甲状腺激素在两栖动物变态过程中的作用

两栖动物的幼体变态是研究甲状腺激素调节组织和器官重构的理想模式。本文主要综述了近年来两栖动物甲状腺激素合成过程中3种脱碘酶D1、D2和D3的特点及其生物学功能;甲状腺激素受体的蛋白结构、类型和机能;以及甲状腺激素对两栖动物幼体变态过程中各个类型组织和器官重构的调节;甲状腺激素、甲状腺激素受体和脱碘酶的互作,并展望了今后的研究方向。     

Gene Regulation by Thyroid Hormone During Amphibian Metamorphosis: Implications on the Role of Cell-Cell and Cell-Extracellular Matrix Interactions

SYNOPSIS. Amphibian metamorphosis is the developmental processinitiated by thyroid hormone which transforms a tadpole intoa frog. This transformation requires extensive remodeling ofalmost every tissue in the animal. One of the more well-studiedtadpole tissues that undergoes remodeling is the small intestine.This tissue requires a shortening in length as well as internalanatomical restructuring to function in the adult frog. Briefly,the tadpole epithelial cells undergo programmed cell death (orapoptosis) and are replaced by a layer of newly formed adultepithelium. About 20 thyroid hormone-regulated genes participatingin this intestinal remodeling have been identified. These genescan be divided into several groups based on the proposed functionsof their products. One of these groups contains several secretedand/or signaling molecules. Most prominent among these are theXenopus homologs of the hedgehog and stromelysin-3 genes. Basedon the expression profiles and cellular localization, hedgehogappears to be involved in adult epithelial morphogenesis. Stromelysin-3may participate in basal lamina modification which is potentiallyinvolved in the apoptosis of the larval epithelium and developmentof the adult epithelium. Here we will review in detail the potentialroles for these secreted factors as well as the proposed molecularmechanisms responsible for their physiological functions. Furthermore,we will examine the effect of these proteins on the extracellularenvironment and how this impacts upon cellular processes involvedin intestinal remodeling.     

Steroids as Potential Modulators of Thyroid Hormone Activity in Anuran Metamorphosis

SYNOPSIS. Anuran metamorphosis is controlled by the complexinteraction of several hormones. Although thyroid hormone isthe major stimulatory hormone in metamorphosis and likely regulatesall of the metamorphic genes directly, other hormones are involvedin regulating thyroid hormone secretion and activity. Corticoids(in particular corticosterone) and the sex steroids (especially17ß estradiol) all potentially regulate thyroid hormoneactivity both by affecting hypothalamic and pituitary controlof thyroid hormone secretion and by interacting with thyroidhormones peripherally. Although there is likely a role for endogenouscorticoids in anuran metamorphosis, the role for endogenousestrogens remains to be shown. The role of these "modulators"must be fully understood, if our understanding of hormonal controlof metamorphosis is to be complete.     

中国林蛙蝌蚪变态过程中甲状腺的发育

两栖动物幼体变态过程是受甲状腺激素所精密调控的。对中国林蛙Rana chensinensis变态前期、临近变态期、变态高峰期和变态完成期的蝌蚪进行了外部形态指标(全长、体长、尾长和后肢长等)的测定,采用解剖和组织学方法对其甲状腺进行了组织学观察,并对蝌蚪外部形态指标与甲状腺形态机能之间的相关性进行了统计分析。结果显示,中国林蛙蝌蚪甲状腺分泌甲状腺激素机能活性的峰值出现于变态高峰期的前肢伸出期。统计分析表明,蝌蚪外部形态指标全长和后肢长的发育信息也可以反映其甲状腺分泌甲状腺激素的机能活性。     

Protein Changes in Regressing Tail Tissue of Xenopus laevis during Natural Metamorphosis

Sodium dodecyl sulfate (SDS) gel electrophoresis was used to study the soluble protein fraction of Xenopus laevis tail tissue during in vivo metamorphosis. Prior to morphological signs of tail regression stage 45, a new subunit protein was resolved. At stage 64 three additional subunit proteins were resolved at the end of tail resorption. Results indicate that the altered balance between protein synthesis and degradation has little effect on the protein subunit population prior to morphological signs of tail regression.     

