Hampered migration of neurons born at and after E15.5

As mentioned before, we suspected either defective migration or alterations in cell fate specification to be a cause of what seems as dispersed and incompact layering in the Sip1

mutant cortices (Fig11a-c). To begin with, we performed BrdU pulse- chase experiments to investigate into the two possibilities. We reasoned that if abnormal lamination is caused by cell fate defects, neurons labeled with BrdU at a certain stage of development should appear at comparable positions within the wildtype and mutant cortices. On the other hand, if neuronal migration is affected, they would end up occupying different positions. In Sip1 mutants, when BrdU was injected at E15.5 and chased to P2, we observed several cells

born at E15.5 that were located at the SVZ/IZ instead of upper cortical layers (Fig11l,m).

While some of these cells did express glial markers like GFAP or Olig2, most others expressed Brn2, expressed by young upper layer neurons before and during migration.

Similarly, most cells born at E17.5 were found to be stuck in the SVZ/IZ/deep layers (Fig11n,o); again, many of these, especially those in the deep cortical layers, expressed Brn2. It therefore seems that radial migration of neurons born at and after E15.5 is hampered in Sip1 conditional mutants. On the other hand, when BrdU injections were done at E12.5/E13.5 and chased to P2, the labeled cells were found to predominantly occupy upper cortical layers in both mutants rather than deep layers. (Fig11p,q).

In order to investigate further into the potential cause of defective migration in Sip1 mutant cortices, we checked the expression of several molecules involved in regulating this process either directly or indirectly. Doublecortin (Dcx) is known to be essential for radial migration especially of upper cortical plate neurons. Both knock down of Dcx as well as double mutation of Dcx and Dclk leads to abnormal migration in the cortex (Bai et al., 2003; Deuel et al., 2006). We did not find any obvious differences in the pattern of expression of Dcx in Sip1 mutants. At E15.5, in both wildtype and Sip1fl/fl-Emx^Cre,strong Dcx expression was seen in the upper part of the cortical plate, while relatively weaker expression was detected in the deeper parts of the CP, basal SVZ and IZ (Fig12i,j).

Fig11. Young neurons born after E15.5 do not migrate properly to their final destination within the cortical plate; defects in Reelin expression might not be the cause. Staining of Wt and mutant cortices for DAPI clearly shows the disorganized stratification of cortical layers at P2 (a-c). Expression of Reelin within the marginal zone is reduced at P2 in Sip1fl/fl-Emx^Cre (d,e) but not in Sip1fl/fl-Nex^Cre (f,g). In Sip1fl/fl-Emx^Cre several Reelin+ cells can be seen within the cortical plate that express Sip1 (e). Co-expression of Reelin and Tbr2 in the putative piriform cortex at E12.5 is reduced in Sip1fl/fl-Emx^Cre (h,i). Ectopically located Reelin+ cells (arrow) can be seen within the cortical plate in Sip1fl/fl-Emx^Cre at E14.5 (j,k). BrdU pulse- chase experiments show that neurons born at and after E15.5 fail to migrate to the appropriate location (arrowheads) and are instead stuck in the IZ/deep layers (arrows). Shown here are E15.5 to P2 chase (l,m) and E17.5 to P2 chase (n,o) in wt and Sip1fl/fl-Emx^Cre. Corroborating the premature specification of upper layer neurons, cells born at E12.5 settle at upper layers in Sip1fl/fl-Emx^Cre (q, arrowhead) by P2, while in Wt they are dispersed mostly through layers 4-6 (p).

Reelin is a secreted signaling molecule that ensures proper migration of young neurons along radial glial fibers. Mutations in Reelin, its receptors VLDLR and ApoER2, and downstream effectors (Dab1) all lead to similar phenotypes that involve a migration delay leading to abnormal neuronal positioning and inverted cortical layers (Tissir and Goffinet, 2003). In Sip1fl/fl-Emx^Cre we did observe a decrease in Reelin expression in comparison to the wildtype (Fig11d,e). Besides the Cajal-Retzius cells of the MZ, several Reelin+ cells were also observed within wildtype and mutant cortical plate. Interestingly, in these mutants almost all Reelin+ cells of the cortical plate were the ones that continued to express Sip1, either because they escaped Cre- mediated recombination, or because they are interneurons that were born in the ganglionic eminences and that migrated tangentially into the neocortex (Fig11d,e). When we checked Reelin expression at earlier stages of development, we detected two major phenotypic effects; 1. At E12.5, Reelin expression is restricted mostly to the putative piriform cortex where they coexpress Tbr2. In Sip1fl/fl-Emx^Cre there were fewer Reelin+ cells in this region (Fig11h,i); 2. At E14.5 cells secreting Reelin are localised to the marginal zone. In Sip1fl/fl-Emx^Cre not only are these cells fewer in number, many of them were localised ectopically in the cortical plate (Fig11j,k). It therefore seems that absence of Sip1 in cortical progenitors leads to a reduction in levels of Reelin, which might in turn lead to the dispersion of cortical layers. However, when Sip1 is ablated from only the postmitotic cells of the cortex (Sip1fl/fl-Nex^Cre ), Reelin expression remains unchanged (Fig11f,g), suggesting that the migration delay in Sip1 mutants is either due to a deficiency in effectors downstream of Reelin or due to a deficiency in alternative pathways like the p35/Cdk5 mediated control of neuronal migration (Gilmore et al., 1998;

Ohshima et al., 2001). Alternatively, such a migration defect could also be caused by a

Fig12. Cortical deletion of Sip1 does not affect radial glial processes or the expression Doublecortin, precluding them as a cause of hampered migration. Expression of Nestin and Blbp is unaltered at E15.5 (a-d) and E16.5 (e-h). Strong expression of Dcx can be seen in the cortical plate close to the pial surface in both Wt (i) and Sip1fl/fl-Emx^Cre (j) at E15.5; there is also weaker expression in the basal SVZ/IZ, which also seems unaffected in the mutant cortex (arrowhead).

disruption in radial glial processes that are often used as a scaffold to migrate on, by several young neurons until they reach their final position in the cortical plate (Nadarajah, 2003). In order to check the integrity of radial glial processes in Sip1 mutants, we labeled them using radial glial markers Nestin and Blbp (Hartfuss et al., 2001; Hockfield and McKay, 1985). We detected no abnormalities in the morphology of these processes in Sip1fl/fl-Emx^Cre at E15.5/E16.5 (Fig12a-h). Radial glial processes, as the name suggests, extend radially from the apical to the pial surface. Although the staining for Nestin is clearly cytoplasmic, Blbp expression can often be detected in the cell bodies of radial glial cells located in the VZ.

3.4 Sip1- mediated signaling in the neocortex