MATERIALS AND METHODS 2.1 Mouse mutants
4.3 Role of Sip1 in establishing the morphology of the neocortex .1 Deletion of Sip1 hampers migration of late born cortical neurons
We have seen that a delay in radial migration of young neurons born at and after E15.5 is seen in both Sip1 conditional mutants, despite the fact that while in Sip1fl/fl-EmxCre, Reelin expression is reduced and might partially explain the phenotype, in Sip1fl/fl-NexCre it remains unaffected (Fig11). It has been shown that overexpression of Reelin in the ventricular zone cells of reeler mice can lead to a rescue of the inverted cortical layers in these mutants, implying that the physical location of the source of Reelin is not so significant as long as there is a supply within the cortex (Magdaleno et al., 2002). The dispersion of Reelin- secreting cells at E14.5, before the onset of the phenotype, therefore, might not really add to the subsequent hampered migration of neurons. Moreover, the morphology of radial glial processes, often used by migrating neurons as a scaffold, is unaffected in Sip1 conditional mutants (Fig12). Altogether, this implies that the general migration delay in these mutants is either due to a deficiency in effectors downstream of Reelin or due to a defect in alternative pathways like the p35/Cdk5 mediated signaling.
In this context, we made an interesting preliminary finding. We found that one of the putative Sip1 targets in the cortex, Itga6 (Integrin alpha 6, a cell- matrix adhesion molecule), is downregulated at E14.5 and upregulated at E16.5 in Sip1fl/fl-EmxCre, as determined by semi- quantitative PCR (Fig13). Though not much work has been published
on putative functions of Itga6 in cortical lamination/migration, there are indications that it might indeed be involved in the same (Georges-Labouesse et al., 1998). In any case, dynamic changes in adhesion properties between cells and the extracellular matrix are expected to influence the rate of migration of young neurons away from the germinal zones (Anton et al., 1999; Schmid et al., 2004). We believe that changes in the level of Itga6 might influence the former, and therefore be partly responsible for the hampered migration of late born upper layer neurons.
Interestingly, the migration of earlier born neurons seems unaltered, if not faster. We have shown that in Sip1 conditional mutants, cell born at E12.5-E13.5 mostly occupy upper cortical layers (Fig11p,q). Coupled to the fact that these cells express upper layer neuronal markers, this particular phenotype seems to be a consequence of premature shift in cell fate specification of cortical progenitors rather than abnormalities in migration of young neurons.
4.3.2 Disorganised stratification of cortical layers is due to both premature production of layer 2-5 neurons as well as delayed migration of late born UL neurons
Compaction of the six different layers of the neocortex is clearly disrupted in both Sip1 conditional knockouts. Defective neurogenesis could be largely responsible for this phenotype. In wildtype embryos, cells born at E12.5/E13.5 are distributed radially throughout the neocortex, but localised mostly to layers 5-6. In Sip1 mutants, however, most of them are located in the upper layers 2-4, possibly due to the premature specification of these neurons. Accompanied by the fact that neurons born at and after E15.5 are fewer and seem to migrate slower in the mutants than in the wildtype, it is not surprising that the final lamination of the neocortex is distorted. In other words, the hampered migration at later stages and cell fate shift at earlier stages, leads to a loss of compaction and hence, dispersion, of cortical layers later.
4.3.3 Thinner cortex and absence of corpus callosum in Sip1 conditional mutants
The reduction in thickness of the Sip1 mutant cortex is intriguing. Considering that in these mutants, early proliferation is not changed and that late proliferation is actually increased, without much difference in the level of apoptosis, it is indeed surprising to find an overall
thinning of the cortex. However, we did observe a premature end to neurogenesis despite the fact that the onset of neurogenesis does not seem to be shifted, based on birthdating of Tbr1+ layer 6 cells, and expression of neuronal marker Hu at E12.5. This in turn implies that the overall duration of neurogenesis is reduced in Sip1 mutants without any changes in the rate of proliferation, mitotic exit, or cell cycle length (Data from collaborators- Seuntjens E, Huylebroeck D), possibly leading to the generation of fewer neurons, and hence a thinner cortex. Also, the extent of apoptosis, though seen to some degree in deep layers at early postnatal stages (Doctoral Thesis, Miquelajauregui A, Mar 2006, University of Goettingen, unpublished data) does not seem to be high enough to result in a thinner cortex. Moreover, although the shift in neuronogenesis of layers 2-5 is followed by premature gliogenesis, the increase in astrocyte production is apparently not sufficient to make up for the overall size reduction. The function of the excess of astrocytes in the mutant cortex is yet to be determined.
It is interesting to note that in contrast to the rest of the neocortex, expression of Sip1 in the cingulate cortex is restricted to deep layer neurons at E17.5, and that absence of Sip1 leads to agenesis of the corpus callosum and formation of a Probst bundle. It has been shown that some of the earliest born cells of the cingulate cortex extend pioneering axons to initiate corpus callosum formation (Koester and O'Leary, 1994; Rash and Richards, 2001).
Together, this suggests that in the cingulate cortex, Sip1 might have a function different from its role in the rest of the dorsal cortex. Moreover, It has been reported previously that neither the integrity of the cortical hem nor BMP signaling at the dorsal telencephalic midline is affected in Sip1 conditional mutants (Doctoral Thesis, Miquelajauregui A, Mar 2006, University of Goettingen, unpublished data).
Besides the corpus callosum, a second thinner bundle of fibres connecting the two cerebral hemispheres is the anterior commissure (AC). It lies anterior to the fornix, and mainly connects the two halves of the olfactory bulb and the temporal cortex. It is often used by callosally projecting neurons as an alternative route to reach the contralateral hemisphere in mutants where the corpus callosum fails to develop (Britanova et al., 2008; Chen et al., 2008; Livy et al., 1997). Several mouse mutants show abnormalities in formation of the AC, usually associated with a general failure of midline crossing (Armentano et al., 2006;
Chen et al., 2007; Deuel et al., 2006). The Slit/Robo family of guidance cues and receptors seem to be essential in establishing a normal AC (Bagri et al., 2002; Kidd et al., 1998).
Most commissural neurons originate from layer 2-4. The lack of anterior commissure in our mutants could be either due to a failure of midline crossing (which would also explain the formation of Probst bundles) or due to the failure of commissural neurons to extend axons towards the midline. The mechanism by which Sip1 might influence axonal crossing at the midline is yet to be determined.