Conserved and divergent expression patterns of markers of axial development in eutherian mammals

Background : Mouse embryos are cup shaped, but most nonrodent eutherian embryos are disk shaped. Extraembryonic ectoderm (ExEc), which may have essential roles in anterior–posterior (A‐P) axis formation in mouse embryos, does not develop in many eutherian embryos. To assess A‐P axis formation in eutherians, comparative analyses were made on rabbit, porcine, and Suncus embryos. Results : All embryos examined expressed Nodal initially throughout epiblast and visceral endoderm; its expression became restricted to the posterior region before gastrulation. Anterior visceral endoderm (AVE) genes were expressed in Otx2‐positive visceral endoderm, with Dkk1 expression being most anterior. The mouse pattern of AVE formation was conserved in rabbit embryos, but had diverged in porcine and Suncus embryos. No structure that was molecularly equivalent to Bmp‐positive ExEc, existed in rabbit or pig embryos. In Suncus embryos, A‐P axis was determined at prehatching stage, and these embryos attached to uterine wall at future posterior side. Conclusions : Nodal, but not Bmp, functions in epiblast and visceral endoderm development may be conserved in eutherians. AVE functions may also be conserved, but the pattern of its formation has diverged among eutherians. Roles of BMP and NODAL gradients in AVE formation seem to have been established in a subset of rodents. Developmental Dynamics 245:67–86, 2016. © 2015 Wiley Periodicals, Inc.     

Postnatal development of the reproductive system in the grey short-tailed opossum, Monodelphis domestica

Postnatal phenotypic sex differentiation has been investigated in a laboratory marsupial, Monodelphis domestica, as part of a larger study to resolve apparent discrepancies between eutherian and marsupial mammals. These include the formation of sex-specific structures in marsupials prior to gonadal differentiation and the retention in both sexes of structures which are sex-specific in eutherians. The time-course and nature of differentiation was investigated in 131 specimens ranging in age from the day of birth to 56 days. Patent wolffian ducts extend to the urogenital sinus in both sexes at birth, while müllerian ducts are identified on day 1 and grow in a cranio-caudal direction to reach the urogenital sinus on day 6. The male müllerian duct shows signs of regression at its cranial end on day 10 and throughout its length on day 12; its lumen has completely disappeared by day 15. By this time the epididymis and vas deferens have developed from the wolffian duct; their histological differentiation occurs between days 26 and 56. Prostatic buds are identifiable in tissue surrounding the male urethra on day 14. In the female, the wolffian duct is larger than the müllerian duct until day 14; thereafter the wolffian duct begins to regress at its cranial end, disappearing by day 17, whereas the müllerian duct begins to enlarge, converging with its fellow at the urogenital sinus by day 19. Lateral vaginae, vaginal culs-de-sac, uteri and oviducts have differentiated from the müllerian ducts by day 25. Gonads of both sexes are elongated in shape at birth, attached along the medial aspect of the large mesonephroi in the abdominal cavity. However, from day 3 onwards the testis becomes more rounded than the ovary. Degeneration of the male mesonephros begins about day 10 and is almost completed by day 19; the female mesonephros is still relatively large at day 14 though it too has almost disappeared by day 19. By postnatal day 13 the abdominal phase of testis descent is underway and the inguinal phase begins at day 15. Testes have reached the scrotal sac by day 24 and achieve their final position at the base of the scrotum by day 28. In summary, postnatal reproductive tract development and gonadal descent has been examined in this important biomedical model, where differentiation of the wolffian and müllerian ducts takes place after gonadal differentiation, according to the normal eutherian pattern.Abbreviations AMH Anti-müllerian hormone - LS Longitudinal section - Md Müllerian duct - PN0 Day of birth - SEM Scanning electron micrograph - SRY Sex-determining region on the Y chromosome - TS Transverse section - Wd Wolffian duct     

Postnatal development of the fore- and hindlimbs in the grey short-tailed opossum,Monodelphis domestica

