Independent differentiation of mammotropes and somatotropes in the chicken embryonic pituitary gland

It has been reported that mammotropes in a rodent pituitary gland are derived from somatotropes via somatomammotropes (SMTs), cells that produce both growth hormone (GH) and prolactin (Prl). However, no studies have been done on the transdifferentiation of somatotropes in the chicken pituitary gland. In this study, in order to determine the origin of mammotropes, we studied detail property of appearance of chicken somatotropes, mammotropes and pit-1 cells and then evaluated the existence of SMTs in the chicken embryonic pituitary gland. Immunohistochemical analysis revealed that GH-immunopositive (GH-ip) cells appeared on embryonic day (E) 14 and were mainly distributed in the caudal lobe, while Prl-immunopositive (Prl-ip) cells appeared in the cephalic lobe of the pituitary gland on E16. In situ hybridization (ISH) and RT-PCR analysis showed that expression of GH and Prl mRNA starts at E12 in the caudal lobe and at E14 in the cephalic lobe respectively. Pit-1 mRNA was first detected on E5 by RT-PCR, and pit-1 mRNA-expressing cells were found in the cephalic lobe on E8. Then with the ontogeny of the chicken, these cells spread into both lobes. Using a double staining method with ISH and immunohistochemistry, we could not detect the existence of SMTs in the chicken embryonic pituitary gland even in the marginal region of either lobe. These results suggest that chicken somatotropes and mammotropes independently appear in different lobes of pituitary gland and that transdifferentiation from somatotropes to mammotropes is not the central route for differentiation of mammotropes in the embryonic chicken pituitary gland.     

Development of gonadotropes in the chicken embryonic pituitary gland

Although a number of immunohistochemical studies have been carried out on the differentiation of chicken gonadotropes during embryogenesis, the temporal and spatial properties of appearance of gonadotropes are not clear. In this study, we studied the appearance and morphological characteristics of gonadotropes in the embryonic and adult chicken anterior pituitary glands using RT-PCR, in situ hybridization and immunohistochemistry. For this purpose, we raised specific antisera against chicken follicle-stimulating hormone beta-subunit (cFSHbeta) and chicken luteinizing hormone beta-subunit (cLHbeta) based on each putative amino acid sequence. RT-PCR analysis revealed that cFSHbeta mRNA was expressed from embryonic day 7 (E7). Chicken FSHbeta mRNA-expressing (-ex) and -immunopositive (-ip) cells started to appear in the ventral part of the caudal lobe in the anterior pituitary gland at E8. Chicken LHbeta-ip cells were also first observed there at E8, but cLH mRNA expression was confirmed from E4 by RT-PCR analysis. The distribution of these chicken gonadotropin-ex and -ip cells spread from the ventral part to dorsal part in the caudal lobe around E10 and subsequently expanded to the cephalic lobe from E12 to E20. These cells were morphologically classified into two types (round- and club-shaped cells). It was found that the density of gonadotropin-ip cells in the caudal lobe was always higher than that in the cephalic lobe throughout the period of development. To the best of our knowledge, this is the first report focusing on the differentiation of chicken gonadotropes by assessment of both protein and mRNA of chicken gonadotropin.     

Using 2-DE for the differentiation of local chicken breeds

The aim of this study was to apply a proteomic approach for the characterisation of local chicken breeds. The experiment involved a total of 29 males of three local Italian chicken breeds: Pépoi, Padovana and Ermellinata di Rovigo. Sarcoplasmic protein fractions of breast muscle were analysed by 2-DE. Image analysis followed by statistical analysis enabled to differentiate groups of individuals based on the similarities of protein expression. Individuals were distinguished into clusters and groups, corresponding to the breed of origin. Distances among individuals, calculated using data on spot volumes, were used to draw a neighbour-joining tree, showing clear individual and breed grouping. The most relevant spots regarding breed differentiation were detected; 11 were identified by MS revealing preliminary evidences on the mechanisms of the breed differentiation process. The results evidenced the ability of proteomic analyses for the characterisation of chicken breeds.     

