Differentiation of Extracutaneous Melanocytes in Embryos of the Turtle,Trionyx sinensis japonicus

The present study investigates the mode of differentiation of neural crest-derived melanocytes in the embryos of the soft-shell turtle, Trionyx sinensis japonicus. DOPA reaction-positive melanoblasts were first detected in 10-day-old embryos. Melanocyte differentiation in terms of pigmentation takes place from the day 16 of development. Melanin pigments were found in the dorsal integument as well as in various extracutaneous tissues such as skeletal muscle, dorsal aorta, peritoneum, blood vessels, choroid, lung, bone marrow, fat tissues and in the connective tissue of the nose. These results suggest the presence of a specific environmental regulation of the melanoblast differentiation in the soft-shell turtle.     

鼋骨骼系统及与中华鳖比较

采用虫蚀法制备了鼋Pelochelys cantorii和中华鳖Pelodiscus sinensis的骨骼标本, 对骨骼系统进行了观察、描述、绘图及比较分析。结果显示, 鼋的骨骼共169枚, 由背甲和腹甲组成的外骨骼、中轴骨和附肢骨组成的内骨骼组成。通过比较鼋和中华鳖的骨骼结构, 发现二者在头骨的吻突长度及第三颈椎结构方面有较大差别。鼋眼眶前部至吻突最前端的长度与头骨总长度比为0.082, 而中华鳖为0.570, 显示中华鳖吻突显著长于鼋。鼋与中华鳖的颈椎骨数目均为9枚, 但中华鳖的第三至第九颈椎的横突要更明显, 第七颈椎的椎体向上显著翘起, 且第九颈椎腹面椎体前端为尖状。从整条颈椎上看, 鼋脊椎长度与其背甲长度之比为0.66, 中华鳖为1.07, 表明中华鳖的颈椎更长; 研究结果丰富了鳖科动物的骨骼学基础数据, 也为鼋物种鉴定、龟鳖动物系统演化及生态适应性提供骨骼学理论依据。     

中华鳖vasa基因克隆及在卵母细胞中的表达分析

研究利用中华鳖为研究模型进行爬行类生殖细胞发育分化成熟等生物学研究,克隆了中华鳖vasa基因的cDNA序列,全长3865 bp,包括5'端非编码区90 bp,3'端非编码区1699 bp,开放阅读框长2076 bp,共编码691个氨基酸。中华鳖Vasa氨基酸序列包含DEAD-box家族蛋白8个保守保守功能域,在N末端有4个RGG重复序列和2个GG富集区,与小鼠Vasa蛋白的同源性较高（72%）。荧光定量PCR的结果表明,中华鳖vasa mRNA主要精巢和卵巢中表达,其他体组织中均难检测到表达。卵巢冰冻切片原位杂交结果显示:中华鳖vasa mRNA在生殖细胞中特异表达;在卵子发生过程中的不同发育期卵母细胞中呈现动态的变化。即vasa mRNA在初级卵母细胞及生长期卵母细胞中表达最强,且均匀分布在细胞质中,随着卵母细胞的逐渐增大,信号逐渐减弱,直至在成熟的卵母细胞中几乎检测不到表达信号,说明vasa可能在中华鳖早期卵母细胞发育中起重要作用。同时,vasa基因可作为中华鳖生殖细胞分子标记物,根据其mRNA的表达水平来鉴别不同发育时期的卵母细胞。研究结果为进一步开展中华鳖胚胎生殖细胞发育及配子生成,特别是研究中华鳖,乃至爬行类原始生殖细胞（Primordial Germ Cells,PGCs）的起源、迁移、分化等研究奠定了基础。     