Marsupials are good experimental animals for developmental studies as their offspring are born at a stage comparable to embryonic stages of eutherian species. The South American opossum, Monodelphis domestica, is particularly useful because of its small size and easy maintenance. This study was carried out to compare development of opossum fore- and hindlimbs during postnatal life, using light microscopy and whole mount alizarin staining. At birth, well-developed mobile forelimbs show cartilage models of bones and myotubular striated muscle fibres. However, hindlimbs are relatively underdeveloped paddle-like outgrowths. Two days later mesodermal condensations form models of the future hindlimb bones and mononucleate myoblast aggregates are present; by 6 days post partum (dpp) the hindlimb has reached a stage of development similar to that of the forelimb at birth. At this stage, periosteal buds have invaded forelimb long bones and nuclei in forelimb muscle fibres have become displaced to the periphery. The 16 dpp hindlimb shows long bones invaded by periosteal buds and closely packed, striated muscle fibres. Epiphyseal plates are now seen in the forelimb long bones and forelimb muscle fibres show mature characteristics. Musculoskeletal development is well correlated with the functional demands of the limbs during postnatal development in the opossum, which provides an excellent model for investigations into the genes and molecules controlling limb development.     

On the prenatal initiation of T cell development in the opossum Monodelphis domestica

Thymus‐dependent lymphocytes (T cells) are a critical cell lineage in the adaptive immune system of all jawed vertebrates. In eutherian mammals the initiation of T cell development takes place prenatally and the offspring of many species are born relatively immuno‐competent. Marsupials, in contrast, are born in a comparatively altricial state and with a less well developed immune system. As such, marsupials are valuable models for studying the peri‐ and postnatal initiation of immune system development in mammals. Previous results supported a lack of prenatal T cell development in a variety of marsupial species. In the gray short‐tailed opossum, Monodelphis domestica, however, there was evidence that αβT cells were present on postnatal day 1 and likely initiated development prenatally. Demonstrated here is the presence of CD3ε⁺ lymphocytes in late‐stage embryos at a site in the upper thoracic cavity, the site of an early developing thymus. CD3ε⁺ cells were evident as early as 48 h prior to parturition. In day 14 embryos, where there is clear organogenesis, CD3ε⁺ cells were only found at the site of the early thymus, consistent with no extra‐thymic sites of T cell development in the opossum. These observations are the first evidence of prenatal T cell lineage commitment in any marsupial.     

Purification and characterization of fibroblast growth factors from the opossum, Monodelphis domestica, brain.

An extract from the brain of the opossum Monodelphis domestica was fractionated by heparin affinity chromatography. A major peak of mitogenic activity (heparin binding growth factor 2, HBGF-2) eluted from heparin-Sepharose between 1.7 and 2.0 M NaCl. Antisera specific for bovine bFGF detected four polypeptides of 17.5–23 kDa in opossum brain HBGF-2 preparations. Opossum brain heparin binding growth factor 1 (HBGF-1), a minor peak of activity, eluted from heparin-Sepharose at 1.1 NaCl and contained a 16.2 kDa protein that cross-reacted with antiserum against bovine aFGF.     

Skeletal fiber types and spindle distribution in limb and jaw muscles of the adult and neonatal opossum,Monodelphis domestica

The South American opossum, Monodelphis domestica, is very immature at birth, and we wished to assess its potential for studies of jaw muscle development. Given the lack of prior information about any Monodelphis fiber types or spindles, our study aimed to identify for the first time fiber types in both adult and neonatal muscles and the location of spindles in the jaw muscles. Fiber types were identified in frozen sections of adult and 6-day-old jaw and limb muscles by using myosin ATPase and metabolic enzyme histochemistry and by immunostaining for myosin isoforms. The distribution of fiber types and muscle spindles throughout the jaw-closer muscles was identified by immunostaining of sections of methacarnoy-fixed, wax-embedded heads. Most muscles contained one slow (type I) and two fast fiber types (equivalent to types IIA and IIX), which were similar to those in eutherian muscle, and an additional (non-IIB) fast type. In jaw-closer muscles, the main extrafusal fiber type was IIM (characteristic of these muscles in some eutherians), and almost all spindles were concentrated in four restricted areas: one in masseter and three in temporalis. Six-day neonatal muscles were very immature, but future spindle-rich areas were revealed by immunostaining and corresponded in position to the adult areas. Extrafusal and spindle fiber types in Monodelphis share many similarities with eutherian mammalian muscle. This finding, along with the immaturity of myosin isoform expression observed 6 days postnatally, indicates that Monodelphis could provide a valuable model for studying early developmental events in the jaw-closer muscles and their spindles. Anat. Rec. 251:548–562, 1998. © 1998 Wiley-Liss, Inc.     