Osteoblastic and osteoclastic differentiation of mononuclear cells facing the resorbing surface of uncalcified cartilage in the tibia of embryonic chick

Summary The resorbing region of uncalcified cartilage in the tibia of embryonic chick was studied using ³H-proline autoradiography, histochemistry, and horseradish-peroxidase tracers.At the cartilage-bone marrow interface, two kinds of cells (A and B) were identified. Type-A cells were elongated, contacted the matrix of the uncalcified cartilage directly, and possessed extensive rough endoplasmic reticulum, one or two juxtanuclear Golgi apparatus and cell membranes exhibiting prominent alkaline phosphatase activity. Type-B cells were round to oval, mononucleate (occasionally binucleate), and contained abundant mitochondria, vacuoles and vesicles, well-developed Golgi apparatus, and lysosomes. The lysosomes and the majority of vacuoles and Golgi lamellae of these cells showed prominent acid phosphatase activity. Type-B cells accumulated more horseradish-peroxidase reaction product in their vacuoles and vesicles than type-A cells. Thick, banded collagen fibrils were occasionally found in the matrix of the resorbing surface. ³H-proline autoradiography revealed small numbers of grains at the cartilage-bone marrow interface.These findings suggest that type-A cells have osteoblastic and type-B cells osteoclastic properties and are precursor cells of osteoblasts and osteoclasts, respectively. The appearance of a mineral phase in the resorbing cartilage is probably important for the differentiation of these cells.     

Detailed analysis of formation of chicken pituitary primordium in early embryonic development

The primordium of the mammalian adenohypophysis derived from Rathke's pouch (RP) is known to be formed by oral ectoderm invagination. However, in the early phase of pituitary development, the detailed process by which the oral ectoderm develops into the adenohypophysis remains largely unknown. Using high-resolution non-radiolabeled in situ hybridization and the BrdU and TUNEL methods, we have examined the detailed expression pattern of factors involved in the formation of RP of chicken and the changes in the mitotic and apoptotic cell regions in RP. In the chicken embryo, Sonic hedgehog (Shh) mRNA was initially expressed in the stomodeal plate but not in the oral ectoderm. After prospective diencephalon had detached from the oral ectoderm, another Shh-expressing region appeared in the most rostral part of the recess. LIM homeobox gene 3 (Lhx3) mRNA first appeared in the anterior area of Rathke's recess, and expression then spread to the caudal region. alphaGSU mRNA-expressing cells were observed at both ends of the Lhx3-expressing region, and thereafter the expression area moved to the posterior region. Furthermore, a close overlap was found between the proliferating region and Lhx3 mRNA-expressing area, and TUNEL-positive cells appeared in Seessel's pouch derived from the foregut. Thus, the primordium of the pituitary gland corresponding to the Lhx3-expressing region is surrounded by the Shh-expressing region, which appears in two steps, and the mass growth and invagination of RP of chicken result from the coordination of the dorsal extension of the anterior region and the ventral extension of the posterior region of RP.     

Ganglioside changes in the chicken optic lobes and cerebrum during embryonic development

