High resolution ultrasound-guided microinjection for interventional studies of early embryonic and placental development in vivo in mice

Background  

In utero microinjection has proven valuable for exploring the developmental consequences of altering gene expression, and for studying cell lineage or migration during the latter half of embryonic mouse development (from embryonic day 9.5 of gestation (E9.5)). In the current study, we use ultrasound guidance to accurately target microinjections in the conceptus at E6.5–E7.5, which is prior to cardiovascular or placental dependence. This method may be useful for determining the developmental effects of targeted genetic or cellular interventions at critical stages of placentation, gastrulation, axis formation, and neural tube closure.     

The functional regeneration of syncytiotrophoblast in cultured explants of term placenta

We have investigated the functional characteristics of term human placental villous explants kept in long-term (7-11 days) culture. Fragments of placental villous tissue (approximately 5-10 mg wet wt) were cultured in supplemented CMRL-1066 culture medium for up to 11 days. After the first day of culture, the syncytiotrophoblast appeared vacuolated and eventually degenerated. However, a new syncytiotrophoblast developed by day 4, being indistinguishable from that of a fresh placenta by 11 days. Release of human chorionic gonadotrophin increased and activity of lactate dehydrogenase in culture medium decreased with culture time. Transport variables were measured over the first 7 days of culture. Basal (86)Rb efflux was reduced with time in culture and was inhibited by Ba2+, suggesting the efflux was mediated by K+ channels. At all stages of culture, (86)Rb efflux was stimulated by ATP, hyposmotic medium, and ANG II. A complex pattern of efflux changes with culture time and type of stimulator was observed, suggesting that several compartments of the tissue contributed to stimulated efflux. This culture system provides opportunities for studies of chronic regulation of placental function.     

遗传谱系示踪技术揭示小鼠胚胎前体心外膜向心外膜转化过程

心外膜的形成是胚胎心脏发育的关键生理过程之一。利用遗传谱系示踪技术示踪观察前体心外膜向心外膜细胞转化过程,具有重要的科学研究价值。本研究拟利用Tbx18+前体/心外膜祖细胞遗传谱系示踪模型,揭示胚胎心外膜的起源及前体心外膜向心外膜转化的过程。利用整胚和切片原位杂交技术揭示,Tbx18 mRNA特异性表达于胚龄（E）9.5 d小鼠胚胎前体心外膜;故Tbx18是前体心外膜的特异性标记基因。利用整胚X-Gal染色,揭示报告基因Lacz在E9.5 d遗传谱系示踪模型鼠胚前体心外膜中大量表达,此时报告基因从前体心外膜逐渐迁移并开始少量表达于心外膜。Lacz在E10～E10.5 d双杂合鼠胚前体心外膜中表达逐渐减少,而在心外膜组织中逐渐增多;在E11.5 d,报告基因在前体心外膜中表达基本消失,而在心外膜组织中大量表达。切片进行X-Gal染色也揭示,报告基因Lacz定位于早期胚胎前体心外膜及心外膜。免疫荧光染色证实,早期胚胎心外膜细胞呈现未分化的祖细胞状态。通过报告基因的表达变化模式揭示,胚胎心外膜的形成经历了启动、转化、完成3个阶段;E9.5～11.5 d左右这个时间段发生的前体心外膜向心外膜转化,可能是心外膜形成的主要来源和形式。