Regulation of proliferation of embryonic heart mesenchyme: role of transforming growth factor-beta 1 and the interstitial matrix

Proliferation of atrioventricular cushion mesenchyme of the embryonic avian heart maintained in three-dimensional aggregate culture is stimulated by interaction with the interstitial matrix. Chicken serum or transforming growth factor-beta 1, which stimulates proliferation, induces matrix deposition in regions of the aggregate showing high labeling indices with tritiated thymidine. Dispersed heart mesenchyme interstitial matrix introduced into serum-free culture is incorporated into the aggregate and stimulates cellular proliferation similar to serum or transforming growth factor-beta 1. Proliferation is reversibly inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro. It is suggested that transforming growth factor-beta 1 stimulates the production of interstitial matrix and that a sufficient stimulus for proliferation in this system is the presence of the matrix, which acts as the adhesive support for cellular anchorage.     

Regional epicardial strain in the embryonic chick heart during the early looping stages

Epicardial strains were measured in Hamburger-Hamilton stage 11 and 12 embryonic chick hearts (1.6-2.0 days of incubation). These stages include part of the early phase of cardiac looping, as the initially straight heart tube bends and twists to form a curved c-shaped tube. By analyzing the motion of microbeads placed on the myocardial surface, we measured strains near the outer curvature, in the central region, and near the inner curvature of the primitive ventricle. No significant differences in strain were found between stages. Relative to end diastole, all three regions shortened by about 10% during systole in the circumferential direction, and the outer curvature shortened longitudinally by about 5%. In contrast, and unlike strains in older hearts, the inner curvature and central regions elongated by approximately 5-10% in the longitudinal direction during systole. These results are consistent with microstructural data and suggest that the material properties of the outer curvature are relatively isotropic, whereas the properties of the central and inner curvature regions are orthotropic, with contractile stress exerted primarily in the circumferential direction.     

Differentiation of ovarian and testicular interstitial cells during embryonic and post-embryonic development in mice

Summary Different steps in mouse ovarian and testicular development have been studied in order to compare the time sequences during the in vivo differentiation of steroidogenic cell populations growing in contact with male and female gonocytes. These time sequences indicated a basic common developmental pattern: early signs of steroid synthesis in the male gonad, but late entering into meiotic prophase of XY germ cells; early meiosis but late steroidogenic activity in the ovary. In both male and female interstitial tissues, signs of involution were found following a period of exponential development.Dedicated to Prof. Dr. med. H. Leonhardt on the occasion of his 60th birthdaySupported by the Deutsche Forschungsgemeinschaft (Pe 104/8)We wish to thank Dr. B. Nabarra, M.-F. Rousseau-Merck and D. Sandoz for helpful advices throughout this study, as well as Mrs. L. Andrianarison and Mrs. R. Sprang for skilful assistance     

Primary mesenchyme cell patterning during the early stages following ingression

Sea urchin primary mesenchyme cells (PMCs) ingress into the blastocoel during an epithelial-to-mesenchymal transition (EMT), migrate along the blastocoelar wall for a period of time, and then settle into a subequatorial ring to form the larval skeleton. Fluorescent-marked blastomeres alone, or in combination with blastomere recombination, were used to track the position of PMCs during the early phases of this movement. Micromeres expressing Golgi-tethered GFP (galtase-GFP) were transplanted onto TRITC-stained hosts (in place of the endogenous micromere) to observe the progeny of a single micromere. Galtase-GFP as a Golgi marker is not transferred between PMCs when the syncytium forms. Thus, the position of cells can be followed relative to beginning position for longer periods than previously reported. The PMC progeny of a single micromere do not disperse upon ingression, but instead remain in a closely associated cluster. Generally, progeny of a single micromere remain in the quadrant of origin. In total, greater than approximately 94% of labeled PMCs remain within the local region of ingression. By contrast, when a transplanted micromere is placed at the vegetal plate after removing all 4 host micromeres, the resultant PMCs ingress and migrate into all 4 quadrants. Similarly, if 1 blastomere is injected at the 2-cell stage, and later the 2 unlabeled micromeres are removed at the 16-cell stage, the remaining PMCs ingress into all 4 quadrants of the vegetal plate. We conclude that the normal restriction of PMCs to a quadrant is due to mechanical constraint from other micromere-PMCs. If a labeled micromere is placed ectopically at the macromere/mesomere boundary, the PMC progeny ingress ectopically and migrate longitudinally along the animal-vegetal axis only. Injection of galtase-GFP into one blastomere at the 4-cell stage shows a 2-step pattern of localization. At late mesenchyme blastula and early gastrula stages, greater than 90% of GFP-expressing PMCs remain in the injected quadrant, while at mid- to late-gastrula stage and beyond, more PMCs are found outside the injected quadrant. The migration that sets up the asymmetry of the larval skeleton first occurs around mid- to late-gastrula stages, when some PMCs from an aboral quadrant migrate to the adjacent oral quadrant. In all, these data combined with previous data suggest that freshly ingressed PMCs migrate along a longitudinal path toward the animal pole and back toward the vegetal pole. Beginning at mid- to late-gastrula stage, PMCs utilize oral-aboral cues from the ectoderm for the first time. At this time, some aboral PMCs migrate into the adjacent oral quadrant to assist in the formation of the ventrolateral cluster.     