Hepatocyte growth factor is crucial for development of the carapace in turtles

Turtles are characterized by their shell, composed of a dorsal carapace and a ventral plastron. The carapace first appears as the turtle-specific carapacial ridge (CR) on the lateral aspect of the embryonic flank. Accompanying the acquisition of the shell, unlike in other amniotes, hypaxial muscles in turtle embryos appear as thin threads of fibrous tissue. To understand carapacial evolution from the perspective of muscle development, we compared the development of the muscle plate, the anlage of hypaxial muscles, between the Chinese soft-shelled turtle, Pelodiscus sinensis, and chicken embryos. We found that the ventrolateral lip (VLL) of the thoracic dermomyotome of P. sinensis delaminates early and produces sparse muscle plate in the lateral body wall. Expression patterns of the regulatory genes for myotome differentiation, such as Myf5, myogenin, Pax3, and Pax7 have been conserved among amniotes, including turtles. However, in P. sinensis embryos, the gene hepatocyte growth factor (HGF), encoding a regulatory factor for delamination of the dermomyotomal VLL, was uniquely expressed in sclerotome and the lateral body wall at the interlimb level. Implantation of COS-7 cells expressing a HGF antagonist into the turtle embryo inhibited CR formation. We conclude that the de novo expression of HGF in the turtle mesoderm would have played an innovative role resulting in the acquisition of the turtle-specific body plan.     

中华鳖GHSR基因SNPs的筛选及生长性状的关联分析

为探究GHSR基因多态性对中华鳖(Pelodiscus sinensis)生长相关性状的影响,采用直接测序法在GHSR基因5'侧翼和3'侧翼上筛选SNPs位点,共检测到5个单核苷酸多态性位点:A335T、G397T、A527G、A13482C和T13526A。随机选取同批繁殖的1冬龄200只中华鳖用直接测序法进行SNPs位点的分型,并分析与生长性状的相关性。检测结果显示,所有SNP位点均符合Hardy-Weinberg平衡状态(p>0.05)。方差分析显示,A336T位点的AT、TT基因型的体重、背甲长、背甲宽和裙边宽4项生长数据均显著高于AA基因型。A13482C位点的AC基因型的体重、背甲长、背甲宽和裙边宽4项数据均显著高于AA基因型(p<0.05)。研究表明,本实验在GHSR基因上获得的这些SNP位点可能影响着中华鳖的生长性状或与之紧密连锁,可为中华鳖分子辅助育种提供助力与参考。     

Mesoderm differentiation in explants of carp embryos

An analysis of carp blastoderm development was carried out in culture after isolation from the yolk cell and its yolk syncytial layer (YSL). The blastoderms were separated from the YSL at four different stages of embryogenesis: the blastula, early epiboly, early gastrula and late gastrula stages. Absence of the YSL in explants was checked by scanning electron microscopy. From observations of living embryos and histological examination of tissues which were formed in explants from all stages studied it was observed that they contained notochordal, muscle and neural tissue as signs of dorsal types of differentiation. Only in explants from the early and late gastrula stages were histotypical tissues organized in an embryonic-like body pattern. The data indicate that mesoderm differentiation in fish embryos is independent from the YSL, contrary to normal pattern formation which needs the presence of the YSL before the onset of gastrulation.     

Protein synthesis in dorsal and ventral regions of Xenopus laevis embryos in relation to dorsal and ventral differentiation

Two-dimensional gel electrophoresis has been used to analyze protein synthesis in dorsal and ventral regions in embryonic stages of Xenopus laevis. Proteins specific either to dorsal or to ventral regions are synthesized for the first time at gastrulation, concomitant with morphological differentiation. The reliability of these proteins as markers of dorsal and ventral differentiation was tested by examining their synthesis in Uv-irradiated embryos, which have severely reduced capacity for dorsal development, reflected in reduced levels of the neuromuscular-specific enzyme acetylcholinesterase, but which continue to synthesize the great majority of proteins at normal rates. Synthesis of dorsal indicator proteins should be reduced or absent in these embryos, whereas ventral indicators should be synthesized at least to the same extent as in control embryos. Some of the putative dorsal and ventral indicators failed this test, but the majority were confirmed as reliable markers of dorsal and ventral differentiation, thus providing a connection between morphology and gene expression in the establishment of the dorsal-ventral axis in X. laevis.     

Effects of local tissue environment on the differentiation of neural crest cells in turtle, with special reference to understanding the spatial distribution of pigment cells

The spatial distribution of neural crest-derived pigment cells in turtles differs markedly from those found in chickens and mice. One hypothesis to explain such differences in the spatial distribution of pigment cells is that local tissue factors interact with neural crest cells, thereby determining their differentiation into pigment-synthesizing cells. It is reported here that local tissue factors in the soft-shell turtle (Trionyx sinensis japonicus) play a critical role in the development of melanophores from neural crest cells during embryogenesis. Undifferentiated neural crest cells derived from trunk neural tubes were co-cultured in vitro with homochronous somites, or with heterochronous dermis, lung or liver for 14 days. Melanophore differentiation from neural crest cells was significantly promoted when co-cultured with cells from lung, somites or dermis, but not when co-cultured with liver cells. These results suggest that local tissue factors stimulate the differentiation of pluripotent neural crest derivatives toward pigment cells. It is proposed that specific environmental cues play an important role in the spatial distribution of pigment cells in a variety of vertebrate species.     