Why do spermatozoa of American marsupials form pairs? A clue from the analysis of sperm-pairing in the epididymis of the grey short-tailed opossum,Monodelphis domestica

In order to understand the evolutionary significance of sperm-pairing in American marsupials, an ultrastructural investigation was made of this process in the South American grey short-tailed opossum, Monodelphis domestica. One epididymis from each animal (5) was fixed for light and electron microscopy and divided into 18 segments. The contralateral tract was divided into similar segments and assessments made of the total number of spermatozoa and the proportion of sperm-pairs. The mean total sperm number was 4.20 ± 0.62 × 10⁶/epididymis. Sperm-pairing commenced around segment 9 in the proximal corpus epididymidis and reached a maximum of 80% in the caudal sperm storage region of the duct. The sperm-pairing process was characterised by four stages. Spermatozoa exhibited parallel alignment as indicated by the positioning of identical cross-sections of sperm heads. This was followed by close apposition with acrosomal faces parallel rather than opposite. Rotation of the sperm heads around each other then apparently occurred as indicated by the morphological alignment of sections of paired sperm heads. Sperm-pairing was complete when the acrosomal faces were precisely aligned and joined. Misalignment and failure to pair was observed in about 20% of spermatozoa in the cauda epididymis. Such a complex sperm-pairing process may ensure that conjugated spermatozoa are precisely aligned so that flagella movement can be accurately coordinated for maximal progressive motility. © 1993 Wiley-Liss, Inc.     

Identification and expression patterns of members of the protease‐activated receptor (par) gene family during zebrafish development

Protease‐activated receptors (PARs) play critical roles in hemostasis in vertebrates including zebrafish. However, the zebrafish gene classification appears to be complex, and the expression patterns of par genes are not established. Based on analyses of genomic organization, phylogenetics, protein primary structure, and protein internalization, we report the identification of four zebrafish PARs: par1, par2a, par2b, and par3. This classification differs from one reported previously. We also show that these genes have distinct spatiotemporal expression profiles in embryos and larvae, with par1, par2a, and par2b expressed maternally and ubiquitously during gastrula stages and their expression patterns refined at later stages, and par3 expressed only in 3‐day‐old larvae. Notably, the expression patterns of zebrafish par1 and par2b resemble those of their mammalian counterparts, suggesting that receptor function is conserved among vertebrates. This conservation is supported by our findings that Par1 and Par2b are internalized following exposure to thrombin and trypsin, respectively. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

结核分枝杆菌lhp基因原核表达载体的构建和表达

目的：构建结核分枝杆菌(MTB)lhp和基因原核表达载体并进行表达。方法：用PCR扩增MTB lhp基因，并克隆入pQE30质粒。测序正确后，再亚克隆入pET32a( )质粒，构建pQE30—CFP10和pET32a( )—CFP10重组体。结果：以重组体分别转化DH5α和BL21(DE3)菌后，经IPTG诱导，pQE30—CFP10未表达目的蛋白；而pET32a( )—CFP10则表达出Mr为20000左右重组蛋白。SDS—PAGE分析显示，IPTG诱导4h重组蛋白的表达量最高：表达蛋白以可溶性非包涵体形式存在于胞质中，表达量占全菌蛋白质的38%，用Western blot证实其具有良好的抗原性。经Ni—NTA柱纯化，获得纯度为93%的重组蛋白。结论：成功地构建原核表达载体pET32a( )—CFP10，并获得重组CFP10蛋白，为MTB重组抗原的应用奠定了基础。     

Peripheral lipopolysaccharide administration transiently affects expression of brain-derived neurotrophic factor, corticotropin and proopiomelanocortin in mouse brain