Summary The developmental profiles of 15 different gangliosides of the optic lobes and cerebrum of the chicken were followed from the 6 th day of incubation to hatching and correlated to morphological development. Five of these gangliosides appearing in both structures between the sixth and tenth day, have not been reported previously in higher vertebrates. Three chromatographed on TLC-plates similarly to G_T3, G_T2, and G_T1c gangliosides, which have been demonstrated in fish brain. One fraction moved just below G_Q1b and is suggested, to contain G_Q1c. These novel gangliosides, which are possibly related to a recently proposed separate and probably phylogenetically older biosynthetic pathway, contained up to 20% of total ganglioside sialic acid. The fifth novel fraction, containing up to 16% of total ganglioside-sialic acid, moved below the penta-sialoganglioside G_P1 and is suggested to contain hexa-sialogangliosides.There were two main changes in ganglioside synthesis, which were identical in both structures.The first occurred from the sixth to the eleventh day, parallel to decreased proliferation, maximal cell migration and neuroblast differentiation, G_D3 and G_D2 decreased rapidly in favour of G_Q1b, G_P1, and to the novel fractions, described above.The second occurred from the eleventh to the eighteenth day, parallel to increased growth and arborization of dendrites and axons as well as functional establishment of synaptic contacts, there was a sharp rise in the amount of G_D1b, G_T1b, and G_D1a. Concomitantly the novel gangliosides decreased. At hatching G_D1a was the predominant ganglioside. G_M3, G_M2, and G_M1 were always minor fractions, each accounting for less than 4% of total ganglioside-sialic acid. G_M4 was never detected, indicating neglegible myelinisation until hatching.Abbreviations NeuAc N-Acetylneuraminic acid - G_M4 I³NeuAc-GalCer - G_M3 II³NeuAc-LacCer - G_M2 II³NeuAc-G_gOse₃Cer - G_M1 II³NeuAc-G_gOse₄Cer - G_D3 II³(NeuAc)₂LacCer - G_D1a IV³NeuAc, II³NeuAc G_gOse₄Cer - G_T3 II³ (NeuAc)₃LacCer - G_D2 II³(NeuAc)₂G_gOse₃Cer - G_D1b II³(NeuAc)₂G_gOse₃Cer - G_D1b II³(NeuAc)₂G_gOse₄Cer - G_T2 II³(NeuAc)₃G_gOse₃Cer - G_T1b IV³NeuAc, II³(NeuAc)₂G_gOse₄Cer - G_T1c II³(NeuAc)₃G_gOse₄Cer - G_Q1b IV³(NeuAc)₂ II³(NeuAc)₂G_gOse₄Cer - G_Q1c IV³NeuAc, II³(NeuAc)₃G_gOse₄Cer - G_P1 IV³(NeuAc)₂, II³(NeuAc)₃G_gOse₄Cer - G_H(?) IV₃(NeuAc)₃, II³(NeuAc)₃G_gOse₄Cer     

Ghrelin promotes the differentiation of human embryonic stem cells in infarcted cardiac microenvironment

Ghrelin is broadly expressed in myocardial tissues, where it exerts different functions. It also has been found to have a wide variety of biological functions on cell differentiation and tissue development. The aim of this study was to investigate the effect of ghrelin on human embryonic stem cell (hESC) differentiation in infarcted cardiac microenvironment. The hESCs grown on feeder layers expressed several pluripotential markers including alkaline phosphatase (AKP). Four weeks after transplantation into rat infarcted hearts, the hESCs and their progeny cells survived and formed intracardiac grafts were 54.7% and 19.6% respectively in ghrelin- and phosphate-buffered saline (PBS)-treated groups. Double immunostaining with anti-human Sox9 and anti-HNA or anti-human fetal liver kinase-1 (Flk1) and anti β-tubulin showed that the human grafts were in development. However, double positive stains were only found in the ghrelin-treated group. In addition, the hESC injection protocol was insufficient to restore heart function of the acute myocardial infarction model. Our study, therefore, provides a new insight of ghrelin on promoting hESC survival and differentiation in rat infarcted cardiac microenvironment. This may give a clue for therapy for myocardial infarction by hESCs or progeny cells.     