The development of the early atrioventricular conduction system in the embryonic heart

Recent electrophysiological evidence indicates that periodic spontaneous depolarizations occur in the primordial heart of the bird (and presumably mammal) even before the myocardial cells can contract, and these are initiated in the primordial sinoatrial region. As contractions are generated, these then establish a peristaltic wave. From that time on, during ontogenesis, the contractile sequence follows a regular pattern of development. As chambers form they contract sequentially in the direction of blood flow, even though, in the twisted configuration, myocardial continuities suggest the possibility of short-circuiting the electrical conduction pathways from atrium to bulbus. This implies that, even at these early stages, the electrical properties of the myocardium are not isotropic, and that specialized conduction pathways must exist. To the present time, electrophysiological techniques have limited the direct evidence that can be obtained on these delicate electrically specialized pathways. However, microscopical techniques have permitted studies on the morphological development of the tissue and of the cells in the various regions of the myocardium. The present paper traces the development of cell morphology in these regions, including the development of structural nodes and proximal ventricular fibre pathways, and from these observations, the manner in which the electrical conduction pathways are believed to develop is suggested.     

胚胎发育早期接种人血清白蛋白诱导鸡的免疫耐受

外源基因的引入及表达常会引发宿主动物强烈的免疫应答,导致蛋白产量的下降或引起炎症反应。本研究试图通过在胚胎发育早期接种异源抗原诱导动物产生免疫耐受来解决这些问题。为了诱导鸡产生免疫耐受,将人血清白蛋白（Human serum albumin,HSA）通过胚胎血管微注射的方式接种到发育65—67h的鸡胚血管中,接种剂量为50μg;通过卵黄注射的方式接种到发育3—7d的鸡胚卵黄中,接种剂量为100μg。孵出的小鸡在3周龄时按照常规免疫方法再次接种同种抗原,收集不同时期的血清样本,用酶联免疫吸附试验（Enzyme-linked immunosorbent assay,ELISA）检测血清中抗-HSA抗体水平。结果表明,两种接种方式均能诱导小鸡产生免疫耐受：在65-67h的胚胎中通过血管微注射法接种抗原诱导小鸡产生免疫耐受的比率为64．52％;通过卵黄注射接种抗原诱导小鸡产生免疫耐受的最佳接种时间为发育的第6d,第5、7d接种对后期血清中抗-HSA抗体形成也有一定抑制作用,但是维持耐受的时间较短,第3、4d接种抗原对诱导小鸡免疫耐受的效果不明显[动物学报51（5）：845—851,2005]。     

Immunolocalization of matricial components during the early stages of chick embryonic liver development

In the chick embryo, the first liver primordium is observed at the end of the second day of incubation. At 3 and 4 days, ultrastructural analysis of the primitive vascular spaces showed that the endothelial limiting plate was constituted by one or several cell layers. At the vascular pole of the hepatoblasts, mesenchymal cells and connective matrix, present as fibrillar and non fibrillar components, were closely associated. At 5 days, some vascular spaces were limited by a simple endothelial layer. The limiting plate was fenestrated and the connective matrix was reduced to rare collagen fibrils and fibers. Collagen types I, III, IV, procollagen type III, fibronectin and laminin were visualized in the perivascular spaces using immunoperoxidase labeling methods. These components were also detected in the endoplasmic reticulum of hepatoblastic, endothelial and mesenchymal cells. All these appeared to be involved in connective matrix synthesis. Comparing 4 and 5 days, we demonstrated that the number of cells showing intracellular labelling of matricial components dropped dramatically at 5 days, indicating a possible decrease of connective matrix synthesis. Quantification of parenchymal and vascular surfaces was carried out using a semi-automatic image analyzer on consecutive parasagittal sections chosen in the axial part of the embryonic liver. These measurements were performed in order to quantitate the vascular distribution pattern during early development of the liver. These combined immunomorphological studies and morphometrical analyses suggest that during embryogenesis of the liver the synthesis of connective matrix precedes and possibly initiates the vascular differentiation.     