Subdivision of the cardiac Nkx2.5 expression domain into myogenic and nonmyogenic compartments

Nkx2.5 is expressed in the cardiogenic mesoderm of avian, mouse, and amphibian embryos. To understand how various cardiac fates within this domain are apportioned, we fate mapped the mesodermal XNkx2.5 domain of neural tube stage Xenopus embryos. The lateral portions of the XNkx2.5 expression domain in the neural tube stage embryo (stage 22) form the dorsal mesocardium and roof of the pericardial cavity while the intervening ventral region closes to form the myocardial tube. XNkx2.5 expression is maintained throughout the period of heart tube morphogenesis and differentiation of myocardial, mesocardial, and pericardial tissues. A series of microsurgical experiments showed that myocardial differentiation in the lateral portion of the field is suppressed during normal development by signals from the prospective myocardium and by tissues located more dorsally in the embryo, in particular the neural tube. These signals combine to block myogenesis downstream of XNkx2.5 and at or above the level of contractile protein gene expression. We propose that the entire XNkx2.5/heart field is transiently specified as cardiomyogenic. Suppression of this program redirects lateral cells to adopt dorsal mesocardial and dorsal pericardial fates and subdivides the field into distinct myogenic and nonmyogenic compartments.     

The epithelium of the dorsal marginal zone of Xenopus has organizer properties.

We have investigated the properties of the epithelial layer of the dorsal marginal zone (DMZ) of the Xenopus laevis early gastrula and found that it has inductive properties similar to those of the entire Spemann organizer. When grafts of the epithelial layer of the DMZ of early gastrulae labelled with fluorescein dextran were transplanted to the ventral sides of unlabelled host embryos, they induced secondary axes composed of notochord, somites and posterior neural tube. The organizer epithelium rescued embryos ventralized by UV irradiation, inducing notochord, somites and posterior neural tube in these embryos, while over 90% of ventralized controls showed no such structures. Combinations of organizer epithelium and ventral marginal zone (VMZ) in explants of the early gastrula resulted in convergence, extension and differentiation of dorsal mesodermal tissues, whereas similar recombinants of nonorganizer epithelium and the VMZ did none of these things. In all cases, the axial structures forming in response to epithelial grafts were composed of labelled graft and unlabelled host cells, indicating an induction by the organizer epithelium of dorsal, axial morphogenesis and tissue differentiation among mesodermal cells that otherwise showed non-axial development. Serial sectioning and scanning electron microscopy of control grafts shows that the epithelial organizer effect occurs in the absence of contaminating deep cells adhering to the epithelial grafts. However, labelled organizer epithelium grafted to the superficial cell layer contributed cells to deep mesodermal tissues, and organizer epithelium developed into mesodermal tissues when deliberately grafted into the deep region. This shows that these prospective endodermal epithelial cells are able to contribute to mesodermal, mesenchymal tissues when they move or are moved into the deep environment. These results suggest that in normal development, the endodermal epithelium may influence some aspects of the cell motility underlying the mediolateral intercalation (see Shih, J. and Keller, R. (1992) Development 116, 901-914), as well as the tissue differentiation of mesodermal cells. These results have implications for the analysis of mesoderm induction and for analysis of variations in the differentiation and morphogenetic function of the marginal zone in different species of amphibians.     

A preprogalanin cDNA from the turtle pituitary and regulation of its gene expression

