The transcription factor Gli3 is an important regulator of the development of the forebrain during embryogenesis. Gli3 is expressed throughout the developing forebrain and Gli3-/-mutant embryos have severe forebrain abnormalities, including the absence of dorsal midline structures such as the hippocampus, and a massively reduced cerebral cortex. We have tried to work out exactly how Gli3 regulates forebrain development. Defects in Gli3-/- embryos may arise in at least two possible ways – Gli3 protein may directly affect the behaviour of cells in which it is expressed (cell-autonomous effects) or it may affect development of neighbouring cells, perhaps through changes in expression of signalling molecules (cell non-autonomous effects). It is likely that a combination of cell autonomous and non-autonomous effects underlies the Gli3-/- phenotype. We generated a set of Gli3-/-:Gli3+/+ chimaeric embryos to separate these effects. In chimaeras with a large contribution from wild type cells, Gli3-/-cells are surrounded by wild type neighbours, so any defects found in mutant cells are most likely due to cell-autonomous effects of Gli3. We found that Gli3-/- cells are able to contribute to all parts of the chimaeric forebrain, including dorsal midline structures that are absent in Gli3-/- mutants, in proportions that are at least as high as those seen in other parts of the embryo. Mutant cells tended to form clumps and segregated from wild-type cells, suggesting altered cell-adhesion properties. Many mutant cells also failed to express appropriate markers of cell fate. Interestingly, the ability of Gli3-/-cells to adopt appropriate fates depended on their location in the chimaeric forebrain. Mutant cells in the ventral telencephalon and in the diencephalon expressed the same marker proteins as their wild-type neighbours, whereas mutant cells close to boundaries between the telencephalon and diencephalon or the dorsal and ventral telencephalon expressed markers found in wild-type cells on the other side of the boundary. As boundaries between emerging forebrain domains commonly act as secondary signalling centres, we hypothesise that our findings indicate a cell-autonomous requirement for Gli3 to allow forebrain cells to respond correctly to signals that promote specific differentiation. These findings have now been published in Developmental Biology.
We further examined development of the dorsal forebrain in Gli3-/- mutants. Previous work from other labs suggested that ventral forebrain areas are expanded in Gli3-/- embryos, at the expense of dorsal forebrain. We found that cerebral cortex is almost entirely missing in mutant embryos and ventral forebrain tissue is joined abnormally to diencephalic tissue. We found that many markers of normal ventral telencephalon are also normally expressed in diencephalon. Thus, much of the expansion of ventral marker gene expression in the mutant forebrain reported by others is actually due to the presence of diencephalic cells. Our findings indicate that Gli3 is of central importance for formation of the telencephalic-diencephalic boundary. We published these findings in J. Neuroscience in 2006.
We also examined early development of the ventral forebrain in Gli3-/- embryos. We found that the mutant ventral telencephalon is expanded at embryonic day (E)10.5 but by E12.5, is smaller than in wild-types. We showed that the relative reduction in size that occurs between E10.5 and E12.5 is probably due to increased apoptosis and premature differentiation of neural precursors. This leads to a distortion of the overall shape of the mutant forebrain, such that the diencephalon extends much further rostrally in mutant embryos. This work was published in J. Comp Neurol.
Finally, we characterised antibodies against two regionally-expressed transcription factors used as tissue-specific markers in the experiments outlined above, leading to publications in BMC Dev Biol and Biochim Biophys Acta.