The effect of ethanol exposure on extraembryonic vascular development in the chick area vasculosa

The effect of ethanol (EtOH) exposure on extraembryonic vascular development was examined using the chick embryo area vasculosa (AV) in shell-less culture. Embryos were placed in cultures at Hamburger Hamilton (HH) stage 11/12 and a single dose of EtOH (10, 30 or 50%) was applied to the center of the blastodisc. Untreated/sodium-chloride-treated controls showed normal embryonic growth and well-developed extraembryonic vessels at 24/48 h of treatment. At doses of 30 and 50%, the mortality rate was significantly increased, and survivors demonstrated significant growth retardation and inhibition of normal vascular development in a dose-dependent manner. Immunostaining for vascular endothelial growth factor (VEGF) showed that mesenchymal cells continued to differentiate into angioblasts to form blood islands, but their assembly into primitive vessels was perturbed in a dose-dependent manner. Northern blot analyses of basic fibroblast growth factor, VEGF, Flt-1 and Flk-1 mRNA expression supported these findings and showed a dose-dependent decrease in EtOH-treated cultures compared to controls. Co-treatment with alpha-tocopherol (0.05 M) or all-trans-retinoic acid (10(-8) M) significantly decreased the mortality rate and improved both embryonic growth and extraembryonic vascular development in the cultures. On the other hand, almost all embryos treated with 10% EtOH survived the first 48 h after treatment. However, the complexity of the vascular tree measured as the relative vasculogenesis index, the surface area of the AV and the mRNA expression of vasculogenic molecules were increased during the first 24 h. This acute effect disappeared 48 h after treatment and the vascular tree continued to develop parallel to the controls. No significant growth retardation was observed in this group. These results suggest that, in terms of extraembryonic vascular development, an early, single, low-dose EtOH exposure may have an acute, short-term positive effect, whereas moderate- or high-dose EtOH exposure may severely perturb this process disabling the necessary absorption of the nutrients for the embryo to develop properly. The mechanisms of action of EtOH on extraembryonic vascular development may involve the establishment of reactive oxygen species, resulting in the initiation of oxidative stress and perturbation of retinoic acid signaling and alterations in the expression of growth-regulatory vasculogenic factors and their receptors.     

Wtap is required for differentiation of endoderm and mesoderm in the mouse embryo.

Wilms' tumor 1-associating protein (WTAP) was previously identified as a protein associated with Wilms' tumor-1 (WT-1) protein that is essential for the development of the genitourinary system. Although WTAP has been suggested to function in alternative splicing, stabilization of mRNA, and cell growth, its in vivo function is still unclear. We generated Wtap mutant mice using a novel gene-trap approach and showed that Wtap mutant embryos exhibited defective egg-cylinder formation at the gastrulation stage and died by embryonic day 10.5. Although they could form extraembryonic tissues and anterior visceral endoderm, Wtap mutant embryos and embryonic stem cells failed to differentiate into endoderm and mesoderm. The chimera analysis showed that Wtap in extraembryonic tissues was required for the formation of mesoderm and endoderm in embryonic tissues. Taken together, our findings indicate that Wtap is indispensable for differentiation of mesoderm and endoderm in the mouse embryo.     

MAP kinase activation in avian cardiovascular development.

Signaling pathways mediated by receptor tyrosine kinases (RTK) and mitogen-activated protein kinase (MAPK) activation have multiple functions in the developing cardiovascular system. The localization of diphosphorylated extracellular signal regulated kinase (dp-ERK) was monitored as an indicator of MAPK activation in the forming heart and vasculature of avian embryos. Sustained dp-ERK expression was observed in vascular endothelial cells of embryonic and extraembryonic origins. Although dp-ERK was not detected during early cardiac lineage induction, MAPK activation was observed in the epicardial, endocardial, and myocardial compartments during heart chamber formation. Endocardial expression of dp-ERK in the valve primordia and heart chambers may reflect differential cell growth associated with RTK signaling in the heart. dp-ERK localization in the epicardium, subepicardial fibroblasts, myocardial fibroblasts, and coronary vessels is consistent with MAPK activation in epicardial-derived cell lineages. The complex temporal-spatial regulation of dp-ERK in the heart supports diverse regulatory functions for RTK signaling in different cell populations, including the endocardium, myocardium, and epicardial-derived cells during cardiac organogenesis.     