Galanin is a hormone 29 or 30 amino acids (aa) long that is widely distributed within the body and exerts numerous biological effects in vertebrates. To fully understand its physiological roles in reptiles, we analyzed preprogalanin cDNA structure and expression in the turtle pituitary. Using the Chinese soft-shell turtle (Pelodiscus sinensis order Testudines), we obtained a 672-base pair (bp) cDNA containing a 99-bp 5'-untranslated region, a 324-bp preprogalanin coding region, and a 249-bp 3'-untranslated region. The open-reading frame encoded a 108-aa preprogalanin protein with a putative 23-aa signal sequence at the NH(2) terminus. Based on the location of putative Lys-Arg dibasic cleavage sites and an amidation signal of Gly-Lys-Arg, we propose that turtle preprogalanin is processed to yield a 29-aa galanin peptide with Gly(1) and Thr(29) substitutions and a COOH-terminal amidation. Sequence comparison revealed that turtle preprogalanin and galanin-29 had 48-81% and 76-96% aa identities with those of other vertebrates, respectively, suggesting their conservative nature. Expression of the turtle galanin gene was detected in the pituitary, brain, hypothalamus, stomach, liver, pancreas, testes, ovaries, and intestines, but not in the adipose or muscle tissues, suggesting tissue-dependent differences. An in vitro study that used pituitary tissue culture indicated that treatment with 17beta-estradiol, testosterone, or gonadotropin-releasing hormone resulted in increased galanin mRNA expression with dose- or time-dependent differences, whereas leptin and neuropeptide Y reduced galanin mRNA levels. These results suggest a hormone-dependent effect on hypophyseal galanin mRNA expression.     

Different spatial distribution of mRNAs for activin receptors (type IIA and IIB) and follistatin in developing embryos of Xenopus laevis

Spatial distribution of mRNAs for activin receptors and follistatin was studied by Northern blot hybridization using RNAs from different parts of dissected Xenopus embryos. mRNAs of two activin receptors (type IIA and IIB) occurred uniformly in pre-gastrular embryos, but occurred in larger amounts in ectoderm (in gastrulae), neural plate (in neurulae) and anterior (head) regions (in tailbud embryos) than in other embryonic regions. By contrast, follistatin mRNA appeared almost exclusively in the dorsal mesoderm including invaginating organizer region at the gastrula stage, in notochord and in dorsal ectoderm at the neurula stage, then in anterior part at the tailbud stage. The localized patterns of the distribution of these mRNAs may be due to the regionally different zygotic expression of genes in embryos at later stages. From the relatively widespread pattern of distribution of their mRNAs, we assume that both type IIA and type IIB activin receptors have broad functions in ectodermal and neural differentiation. On the other hand, follistatin mRNA showed quite a restricted pattern of expression, and therefore, we assume that follistatin may have functions more specifically related to the sites of expression of its mRNA. Thus, follistatin may be involved in the differentiation of notochord itself and/or directly be responsible for organizer functions such as neural induction and subsequent differentiation of induced neural tissues at the gastrula and later stages.     

Conserved pattern of tangential neuronal migration during forebrain development

Origin, timing and direction of neuronal migration during brain development determine the distinct organization of adult structures. Changes in these processes might have driven the evolution of the forebrain in vertebrates. GABAergic neurons originate from the ganglionic eminence in mammals and migrate tangentially to the cortex. We are interested in differences and similarities in tangential migration patterns across corresponding telencephalic territories in mammals and reptiles. Using morphological criteria and expression patterns of Darpp-32, Tbr1, Nkx2.1 and Pax6 genes, we show in slice cultures of turtle embryos that early cohorts of tangentially migrating cells are released from the medial ganglionic eminence between stages 14 and 18. Additional populations migrate tangentially from the dorsal subpallium. Large cohorts of tangentially migrating neurons originate ventral to the dorsal ventricular ridge at stage 14 and from the lateral ganglionic eminence from stage 15. Release of GABAergic cells from these regions was investigated further in explant cultures. Tangential migration in turtle proceeds in a fashion similar to mammals. In chimeric slice culture and in ovo graft experiments, the tangentially migrating cells behaved according to the host environment - turtle cells responded to the available cues in mouse slices and mouse cells assumed characteristic migratory routes in turtle brains, indicating highly conserved embryonic signals between these distant species. Our study contributes to the evaluation of theories on the origin of the dorsal cortex and indicates that tangential migration is universal in mammals and sauropsids.     

