Henk Roelink

Wnt signaling is required at distinct stages of development for the induction of the posterior forebrain

One of the earliest manifestations of anteroposterior pattering in the developing brain is the restricted expression of Six3 and Irx3 in the anterior and posterior forebrain, respectively. Consistent with the role of Wnts as posteriorizing agents in neural tissue, we found that Wnt signaling was sufficient to induce Irx3 and repress Six3 expression in forebrain explants. The position of the zona limitans intrathalamica (zli), a boundary-cell population that develops between the ventral (vT) and dorsal thalamus (dT), is predicted by the apposition of Six3 and Irx3 expression domains. The expression patterns of several inductive molecules are limited by the zli, including Wnt3, which is expressed posterior to the zli in the dT. Wnt3 and Wnt3a were sufficient to induce the dT marker Gbx2 exclusively in explants isolated posterior to the presumptive zli. Blocking the Wnt response allowed the induction of the vT-specific marker Dlx2 in prospective dT tissue. Misexpression of Six3 in the dT induced Dlx2 expression and inhibited the expression of both Gbx2 and Wnt3. These results demonstrate a dual role for Wnt signaling in forebrain development. First, Wnts directed the initial expression of Irx3 and repression of Six3 in the forebrain, delineating posterior and anterior forebrain domains. Later, continued Wnt signaling resulted in the induction of dT specific markers, but only in tissues that expressed Irx3.

Evidence for a role of vertebrate Disp1 in long-range Shh signaling

Dispatched 1 (Disp1) encodes a twelve transmembrane domain protein that is required for long-range sonic hedgehog (Shh) signaling. Inhibition of Disp1 function, both by RNAi or dominant-negative constructs, prevents secretion and results in the accumulation of Shh in source cells. Measuring the Shh response in neuralized embryoid bodies (EBs) derived from embryonic stem (ES) cells, with or without Disp1 function, demonstrates an additional role for Disp1 in cells transporting Shh. Co-cultures with Shh-expressing cells revealed a significant reduction in the range of the contact-dependent Shh response in Disp1−/− neuralized EBs. These observations support a dual role for Disp1, not only in the secretion of Shh from the source cells, but also in the subsequent transport of Shh through tissue.

Transient expression of the proto-oncogene int-1 during differentiation of P19 embryonal carcinoma cells.

In mouse embryos, the int-1 proto-oncogene is transiently expressed in areas of the developing neural system. Retinoic acid-treated P19 embryonal carcinoma cells have often been used as an in vitro model for the molecular basis of neural development. We shown here that int-1 is transiently expressed in differentiated P19 cells. The time course and retinoic acid dose dependence of int-1 expression suggest that the gene is specifically expressed during early neural differentiation. P19 cells may be a useful model to assist in the study, at the cellular level, of the role of int-1 in neural development.

Using mRNA in Situ Hybridization to Localize Wnt-3 and Wnt-3A Expression in Developing Neural Tube

Publisher Summary This chapter discusses the using mRNA in situ hybridization to localize Wnt-3 and Wnt3A expression in developing neural tube. RNA in situ hybridization allows localization of specific mRNAs in tissue sections. In this procedure, radioactively labeled probes are hybridized to mRNA. Distribution of the hybridized probes is visualized after autoradiography to a photographic emulsion coating. The hybridized section is counter-stained and a microscopic analysis of the localization of the hybridization signal can reveal expression at the level of a few cells. The whole procedure can take, depending on the exposure times, from 2 to over 4 weeks. In a study described in the chapter, it was found that Wnt-3 gene is broadly expressed in the dorsal portion of the neural tube, with a rostral boundary in the dorsal thalamus. Within the telencephalon, Wnt-3 A was highly expressed in areas just dorsolateral of the telencephalic flexure. At day 12.5, the expression pattern of Wnt-3 and Wnt-3A was similar to the pattern found in day-11.5 embryos. In the brain, Wnt-3A and the spinal cord expression was observed in the dorsal midline up to the diencephalon, except in the hindbrain, where, as at earlier times, a band of midline expression bifurcated to accommodate the diencephalic roof.