Peripheral inflammation induced by intraperitoneal (i.p.) injection of Lipopolysaccharide (LPS) is known to cause functional impairments in the brain affecting memory and learning. One of mechanisms may be the interference with neurotrophin (NT) expression and function. In the current study we administered a single, high dose of LPS (3mg/kg, i.p.) into mice and investigated changes in brain-derived neurotrophic factor (BDNF) gene expression within 1-6 days after LPS injection. Crude synaptosomes were isolated from brain tissue and subjected to Western-blot analyses. We found transient reductions in synaptosomal proBDNF- and BDNF protein expression, with a maximal decrease at day 3 as compared to saline injected controls. The time course of reduction of BDNF mRNA in whole brain extracts parallels the decrease in protein levels in synaptosomes. LPS effects in the central nervous system (CNS) are known to crucially involve the activation of the hypothalamic-pituitary-adrenal (HPA) axis. We analysed the time course of corticotropin releasing hormone (CRH)- and proopiomelanocortin (POMC) mRNA expression. As observed for BDNF-, CRH- and POMC mRNA levels are also significantly reduced on day 3 indicating a comparable time course. These results suggest that peripheral inflammation causes a reduction of trophic supply in the brain, including BDNF at synaptic sites. The mechanisms involved could be a negative feedback of the activated HPA axis.     

与原发性食管癌进展相关基因的筛选

目的 应用基因微矩阵技术分析原发性食管癌基因表达谱,筛选与食管癌进展相关基因。方法 利用激光捕获微切割-T7 RNA聚合酶体外扩增联合cDNA芯片技术对15例原发性食管癌的基因表达谱进行筛选,并通过比较组间差异基因筛选出与肿瘤进展相关的基因。结果 在886条目的基因中,筛选出34条基因在Ⅰ/Ⅱ和Ⅲ/Ⅳ组间存在显著性差异(p<0.05),其中细胞周期调节类基因与早期食管癌发生有关,而细胞外基质类、黏附分子类基因主要参与食管癌的浸润和转移。结论 多个基因表达变化在食管癌发生中起作用,而进展相关基因的筛选为其临床诊治提供了新的思路和线索。     

参芦总皂甙通过cAMP cGMP对人淋巴细胞E—玫瑰花结形成的影响

应用参芦总皂甙对人外周血白细胞内环核苷酸含量的影响,及其与E—玫瑰花结形成之间的关系,并与外源性cAMP、cGMP、茶硷对E—玫瑰花结形成影响进行了比较。发现参芦总皂甙能明显提高淋巴细胞内cAMP含量,同时提高cGMP含量,尤以cAMP为著、参芦总皂甙对淋巴细胞作用时间不同,其形成的总T花结亦不同。     

亚甲蓝对APP/PS1转基因小鼠学习记忆及海马结构内GFAP表达的影响

目的:观察亚甲蓝对APP/PS1转基因小鼠学习记忆及胶质纤维酸性蛋白(Glial fibrillary acidic pro-tein,GFAP)在海马结构表达变化的影响。方法:20只3月龄APP/PS1小鼠,随机分2组,每组10只,对照组:自由饮水;治疗组:根据小鼠饮水量将亚甲蓝加入日常饮水中(25 mg/kg/d)连用4个月至7月龄。水迷宫测试观察其行为学的改变,免疫组化、Western Blot和TUNEL染色法观察GFAP在海马结构的表达及神经元的凋亡情况。结果:水迷宫测试结果显示亚甲蓝喂养组APP/PS1转基因小鼠第2～4 d的平均潜伏期显著低于对照组小鼠的潜伏期,说明治疗组与对照组相比在7个月时出现明显差异(P<0.05);免疫组化和Western结果显示治疗组海马结构内的GFAP在APP/PS1转基因小鼠的表达下调(P<0.05)。TUNEL染色法显示治疗组海马CA1、CA3区和齿状回TUNEL阳性细胞数较对照组明显减少(P<0.05)。结论:亚甲蓝能够下调APP/PS1小鼠海马结构内GFAP蛋白的表达,并通过抑制海马结构神经元的凋亡,提高APP/PS1小鼠的认知能力。     

