Postimplantation mouse embryos cultured in vitro : Assessment with whole-mount immunostaining and in situ hybridization

Van Maele, Geneviève [UCL] Clotman, Frédéric [UCL] Gofflot, Françoise [UCL] Bosschaert, Johan Picard, Jacques [UCL]

The postimplantation embryos of rodents have been particularly convenient to study in culture using the whole embryo culture (WEC) system developed by New. Two serious limitations of the method will be illustrated in the present paper and proposals will be made to improve the quality of the information. The first limitation is that the developmental period amenable to culture has not been significantly extended in recent years. In the present paper, we show that the culture of mouse presomitic stages for 48 h leads to poorly reproducible results and frequent dysmorphogenic embryos. We also show that early somite stages cultured for 54 h or less have a normal growth and differentiation. In contrast, the culture of these embryos for 72 h results in subtle abnormalities of the head and the first branchial arch. The second limitation is that the gross morphology and histology are often not informative enough to distinguish between overall toxicity and developmental toxicity. We suggest some improvements by the association of WEC with two specific techniques: 1) whole-mount immunostaining of sensory ganglia and nerves and 2) in situ hybridization on histological sections using molecular probes for some developmental genes. Embryos reaching about the 30 somite stage at the end of the culture were processed for whole-mount immunostaining of sensory ganglia and nerves. We show that these structures are very sensitive to the noxious effects of HgCl2 and valproate. Both developmental retardations and dysmorphogeneses of the cervical ganglia and nerves were observed. Embryos were also exposed in vitro to low concentrations of all-trans-retinoic acid (AT-RA) and processed for in situ hybridization with radiolabeled anti-sense RNA probes for the Hoxb-1 and Hoxb-2 developmental genes. Three-dimensional reconstructions of the expression domains were performed. The data show that AT-RA induces ectopic expression domains of Hoxb-1. Our experiments demonstrate that techniques such as immunostaining and in situ hybridization can significantly expand the information obtained from whole postimplantation embryo culture.