Insertional mutation of a gene involved in growth regulation of the early mouse embryo.

A transgenic mouse strain derived from embryonic stem (ES) cells infected with multiple copies of a retroviral vector carries a recessive insertional mutation resulting in prenatal lethality. A detailed histological analysis of developing embryos has shown that the mutation results in hyperplasia of both embryonic and extraembryonic ectoderm and failure of mesoderm formation in the egg cylinder stage embryo. The number of cells in each lineage of normal and mutant embryos was estimated using stereological analysis of serial sections taken from implantation sites. We observed a 2-fold increase in the number of embryonic ectoderm cells in mutant embryos at 7.5 days postcoitum (dpc). In addition, we found that mutant embryonic ectoderm cells are only 0.6 times as large as normal cells. The number of extraembryonic ectoderm cells in mutant embryos at 7.5 dpc is also increased, by almost 4-fold. Mutant extraembryonic ectoderm cells are also smaller than normal, being only two-thirds the size of wild-type cells. The mutant phenotype suggests that the gene identified by this insertional mutation plays an important role in the growth control of early embryonic lineages.     

Segregation of arterial and venous markers in subpopulations of blood islands before vessel formation.

The neuropilin-1 (np1) and the neuropilin-2 (np2) receptors bind vascular endothelial growth factor (VEGF) and class-3 semaphorins. They form complexes with VEGF tyrosine-kinase receptors or alternatively with type-A plexins to transduce respective VEGF or semaphorin signals. We have compared the expression patterns of np1, np2, plexin-A1, and plexin-A2 in the emerging vasculature of chick embryos. Double in situ hybridization reveals that six-somite embryos contain intermingled extraembryonic blood island (BI) subpopulations that express np1 or np2 as well as a BI subpopulation that coexpresses both neuropilins. In 13-somite embryos, which already contain an extraembryonic vascular plexus, the expression of np1 and np2 is segregated between the arterial and venous parts of the plexus, despite the absence of blood flow. However, the arterial marker ephrin-B2 was not yet expressed in the plexus at this stage. In 26-somite embryos, which possess a functional vascular system, np1 and np2 are differentially expressed in arteries and veins as previously reported. At this stage, posterior BIs expressing np2 appear to undergo fusion to form the posterior sinus vein and its tributaries, suggesting that the venous identity of these veins may be established before their formation. The neuropilin coreceptor plexin-A2 was expressed in extraembryonic veins but not in extraembryonic arteries. In contrast, within the embryo, plexin-A2 expression was observed in the dorsal aorta as well as in the cardinal vein. Semaphorin-3F (s3f), an np2 ligand, bound to np2-expressing cells in 26-somite embryos regardless of the presence or absence of plexin-A1 or plexin-A2. Of interest, even though s3f binds to np1 in vitro, np1-expressing arteries fail to bind s3f in whole-mount binding experiments.     

Relationship Between Physiological Umbilical Herniation and Liver Morphogenesis During the Human Embryonic Period: A Morphological and Morphometric Study

It is widely hypothesized that physiological umbilical herniation (PUH) in humans occurs, because the liver occupies a large space in the abdominal cavity, which pushes the intestine into the extraembryonic coelom during the embryonic period. We have recently shown the presence of the intestinal loop in the extraembryonic coelom in embryos with liver malformation. Here, we analyzed the relationship between the liver and the PUH at Carnegie stage 21 of four embryos with liver malformation, including two with hypogenesis (HY1, HY2) and two with agenesis (AG1, AG2), using phase-contrast X-ray computed tomography and compared them with two control embryos. The intestinal loop morphology in the malformed embryos differed from that in the control embryos, except in HY1. The length of the digestive tract in the extraembryonic coelom of the embryos with liver malformation was similar to or longer than that of the controls. The rate of intestinal loop lengthening in the extraembryonic coelom compared with that of the total digestive tract in all embryos with liver malformation was similar to or higher than that of the controls. The estimated total abdominal cavity volume in the embryos with liver malformation was considerably smaller than that of the controls, while the intestinal volume was similar. The cardia and proximal portion of the pancreas connecting to the duodenum were located at almost identical positions in all the embryos, whereas other parts of the upper digestive tract deviated in the embryos with abnormal livers. Thus, our results provided evidence that PUH occurred independently of liver volume. Anat Rec, 302:1968–1976, 2019. © 2019 American Association for Anatomy     

Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development

In vitro culture of mouse embryos results in loss of imprinting. The aim of the present study was to examine how two of the techniques commonly used during assisted reproduction, namely embryo culture and embryo transfer, affect genomic imprinting after implantation in the mouse. F1 hybrid mouse embryos were subjected to three experimental conditions: control (unmanipulated), embryo transfer and in-vitro-culture followed by embryo transfer. Concepti were collected on d9.5 of development and allelic expression determination of ten imprinted genes (H19, Snrpn, Igf2, Kcnq1ot1, Cdkn1c, Kcnq1, Mknr3, Ascl2, Zim1, Peg3) was performed. Although control concepti had monoallelic imprinted gene expression in all tissues, both manipulated groups had aberrant expression of one or more imprinted genes in the yolk sac and placenta. Culture further exacerbated the effects of transfer by increasing the number of genes with aberrant allelic expression in extraembryonic, as well as embryonic tissues. Additionally, placentae of both groups of manipulated concepti exhibited reduced levels of Igf2 mRNA and increased levels of Ascl2 mRNA when compared with their unmanipulated counterparts. Furthermore, we show that biallelic expression of Kcnq1ot1 coincided with loss of methylation on the maternal allele of the KvDMR1 locus, a phenotype often associated with the human syndrome Beckwith-Wiedemann. In conclusion, our results show that even the most basic manipulation used during human-assisted reproduction, namely, embryo transfer, can lead to misexpression of several imprinted genes during post-implantation development. Additionally, our results serve as a cautionary tale for gene expression studies in which embryo transfer is used.     

斑马鱼胚胎发育过程中miR-9、miR-219及Dicer基因的时序性表达

目的探讨斑马鱼胚胎发育过程中miR-9、miR-219及Dicer基因时序性表达规律。方法取正常发育至受精后4、8、12、16、20、24、36、48和72 h(hpf)斑马鱼胚胎各约40枚,用Trizol法分别提取总RNA,茎环法反转录为c DNA,用Real-time PCR检测胚胎miR-9、miR-219及Dicer表达量。结果 miR-9、miR-219及Dicer表达水平在胚胎发育过程中呈现峰谷变化,不同发育阶段之间总的差异有统计学意义(P0.05)。与胚胎发育全程的基因平均表达水平相比,miR-9在8 hpf之前表达量较低(P0.05),36 hpf后表达量持续增高,48 hpf和72 hpf时表达量显著高于平均水平(P0.01)。miR-219在8 hpf之前表达量也处于较低水平(P0.05),20 hpf后迅速增高,24hpf时达到峰值,并持续到48 hpf(P0.01)。Dicer在胚胎早期(4 hpf)就有明显表达(P0.05),随后表达水平迅速下降,16 hpf时达到低点(P0.05),然后逐渐回升,直至胚胎末期。结论斑马鱼胚胎发育过程miR-9、miR-219及Dicer时序性表达有一定规律,miR-9主要在胚胎后期表达,miR-219主要在胚胎中期表达,Dicer主要在胚胎早期表达,其次在胚胎后期,提示miR-9、miR-219及Dicer参与胚胎发育调控过程。     

Mouse choroideremia gene mutation causes photoreceptor cell degeneration and is not transmitted through the female germline

Choroideremia (CHM) is an X-linked progressive eye disorder which results from defects in the human Rab escort protein-1 (REP-1) gene. A gene targeting approach was used to disrupt the mouse chm/rep-1 gene. Chimeric males transmitted the mutated gene to their carrier daughters but, surprisingly, these heterozygous females had neither affected male nor carrier female offspring. The targeted rep-1 allele was detectable, however, in male as well as female blastocyst stage embryos isolated from a heterozygous mother. Thus, disruption of the rep-1 gene gives rise to lethality in male embryos; in female embryos it is only lethal if the mutation is of maternal origin. This observation can be explained by preferential inactivation of the paternal X chromosome in murine extraembryonic membranes suggesting that expression of the rep-1 gene is essential in these tissues. In both heterozygous females and chimeras the rep-1 mutation causes photoreceptor cell degeneration. Consequently, conditional rescue of the embryonic lethal phenotype of the rep-1 mutation may provide a faithful mouse model for choroideremia.      

