、消光系数等差别不大,其中5个牡丹SWEET基因编码的蛋白为不稳定蛋白;亚细胞定位预测这10个牡丹SWEET基因编码的蛋白均定位在细胞膜;进化树分析显示,牡丹SWEET基因与拟南芥亲缘关系更近;结构分析表明,牡丹SWEET蛋白结构在进化过程中非常保守,这10个牡丹SWEET蛋白同时具有5个相同的Motif且均含2个MtN3 saliva结构域。(3)可溶性糖含量分析显示,从小风铃期到盛花期,牡丹花瓣中蔗糖含量不断降低,果糖和葡萄糖含量不断升高,均在盛花期达到峰值。(4)qRT-PCR分析显示,PsSWEET4盛花期表达量是小风铃期的34倍,PsSWEET8盛花期表达量是小风铃期的60倍。结合这2个基因表达与进化关系及可溶性糖变化值,初步推测PsSWEET4和PsSWEET8在开花过程中可能通过调控果糖、葡萄糖的转运而间接调控开花过程;该结果为进一步研究PsSWEET基因在牡丹生长发育过程中的功能奠定了基础。     

Gene expression analysis of Six3, Pax6, and Otx in the early development of the stalked crinoid Metacrinus rotundus

The stalked crinoid, Metacrinus rotundus, is one of the most basal extant echinoderms. Here, we show the expression patterns of Six3, Pax6, and Otx in the early development of M. rotundus. All three genes are highly expressed in stages from the gastrula to the auricularia larval stage. Ectodermal expression of MrOtx appears to be correlated with development of the ciliary band. These three genes are expressed sequentially along the embryonic body axis in the anterior and middle walls of the archenteron in the order of MrPax6, MrSix3, and MrOtx. The anterior, middle, and posterior parts of the archenteron in the late gastrula differentiate into the axo-hydrocoel, the enteric sac, and somatocoels at later stages, respectively. The three genes are expressed sequentially from the tip of the axo-hydrocoel to the bottom of enteric sac in the order of MrSix3, MrPax6, and MrOtx at the later stages. This suggests that these genes are involved in patterning of the larval endo-mesoderm in stalked crinoids. The present results suggest that radical alterations have occurred in the expression and function of homeobox genes in basal echinoderms.