Ligh- and electron-microscopic immunocytochemistry of somatotropes in the anterior pituitary gland of European ferret,Mustela putorius furo

Light-microscopic immunocytochemistry of ferret anterior pituitary revealed the localization of somatotropes in the pars distalis, but no immunoreactive cells were detected in the pars tuberalis. Ultrastructural studies by superimposition immunocytochemistry and immuno-electron microscopy, clucidated the morphological heterogeneity of these somatotropic cells. They were classified into 2 subtypes on the basis of size of the secretory granules. Type-I cells with small granules (mean diameter, 192 nm), were considered to be the immature somatotrop, while Type-II cells, with comparatively larger secretory granules (mean diameter, 257 nm), were considered to be the matured form of Type-I cells and the typical somatotropic cell-type, and were much more predominant than the Type-I cells. The fact that Type-II cells had a distinct Golgi zone and many mitochondria, while in Type-I cells the intracellular organelles were generally less developed, supports this suggestion. In addition to these two extreme subtypes, several intermediate forms were also encountered that may represent different transitional phases during the conversion of Type I to Type II. Protein A-gold immuno-electron microscopy illustrated the specific localization of growth hormone over the granules, with no labelling over any other cytoplasmic organelles of the 2 somatotrope subtypes.     

A new intracellular serine protease inhibitor expressed in the rat pituitary gland complexes with granzyme B

We have cloned a novel serpin (raPIT5a) from a rat pituitary cDNA library which is structurally related to members of the ovalbumin subfamily of serine protease inhibitors. This new cDNA encodes a 374-amino acid protein, designated raPIT5a. raPIT5a was expressed in specific cells in the intermediate and anterior lobes of the pituitary. Recombinant raPIT5a was not secreted suggesting raPIT5a functions to inhibit intracellular proteases. Recombinant raPIT5a formed an SDS-stable complex with human granzyme B, a serine protease which induces apoptosis by activating members of the caspase enzyme family. These data suggest raPIT5a may have a role in regulating granzyme B or related enzymes and apoptosis in the pituitary gland.     

组蛋白修饰对胚胎干细胞分化过程[]的调控机制

选用人类胚胎干细胞系和由人类胚胎干细胞系分化来的神经干细胞系为研究对象,分析组蛋白修饰对胚胎干细胞分化过程的调控作用。得到了两种细胞系差异表达基因转录起始位点侧翼区域内八种组蛋白修饰的分布模式,以及组蛋白修饰功能簇。研究表明在两类细胞系中,八种组蛋白修饰谱分布模式一致,且呈现两种分布类型; H3K27ac,H3K4me3和H3K9ac组成的功能簇是保守的;H3K27me3,H3K36me3和H3K79me1组成的功能簇以及H3K9me3和H3K27me3组成的功能簇在胚胎干细胞向神经干细胞分化的过程中消失。结果揭示了组蛋白修饰对胚胎干细胞系向神经干细胞系分化过程的部分调控机制,为该分化过程分子调控机制的研究提供部分重要的理论基础。     

Neural differentiation from embryonic stem cells in vitro: An overview of the signaling pathways

Neurons derived from embryonic stem cells (ESCs) have gained great merit in both basic research and regenerative medicine. Here we review and summarize the signaling pathways that have been reported to be involved in the neuronal differentiation of ESCs, particularly those associated with in vitro differentiation. The inducers and pathways explored include retinoic acid, Wnt/β-catenin, transforming growth factor/bone morphogenetic protein, Notch, fibroblast growth factor, cytokine, Hedgehog, c-Jun N-terminal kinase/mitogen-activated protein kinase and others. Some other miscellaneous molecular factors that have been reported in the literature are also summarized and discussed. These include calcium, calcium receptor, calcineurin, estrogen receptor, Hox protein, ceramide, glycosaminioglycan, ginsenoside Rg1, opioids, two pore channel 2, nitric oxide, chemically defined medium, cell-cell interactions, and physical stimuli. The interaction or crosstalk between these signaling pathways and factors will be explored. Elucidating these signals in detail should make a significant contribution to future progress in stem cell biology and allow, for example, better comparisons to be made between differentiation in vivo and in vitro. Of equal importance, a comprehensive understanding of the pathways that are involved in the development of neurons from ESCs in vitro will also accelerate their application as part of translational medicine.     

