Patroonvorming van de achterzijde van de neurale plaat
02 / 2002 - onbekend
Website Hubrecht Laboratorium
The intention for this main line of research is to elucidate molecular cascades which pattern the main body axis in vertebrates. We use Xenopus as the main experimental system. A specific biological focus is investigating the identities and modes of action of signalling pathways which specify different a/p levels in the very early neural plate during gastrulation and subsequent stages, where there is evidence for two classes of signals. Activation signals from anterior dorsal mesoderm induce gastrula ectoderm to differentiate to anterior neural plate. Transformation signals from posterior mesoderm posteriorise anterior neural plate, thus generating the axial pattern of the central nervous system. Recently, we have focused on regionalisation of the posterior neural plate. The following aspects have been investigated. 1. The importance of known signalling pathways for posterior neural patterning: retinoids, FGFs and wnts. Nine years ago, we discovered that retinoids posteriorise the early vertebrate neural plate (Durston et al., Nature, 340, 140, 1989). Subsequent investigations by ourselves (gain and loss of function experiments using receptors and binding proteins, partly collaboration with P.T. van der Saag) (Dekker et. al, Development, 120, 973, 1994, van der Wees et al, Development, 125, 545, 1998) and by others indicate a role for retinoids in hindbrain patterning. Our findings fit involvement of a retinoid gradient (Godsave et al, Dev. Dynamics in press). We identified two new endogenous retinoid ligands from the gastrula embryo (Pijnappel et al. Nature, 366, 340, 1993, PNAS. US. accepted). One, which is extraordinarily active and probably mediates axial patterning in vivo, has novel molecular properties, being a RAR/RXR synergist, which selectively activates RXRs in RAR/RXR heterodimers. We are investigating localisation/function of a P450 enzyme which may generate this ligand (de Roos et al, Mech Dev.,82, 205, 1999). Investigations by ourselves (Godsave et al., Int. J. Dev. Biol., 41, 57, 1997) and others implicate FGFs in specifying the very posterior (spinal cord) region of the neural plate. Results from two other groups implicate wnts in posterior neural patterning. We (with O. Destrée) are now investigating this aspect (see below). 2. Hox induced signalling. Signals regulating posterior neural patterning act partly by regulating Hox gene expression. Some Hox genes are direct retinoid targets in the neural plate. Early development of the Xenopus neural plate is characterised by a spatially colinear Hoxb expression sequence which arises in a temporal sequence which is also colinear with the genomic Hox sequence (Dekker et al Development Suppl. 195, 1992, Mech. of Devl. 40, 3, 1993, Godsave et al, Dev. Biol. 166, 465, 1994). Our present investigations focus on this early Hox establishment phase. We discovered (for the first time in a vertebrate), that a subset of Hox genes including Hoxb-4, interact in very early neural plate via cell to cell signalling (to induce non cell autonomous changes in the Hox code). They (a) autoregulate their own expression, (b) colinearly activate expression of more 5' posterior Hox genes, (c) colinearly repress expression of more 3' anterior Hox genes (submitted for publication). Our main drive now is to determine the functions and mechanisms of these interactions. This involves screening for Hoxb-4 downstream targets (in connection with investigating the signalling mechanism), application of Xenopus transgenesis (with O. Destrée) to investigate relevance of identified enhancers for posterior vertebrate Hox genes (collaboration with J. Deschamps, R. Krumlauf), investigating leads which indicate to us that (time/space dependent) cofactor interactions with particular functional domains in Hox proteins are of central importance for these interactions. 3. New genes. We have screened/are screening for new neural patterning genes. We used expression cloning, as a functional screen for genes which modulate anteroposterior neural patterning; this approach identified a new pathway mediating the early morphogenetic (convergence extension) difference between the brain and the posterior neural plate (Morgan et al., Mech. Dev., 85, 97, 1999 and in press). We and O. Destrée (see below) are using subtractive PCR/differential display to identify new targets in patterning cascades. Our interest is Hoxb-4 targets. One putative target is a wnt. We will (with O. Destrée) investigate whether wnts mediate non cell autonomous vertebrate Hox/Hox interactions. Based on findings in Drosophila concerning wg/en and wg/ubx and in vertebrates concerning wnt/en, we are interested in the idea that autoregulatory circuits involving different Q50 homeoproteins and wnt signalling pathways organise different anteroposterior levels in the neural plate (Morgan et al., Trends in Genetics., in press). At a later stage, we will (with O. Destrée) set up appropriate two hybrid screens, to identify partners for Hoxb-4 and its cofactors.