Spatial and temporal patterns of expression of melanocortin type 2 and 5 receptors in the fetal mouse tissues and organs

In the present study to analyze the role of ACTH in fetal tissues and organs, we observed the expression of melanocortin type 2 (MC2) and 5 (MC5) receptors in ICR mouse embryos from E11.5 to E18.5 by immunohistochemistry. In the adrenal gland and testis, both receptors were expressed from E13.5 to E18.5. In the genital ridge and the ovary, melanocortin type 2 receptors (MC2R) was detected from E11.5 to E12.5 and from E13.5 to E18.5, respectively, while melanocortin type 5 receptors (MC5R) was not detected. In the mesonephros, MC2R and MC5R were expressed from E11.5 to E12.5, and in the metanephros, MC2R and MC5R were expressed from E12.5 to E18.5 and from E14.5 to E18.5, respectively. In the lung, MC2R was expressed from E11.5 to E14.5, but MC5R was not expressed at all. In blood cells, MC5R was detected at all stages examined, while MC2R was detected at none. MC2R was observed in the brain and spinal cord from E11.5 to E13.5, while MC5R was detected only in the telencephalon and only from E16.5 to E18.5. At different temporal patterns, MC2R, but not MC5R, was detected in the choroid plexus, while MC5R, but not MC2R, was expressed in the liver and in the nasal epithelium, and both MC2R and MC5R were expressed in the dorsal root ganglion and the trigeminal ganglion. These findings show the spatio-temporal specific expression patterns of MC2R and MC5R in the mouse embryo and suggest that ACTH may be related to histogenesis and/or prenatal function of various tissues and organs via MC2R and/or MC5R.     

Genetic changes of CDH1, APC, and CTNNB1 found in human brain tumors

This paper focuses on changes in E-cadherin (CDH1), adenomatous polyposis coli (APC), and beta-catenin (CTNNB1) in 50 tumors of the central nervous system. All gene products are components of adherens junctions, but are also involved in wnt signaling. The results of our analysis showed LOH of CDH1 gene in 31% of meningiomas examined (significant correlation; p=0.002). LOH was noted in a single case of germinoma, while other tumor types did not demonstrate any change in CDH1. Fourteen samples (29.2%) with changes in APC gene were observed. The changes were seen in 33.3% of glioblastomas and in 27% of meningiomas; LOH occurred in five informative astocytomas (20%) and in six informative neurinomas (17%). One oligoastrocytoma showed LOH at exon 11, and one medulloblastoma had allelic imbalance at both exons. Five samples (10%) showed heteroduplexes in exon 3 of beta-catenin. Potential mutations were confined to two meningiomas, one astrocytoma, one glioblastoma, and one germinoma. Our results suggest that genetic changes in wnt components are involved in brain tumor genesis. Changes in E-cadherin are involved in meningiomas, while changes in APC gene occur in different tumor types, with glioblastomas showing the highest percentage.     

Diversification of the expression patterns and developmental functions of the dishevelled gene family during chordate evolution

Dishevelled (Dvl) proteins are key transducers of Wnt signaling encoded by members of a multi‐gene family in vertebrates. We report here the divergent, tissue‐specific expression patterns for all three Dvl genes in Xenopus embryos, which contrast dramatically with their expression patterns in mice. Moreover, we find that the expression patterns of Dvl genes in the chick diverge significantly from those of Xenopus. In addition, in hemichordates, an outgroup to chordates, we find that the one Dvl gene is dynamically expressed in a tissue‐specific manner. Using knockdowns, we find that Dvl1 and Dvl2 are required for early neural crest specification and for somite segmentation in Xenopus. Most strikingly, we report a novel role for Dvl3 in the maintenance of gene expression in muscle and in the development of the Xenopus sclerotome. These data demonstrate that the expression patterns and developmental functions of specific Dvl genes have diverged significantly during chordate evolution. Developmental Dynamics 238:2044–2057, 2009. © 2009 Wiley‐Liss, Inc.