Lecithinized brain-derived neurotrophic factor promotes the differentiation of embryonic stem cells in vitro and in vivo

The addition of lecithin molecules to brain-derived neurotrophic factor (BDNF) has been reported to markedly enhance its pharmacological effect in vivo. In the current study, we show that lecithinized BDNF (PC-BDNF) has a higher affinity than BDNF for neural precursor cells. Although BDNF only slightly increased the expression of the genes for Mash-1, p35, 68 kDa neurofilament, and TrkB receptor, PC-BDNF caused a significant increase in their expression. PC-BDNF also increased the level of neurofilament protein and dramatically increased TrkB mRNA gene expression, which was followed by a sustained activation of the p42/p44 extracellular-regulated kinases. Finally, transplantation of PC-BDNF-treated cells was more effective than BDNF-treated cells at improving impaired motor function caused by spinal cord injury. These findings showed that PC-BDNF has a better potential than BDNF for promoting neural differentiation, partly due to a higher cellular affinity. Furthermore, PC-BDNF-treated cells could be useful for transplantation therapy for central nervous system injuries.     

应用抑制消减杂交分离雌、雄鸡胚性分化早期的差异表达基因

分离不同性别的鸡胚性分化早期差异表达基因,可为禽类性别决定和性分化机制研究提供基本信息。本研究分别以孵化3.5-6d的雌、雄鸡胚性腺为材料,利用抑制性消减杂交技术成功构建了雌-雄鸡胚间正、反向消减cDNA文库,并利用斑点印迹杂交从中筛选出了39个性别差异表达的阳性cDNA克隆。以持家基因GAPDH为参照指标检测消减文库的消减效率,结果发现两个文库的消减效率均高达25倍。插入片段PCR鉴定结果显示,消减文库中cDNA插入片段的长度主要分布于250-750bp之间。分别对雌、雄鸡胚消减文库中的252和168个cDNA克隆进行斑点杂交筛选,再随机从两个消减文库中共抽取39个阳性差异表达克隆进行序列测定及序列比对分析。结果表明:这39个cDNA克隆分别代表了定位于鸡不同染色体上的18个已知功能的基因和11个假定基因;参照哺乳动物同源基因的功能,所得的18个已知差异基因可能参与多种生物反应过程。用半定量RT-PCR方法对雌、雄鸡胚消减文库中各5个基因的表达情况进行进一步验证,发现除雌性库中的一个基因外其它9个基因均有较明显的性别差异表达。这些性别差异表达基因的获得为进一步研究鸡胚性腺发育中的基因表达调控奠定了基础     

Nitric oxide drives embryonic myogenesis in chicken through the upregulation of myogenic differentiation factors

The muscle-specific variant of neuronal nitric oxide (NO) synthase (NOS-I), is developmentally regulated in mouse suggesting a role of NO during myogenesis. In chick embryo, a good model of development, we found that the expression of NOS-I is up-regulated, but only in the early phase of development. Through a pharmacological intervention in ovo we found that NO signalling plays a relevant role during embryonic development. The inhibition of NOS-I decreased the growth of embryo, in particular of muscle tissue, while the restoring of physiological NO levels, via administration of a NO donor, reversed this effect. We found a selective action of NO, produced by NOS-I, on regulatory factors involved in myogenic differentiation in the early phase of chick embryo development: inhibition of NO generation leads to a decreased expression of the Myocyte enhancer factor 2a (Mef2a), Mef2c, Myogenin and Myosin, which was reversed by the administration of a NO donor. NO had no effects on Myf5 and MyoD, the myogenic regulatory factors necessary for myogenic determination. The action of NO on the myogenic regulatory factors was mediated via generation of cyclic GMP (cGMP) and activation of the cGMP-dependent protein kinase G (PKG). Finally we found in myoblasts in vitro that the activation of Mef2c was the key event mediating the NO-induced modulation of myogenesis.     