Sexual differentiation in the spiny softshell turtle (Apalone spinifera), a species with genetic sex determination

It is hypothesized on the basis of sex determination theory that species exhibiting genetic sex determination (GSD) may undergo sexual differentiation earlier in development than species with environmental sex determination (ESD). Most turtle species exhibit a form of ESD known as temperature-dependent sex determination (TSD), and in such species the chronology of sex differentiation is well studied. Apalone spinifera is a species of softshell turtle (Trionychidae) that exhibits GSD. We studied sexual differentiation in this species in order to facilitate comparison to TSD species. Eggs were incubated at two different temperatures and embryos were harvested at various stages of mid to late development. Gonad length was measured with image analysis software, then prepared histologically. Indifferent gonads have differentiated in stage 19 embryos. Histological details of gonadogenesis follow the same pattern as described for other reptiles. Regression of the male paramesonephric duct closely follows testicular differentiation. Gonad lengths are longer at the warmer incubation temperature, and ovaries are generally longer than testes at each stage and for each temperature. Although sexual differentiation takes place at about the same stage as in other turtles with TSD (18-20), in A. spinifera this differentiation is irreversible at this stage, while in some of the TSD species sex is reversible until about stage 22. This immutable, definitive sexual differentiation may support the hypothesis of an accelerated chronology of sex differentiation for this species. We also note that sexual dichromatism at hatching is known in this species and may provide additional evidence of early differentiation. J. Exp. Zool. 290:190-200, 2001.     

Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice

During embryogenesis, the pancreas arises from dorsal and ventral pancreatic protrusions from the primitive gut endoderm upon induction by different stimuli from neighboring mesodermal tissues. Recent studies have shown that Retinoic Acid (RA) signaling is essential for the development of the pancreas in non-mammalian vertebrates. To investigate whether RA regulates mouse pancreas development, we have studied the phenotype of mice with a targeted deletion in the retinaldehyde dehydrogenase 2 (Raldh2) gene, encoding the enzyme required to synthesize RA in the embryo. We show that Raldh2 is expressed in the dorsal pancreatic mesenchyme at the early stage of pancreas specification. RA-responding cells have been detected in pancreatic endodermal and mesenchymal cells. Raldh2-deficient mice do not develop a dorsal pancreatic bud. Mutant embryos lack Pdx 1 expression, an essential regulator of early pancreas development, in the dorsal but not the ventral endoderm. In contrast to Pdx 1-deficient mice, the early glucagon-expressing cells do not develop in Raldh2 knockout embryos. Shh expression is, as in the wild-type embryo, excluded from the dorsal endodermal region at the site where the dorsal bud is expected to form, indicating that the dorsal bud defect is not related to a mis-expression of Shh. Mesenchymal expression of the LIM homeodomain protein Isl 1, required for the formation of the dorsal mesenchyme, is altered in Raldh2--/-- embryos. The homeobox gene Hlxb9, which is essential for the initiation of the pancreatic program in the dorsal foregut endoderm, is still expressed in Raldh2--/-- dorsal epithelium but the number of HB9-expressing cells is severely reduced. Maternal supplementation of RA rescues early dorsal pancreas development and restores endodermal Pdx 1 and mesenchymal Isl 1 expression as well as endocrine cell differentiation. These findings suggest that RA signaling is important for the proper differentiation of the dorsal mesenchyme and development of the dorsal endoderm. We conclude that RA synthesized in the mesenchyme is specifically required for the normal development of the dorsal pancreatic endoderm at a stage preceding Pdx 1 function.     

Role of BMP-4 in the inducing ability of the head organizer in Xenopus laevis

BMP-4 has been implicated in the patterning of the Dorsal-Ventral axis of mesoderm and ectoderm. In this study, we describe the posteriorizing effect of BMP-4 on the neural inducing ability of dorsal mesoderm (dorsal lip region) in Xenopus gastrulae. Dorsal lip explants dissected from stage 10.25 embryos retained anterior inducing ability when precultured for 6 hrs until sibling embryos reach stage 12. When the dorsal lips from stage 10.25 embryos were treated with a range of BMP-4 concentrations, posterior tissues were induced in adjacent ectoderm in a dose-dependent manner. Thus activin-treated explants able to act as head inducers can also induce posterior structures in the presence of BMP-4. To investigate whether BMP-4 directly affects the inducing ability of dorsal mesoderm, we blocked the BMP-4 signaling pathway by injection of mRNA encoding a truncated form of the BMP-4 receptor (tBR) mRNA. Under these conditions, activin-treated explants induced anterior tissues following BMP-4 treatment. Taken together, these results indicate that BMP-4 may affect the head inducing ability of dorsal mesoderm and confer trunk-tail inducing ability during Xenopus gastrulation.     

Sequence of a cDNA encoding turtle high mobility group 1 protein

In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.