大鼠海马发育过程中Rho GTPases相关信号分子mRNA的表达

目的探讨Rho GTPases相关信号分子的发育规律。方法RT-PCR法检测大鼠发育不同阶段Rho-A、Rac-1、CRMP-1和Tub β3mRNA的表达。结果RT-PCR实际扩增长度与设计长度相吻合。内参照β-actin的PCR产物电泳条带在各个阶段均呈高表达。Rho-A和Rac-1的表达有相似的变化规律,随着海马发育呈明显的阶段性,表达的高峰在胚胎、幼年和老年阶段,而新生阶段和成年阶段处于相对低表达阶段;Rac-1的表达量在海马发育的各个阶段均超过Rho-A的表达量。CRMP-1和Tubβ3的表达从胚胎到成年的各个阶段与CRMP-1的表达具有相似的变化趋势,以胎鼠、新生大鼠和幼年大鼠表达较多,但老年阶段CRMP-1的表达较成年阶段显著升高(P<0.05),而Tubβ3在老年阶段停留在成年阶段的水平。结论大鼠海马发育过程中,Rho-A和Rac-1呈阶段性高表达,CRMP-1和Tubβ3的表达从胚胎到成年逐渐减少,但老年阶段CRMP-1表达再次增多。     

Neurocan in the embryonic avian heart and vasculature

The chondroitin sulfate proteoglycan (CSPG) neurocan was previously considered to be nervous-system specific. However, we have found neurocan in the embryonic heart and vasculature. In stage 11 quail embryos, neurocan was prominently expressed in the myocardium, dorsal mesocardium, heart-forming fields, splanchnic mesoderm, and vicinity of the extraembryonic vaculature, and at lower levels in the endocardium. A comparison of neurocan staining with QH1 staining of vascular endothelial cells demonstrates that neurocan is frequently expressed by cells adjacent to endothelial cells, rather than by endothelial cells themselves. In some cases, a dispersed subset of cells are neurocan-positive in a field of cells that otherwise appear uniform in morphology. Later in development, neurocan expression becomes relatively limited to the nervous system. However, even in 10-day embryos, neurocan is expressed in the chorio-allantoic membrane in the tissue that separates closely packed, small-diameter blood vessels. In summary, our results suggest that neurocan may function as a barrier that regulates vascular patterning during development.     

Proteins in the extraembryonic matrix of preimplantation rabbit embryos

Preimplantation embryos of several species are surrounded by an extraembryonic matrix (often simply named zona pellucida) until briefly before implantation. All signals of the early embryo-maternal dialogue have to pass this matrix and therefore are detectable inside. We investigated the protein pattern of the extraembryonic matrices of 3-6-day-old rabbit embryos by two-dimensional gel-electrophoresis. Using (35)S-methionine incorporation, embryonic proteins were labelled and could be distinguished from maternal proteins. Furthermore, the presence of three different proteins (insulin-like growth factor-binding protein-3, uteroglobin, haptoglobin) within the matrices of day-6 embryos was investigated by Western blot analysis. The pattern and numbers of protein spots detected was clearly dependent on the time of embryonic development. Of all proteins detected, 19.3% and 33% are of embryonic origin (day 5 and day 6, respectively). At day 4 the zona proteins are no longer detectable, reflecting the degradation of the zona pellucida. From day 4 to day 5 proteins detectable within the extraembryonic matrices increase enormously. This demonstrates that embryo-maternal signalling accelerates at least 2 days before implantation. Insulin-like growth factor-binding protein-3, uteroglobin and haptoglobin are part of the early signalling as shown by Western blot analysis. Insulin-like growth factor-binding protein-3 could be detected as one spot at 38 kDa pI 6.1, uteroglobin at 8 kDa pI 6.0 and haptoglobin as two spots/isoforms at 36/38 kDa pI 5.8 and pI 6.0. These results demonstrate that extraembryonic matrices serving as a mailbox are a valuable tool for investigating early embryo-maternal signalling.