Second heart field and the development of the outflow tract in human embryonic heart

The second heart field (SHF) is indicated to contribute to the embryonic heart development. However, less knowledge is available about SHF development of human embryo due to the difficulty of collecting embryos. In this study, serial sections of human embryos from Carnegie stage 10 (CS10) to CS16 were stained with antibodies against Islet‐1 (Isl‐1), Nkx2.5, GATA4, myosin heavy chain (MHC) and α‐smooth muscle actin (α‐SMA) to observe spatiotemporal distribution of SHF and its contribution to the development of the arterial pole of cardiac tube. Our findings suggest that during CS10 to CS12, SHF of the human embryo is composed of the bilateral pharyngeal mesenchyme, the central mesenchyme of the branchial arch and splanchnic mesoderm of the pericardial cavity dorsal wall. With development, SHF translocates and consists of ventral pharyngeal mesenchyme and dorsal wall of the pericardial cavity. Hence, the SHF of human embryo shows a dynamic spatiotemporal distribution pattern. The formation of the Isl‐1 positive condense cell prongs provides an explanation for the saddle structure formation at the distal pole of the outflow tract. In human embryo, the Isl‐1 positive cells of SHF may contribute to the formation of myocardial outflow tract (OFT) and the septum during different development stages.     

Essential function of PTP-PEST during mouse embryonic vascularization, mesenchyme formation, neurogenesis and early liver development

PTP (protein-tyrosine phosphatase)-PEST is a ubiquitously expressed cellular regulator of integrin signalling. It has been shown to bind several molecules such as Shc, paxillin and Grb2, that are involved downstream of FAK (focal adhesion kinase) pathway. Through its specific association to p130cas and further dephosphorylation, PTP-PEST plays a critical role in cell-matrix interactions, which are essential during embryogenesis. We report here that ablation of the gene leads to early embryonic lethality, correlating well with the high expression of the protein during embryonic development. We observed an increased level of tyrosine phosphorylation of p130cas protein in E9.5 PTP-PEST(-/-) embryos, a first evidence of biochemical defect leading to abnormal growth and development. Analysis of null mutant embryos revealed that they reach gastrulation, initiate yolk sac formation, but fail to progress through normal subsequent developmental events. E9.5-10.5 PTP-PEST(-/-) embryos had morphological abnormalities such as defective embryo turning, improper somitogenesis and vasculogenesis, impaired liver development, accompanied by degeneration in both neuroepithelium and somatic epithelia. Moreover, in embryos surviving until E10.5, the caudal region was truncated, with severe mesenchyme deficiency and no successful liver formation. Defects in embryonic mesenchyme as well as subsequent failure of proper vascularization, liver development and somatogenesis, seemed likely to induce lethality at this stage of development, and these results confirm that PTP-PEST plays an essential function in early embryogenesis.     

L-DOPA supply to the neuro retina activates dopaminergic communication at the early stages of embryonic development

DOPA decarboxylase (DDC; aromatic-l-amino acid decarboxylase; EC 4.1.1.28) is absent in retinas from 6-day-old chicken embryos (E6) but is expressed in retina of E8 embryos, in the presumptive outer plexiform layer. Thereafter, DDC appears in cell bodies of presumptive amacrine cells. The dopamine (DA) content of E9/10 and E15/16 retinas, pre-incubated with l-DOPA for 1 h, increased 250- and 600-fold, respectively, showing that DDC is active since early in development. Intercellular communication, measured by endogenous cyclic AMP accumulation, was observed when retinas from E9/10 to E15/16 were pre-incubated for 1 h with 1 mm l-DOPA, washed and followed by incubation in the presence of 0.5 mm 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. Cyclic AMP accumulation was prevented when pre-incubation with l-DOPA was carried out in the presence of carbidopa. Moreover, the accumulation of cyclic AMP was inhibited by SCH 23390 (2 micro m). The incubation of retinas in medium previously conditioned by retina-pigmented epithelium (RPE) also increased its cyclic AMP content with the characteristics described for l-DOPA. Our results show that dopaminergic communication takes place in the embryonic retina, before tyrosine hydroxylase expression, provided l-DOPA is supplied to the tissue. It also shows that RPE is a potential source of l-DOPA early in development.     

Local expression of cytokeratins 8, 17 and 18 in the mesenchyme and smooth muscles in the early stages of human organogenesis

Expression of cytokeratins 7, 8, 17, 18 in human embryos and fetuses of 6.5-13 weeks was studied using light and electron immunocytochemistry and immunoelectroblotting with the monoclonal antibodies. Cytokeratins 8 and 18 were expressed in 6.5-8 week old embryos not only in epithelium but also in mesenchyme of allantois, urogenital sinus, Wolffian and Mullerian ducts, mesentery, urinary bladder and certain regions of colon, rectum and atrium cordis walls. Furthermore, starting from the 10th week smooth-muscle cells of ring layer in caudal part of rectum bound antibodies against cytokeratin 17 in addition to those against cytokeratins 8 and 18. Corresponding mesenchymal and smooth-muscle cells of adult individuals did not react with either of them. Cytokeratins were still synthesized when mesenchymal cells of embryonic intestine wall were cultivated in vitro. Intermediate filaments of these cells contain cytokeratins 8 and 18, as demonstrated by electron immunocytochemistry and immunoelectroblotting. Thus, the expression of cytokeratins is not restricted to adult and embryonic epithelial tissues but is also characteristic of mesenchyme and smooth muscle differentiation in human embryos and fetuses.