Steroid Hormone Regulation and Tissue-Specific Expression of the Human GnRH Gene in Cell Culture and Transgenic Animals

In order to study the molecular mechanisms involved in the control of GnRH gene expression, the human GnRH gene was cloned and characterized. The gene was expressed in cells obtained from CNS tumors in transgenic mice generated utilizing 1131 bp of 5' flanking GnRH DNA fused to the simian virus 40 large T antigen. We have shown a stimulatory estrogen response element in the human GnRH gene by transient transfection studies. DNase I footprinting and an avidinbiotin DNA binding assay demonstrated that the human GnRH gene bound ER. The GN cell line was found to have nuclear ERs utilizing an ¹²⁵I estradiol binding study and by in situ hybridization histochemistry. In order to study GnRH expression in vivo, either 5000 or 484 bp of GnRH flanking DNA was fused to the luciferase (Luc) reporter gene, and transgenic mice generated. Expression in the transgenic animals was found in the hypothalamus of animals bearing the -500Luc transgene, but not in animals bearing the -484Luc transgene. The transgenic mice expressing the -5000Luc gene were gonadectomized resulting in a 20-30% increase in hypothalamic Luc expression in the males and a 65% increase in females, while mice who were gonadectomized and replaced with testosterone (males) or E₂ (females) showed a 50% decrease in Luc expression over control levels. Thus, these studies present in vitro evidence of E₂ modulation of GnRH gene expression and an in vivo model in which sensitive studies of GnRH regulation and expression can be performed.     

Thyroid Hormone-Regulated Wnt5a/Ror2 Signaling Is Essential for Dedifferentiation of Larval Epithelial Cells into Adult Stem Cells in the Xenopus laevis Intestine

Background and Aims

Amphibian intestinal remodeling, where thyroid hormone (T3) induces some larval epithelial cells to become adult stem cells analogous to the mammalian intestinal ones, serves as a unique model for studying how the adult stem cells are formed. To clarify its molecular mechanisms, we here investigated roles of non-canonical Wnt signaling in the larval-to-adult intestinal remodeling during Xenopus laevis metamorphosis.

Methods/Findings

Our quantitative RT-PCR (qRT-PCR) and immunohistochemical analyses indicated that the expressions of Wnt5a and its receptors, frizzled 2 (Fzd2) and receptor tyrosine kinase-like orphan receptor 2 (Ror2) are up-regulated by T3 and are spatiotemporally correlated with adult epithelial development in the X. laevis intestine. Notably, changes in morphology of larval absorptive epithelial cells expressing Ror2 coincide well with formation of the adult stem cells during metamorphosis. In addition, by using organ cultures of the tadpole intestine, we have experimentally shown that addition of exogenous Wnt5a protein to the culture medium causes morphological changes in the larval epithelium expressing Ror2 even in the absence of T3. In contrast, in the presence of T3 where the adult stem cells are formed in vitro, inhibition of endogenous Wnt5a by an anti-Wnt5a antibody suppressed the epithelial morphological changes, leading to the failure of stem cell formation.

Significance

Our findings strongly suggest that the adult stem cells originate from the larval absorptive cells expressing Ror2, which require Wnt5a/Ror2 signaling for their dedifferentiation accompanied by changes in cell morphology.     

Immunolocalization of Fibronectin and Laminin in the Amphibian Intestine During Spontaneous and Triiodothyronine-Induced Metamorphosis

Fibronectin and laminin were detected by indirect immunofluorescence in the intestine of Alytes obstetricans (anuran amphibian) during triiodothyronine (T3)-induced metamorphosis and spontaneous post-embryonic development. Fibronectin was first detected between a small number of connective tissue cells. As T3-treatment and spontaneous development progressed, fibronectin became detectable as a fine network extending throughout the whole thickness of the connective tissue and particularly in the core of the developing epithelial folds. During the first week of T3-treatment and throughout the spontaneous larval period, laminin was present as a linear band within the basement membrane. Between day 6 and 12 of hormonal treatment, an increase in the laminin fluorescent staining was noted. After hormonal treatment for two weeks and at the end of spontaneous metamorphosis, laminin staining was localized within the basement membrane of the folded epithelium and around muscle fibers. These observations indicate that variations in the density and distribution of extracellular matrix molecules are closely related spatiotemporarily to the structural changes occurring in the connective and muscle tissues of the intestine during metamorphosis.     

Thyroid Hormone Induces Apoptosis in Primary Cell Cultures of Tadpole Intestine: Cell Type Specificity and Effects of Extracellular Matrix

Thyroid hormone (T₃ or 3,5,3′-triiodothyronine) plays a causative role during amphibian metamorphosis. To investigate how T₃ induces some cells to die and others to proliferate and differentiate during this process, we have chosen the model system of intestinal remodeling, which involves apoptotic degeneration of larval epithelial cells and proliferation and differentiation of other cells, such as the fibroblasts and adult epithelial cells, to form the adult intestine. We have established in vitro culture conditions for intestinal epithelial cells and fibroblasts. With this system, we show that T₃ can enhance the proliferation of both cell types. However, T₃ also concurrently induces larval epithelial apoptosis, which can be inhibited by the extracellular matrix (ECM). Our studies with known inhibitors of mammalian cell death reveal both similarities and differences between amphibian and mammalian cell death. These, together with gene expression analysis, reveal that T₃ appears to simultaneously induce different pathways that lead to specific gene regulation, proliferation, and apoptotic degeneration of the epithelial cells. Thus, our data provide an important molecular and cellular basis for the differential responses of different cell types to the endogenous T₃ during metamorphosis and support a role of ECM during frog metamorphosis.