Structural and functional differentiation of the embryonic chick pineal organ in vivo and in vitro

Summary The development of sensory structures in the pineal organ of the chick was examined by means of scanning electron microscopy from embryonic day 10 through day 12 post-hatching. At embryonic day 10, the wall of the tubules within the pineal primordium is composed of cells with unspecialized luminal surface. Differentiation of sensory structures starts at embryonic day 12 when pinealocytes and supporting cells can be distinguished. Pinealocytes are recognized by virtue of an inner segment only rarely endowed with a cilium, whereas supporting cells exhibit numerous short microvilli. Further differentiation of the sensory apparatus is achieved by development of an oval-shaped, biconcave swelling at the tip of the cilium, 1×2 m in size, and a collar of long microvilli at the base of the inner segment. Membrane specializations of sensory cilia, however, were not detected. Since during embryonic life new tubules and follicles are continuously formed, all stages of differentiation of sensory structures are found in the chick pineal organ during the second half of the incubation period and the first two weeks after hatching. In 200-m-thick Vibratome sections of chick-embryo pineal organs cultured in medium BM 86 Wissler for periods up to 13 days the cytodifferentiation parallels the development in vivo. Using an organ-culture system the 24-h release of melatonin into the culture medium was measured by means of radioimmunoassay after solid-phase extraction. At embryonic day 10, the 24-h secretion of melatonin was at the lower range of detection of the RIA (5 pg). The rapid increase in 24-h secretion in melatonin until hatching (50 g) is approximated by an exponential curve.Preliminary results of this study were reported at the Versammlung der Anatomischen Gesellschaft in Lübeck, 1986 (Möller 1987). Supported by the Deutsche Forschungsgemeinschaft (MO234/9-2)     

The differentiation of the decidua and the distribution of metrial gland cells in the pregnant mouse uterus

Summary A study was made with the light microscope of the cellular changes involved in the formation of the decidua in the pregnant mouse uterus up to day 11 of pregnancy. The differentiation sequence was similar to that found by previous workers but the morphology and development of the basal zone is described in more detail. In addition, the morphology of glycogen rich cells in an area termed the lateral decidual zone is described. The distribution of granulated metrial gland cells and their precursors is described. These cells are first found in the uterine stroma before the blastocyst has implanted. Later they occur in the decidua and in the circular smooth muscle zone beneath the mesometrial triangle prior to the formation of the metrial gland. Granulated metrial gland cells are also found in the maternal blood spaces of the implantation site.     

Temporal changes in the carbohydrates expressed on BG01 human embryonic stem cells during differentiation as embryoid bodies

Cell surface carbohydrates present on BG01 human embryonic stem cells after 28 days of differentiation were examined using two classes of carbohydrate binding proteins: lectins and antibodies specific for carbohydrate epitopes. Specificity of lectin staining was verified using carbohydrate ligands to block lectin interaction, glycohydrolases to cleave specific sugar residues that are receptors for these proteins, and periodate oxidation to destroy susceptible sugar residues. Specific antibodies were used to identify various tissue types and germ layers present in the 12- and 28-day differentiating embryoid bodies. Results from 12 and 28-day differentiated embryoid bodies were compared to determine changes over time. A slight increase in the sialylation of α-GalNAc was seen between 12 and 28 days of differentiation due to the presence of sialyl Tn and/or other sialylated α-GalNAc residues. Increases were also observed in GalNAc, the T antigen (Gal β1,3 GalNAc), and difucosylated LacNAc residues during this time interval. Additionally, some distinct differences in the pattern of lectin staining between 12 and 28 days were observed. Not unexpectedly, the presence of most differentiated cell-types increased during this time period with the exception of neural progenitors, which decreased. Undifferentiated cells, which were prevalent in the 12-day EBs, were undetectable after 28 days. We conclude that several changes in glycosylation occurred during the differentiation of embryonic stem cells, and that these changes may play a role in embryonic development. Lectin abbreviations can be found in Table 1.