P2 An immunocytochemical and in situ hybridisation study of embryonic development of the human diencephalon.
Authors: CLOWRY G. , LISGO S. , HAGAN D.‐M. , ROBSON S. , SRACHAN T. , LINDSAY S. 
Published: March 2003

In order to understand gene expression patterns in human embryos for a number of genes known to be of importance to normal brain development and to underlie certain developmental disorders, we have supplemented our in situ hybridisation studies with immunostaining for a number of developmental markers. Here, we present expression patterns for the transcription factors PAX6, PAX3 and OTX2, the ventralising signalling molecule sonic hedgehog (SHH) and immunostaining for nestin (a radial glia marker), β‐tubulin (marker for postmitotic neurons), MAP2 (a marker for differentiating neurons) and GAP43 (a marker for growing axons) in the human diencephalon between Carnegie stages 15 and 21 (33–52 postovulatory days). Human embryos were taken with maternal consent following termination according to Local Ethics Committee Regulations. They were fixed by immersion in paraformaldehyde, embedded in paraffin and oblique transverse serial sections were cut. In situ hybridisation was carried out using radioactively labelled RNA probes, and immunocytochemistry was performed with well characterised, commercially available antibodies using standard methods. SHH expression was confined to the basal plate with the exception of the zona limitans intrathalamica (ZLI) where a band of expression formed a boundary between the alar plates of prosmeres 2 (dorsal thalamus) and 3 (ventral thalamus). The ZLI was bounded by PAX6 and OTX2 hybridisations and was the site of unusually strong β‐tubulin immunoreactivity in the ventricular layer. Nestin immunoreactivity was strong in the ventricular zone and also in alar plate regions where a radial arrangement of glial fibres could be seen. In basal plate regions nestin positive glial fibres were not so evident. However MAP2 immunoreactivity was prevalent in basal regions showing that neuronal development is further advanced compared to alar regions. GAP43 immunostaining indicated that development of longitudinal axon pathways occurs predominantly through the ventral regions of the forebrain at these stages. Ventral thalamus development precedes dorsal thalamus development as shown by its thinner ventricular layer and a thicker intermediate layer that is also strongly MAP2 immunoreactive. The outgrowth of the thalamocortical projection from dorsal thalamus through the ventral thalamus had not begun at this age, judging by the lack of GAP43 immunoreactivity. In addition PAX6 expression was seen in both intermediate and ventricular layers in the ventral thalamus. PAX3 expression was confined to the roofplate region of prosomeres 1 and 2 corresponding to the pretectum and epithalamus. Therefore our results indicate that by CS21, human diencephalon development is less a advanced than the mouse diencephalon at the generally accepted equivalent stage (embryonic day 13–13.5). However development of the human diencephalon was found broadly to conform to the prosomeric model of forebrain development of Puelles & Rubinstein (Puelles, Trends Neurosci.16, 1993).