Sip1 controls sequential cell fate switch in cortical progenitors during the course of embryonic development

On the basis of our analysis of two different cortex- specific conditional knockouts of Sip1, we have been able to demonstrate that this transcriptional repressor plays a key role in regulating the timing of cell fate switch in the neocortex by controlling the activity of certain signaling pathways.

Sip1 deficient neocortex is thinner, possibly due to fewer neurons, accompanied by reduced layers 5 and 6 and a concomitant expansion of layers 2-4 at prenatal stages of development. Possible reasons would include alterations in proliferation, cell cycle kinetics, apoptosis, neuronal fate specification and/or differentiation.

Our analyses have shown that specific deletion of Sip1 in the postmitotic compartment of the neocortex leads to premature specification of cortical progenitors towards an upper layer lineage at early stages, and a glial lineage at later stages of corticogenesis (Fig21).

While the dynamics of generation of the early born layer 6 neurons is not affected in Sip1 conditional mutants, Ctip2+ layer 5 neurons, Foxp1+ layer 3-5 neurons, and Satb2+ and Brn2+ layer 2-4 neurons are all generated prematurely (Fig8). Their peak of production is shifted temporally by at least one day. It is important to note that for all of these cell types, irrespective of their usual time of generation, in Sip1 mutants the peak of production is shifted to earlier stages (E12.5-E13.5). On the other hand, we detected no alteration in either the extent of proliferation or the rate of mitotic exit at these stages. In fact, expression of Hu (a marker of young postmitotic neurons) at E12.5 and E13.5 indicates no changes in either the thickness or the cell density of the mutant cortical plate (Fig6). This implies, together with the results of Tbr1 birthdating, that the onset of neurogenesis in Sip1 mutants is not altered. Furthermore, we have also determined the function of Sip1 in controlling the timing and extent of astrocyte production in the neocortex. Our pulse chase experiments prove increased and earlier specification of both Olig2+ as well as GFAP+

glial cells in the mutants (Fig9). Altogether, we believe that the onset of neurogenesis in the cortex is not affected by Sip1 deletion; however, once the process commences, Sip1 plays a major role in the temporal control of the sequential generation of different cell types, irrespective of the number of each type generated.

From our extensive analysis of proliferation and cell cycle kinetics in Sip1 mutants, we can conclude that Sip1 has no influence on mitotic divisions of early cortical progenitors.

However, Sip1 does induce expansion of the pool of glial progenitors (expressing Blbp/

Olig2) at E17.5 in a non- cell autonomous manner (Fig7).

Apoptosis is not responsible for the reduction of deep layer neurons in Sip1 mutants.

Although it has been shown before that apoptotic cell death increases in layer 6 in Sip1 mutant cortices at early postnatal stages (Doctoral Thesis, Miquelajauregui A, Mar 2006, University of Goettingen, unpublished data) we did not detect any such effect during early embryonic development, when the reduction in deep layers is already obvious. This suggests that apoptotic cell death is not the primary reason behind the appearance of fewer layer 5/6 neurons in mutant cortices.

The fact that GFAP+ astrocytes are detected in mutant cortices not before their usual time of appearance, albeit at higher numbers, indicates that the function of Sip1 might be limited to influencing the commitment of progenitors towards a certain lineage and inducing them to expand their pool size, but that the final differentiation takes place at its

Fig21. A model proposing Sip1- mediated non- cell autonomous control of cortical progenitor fate. At early stages of corticogenesis (E13.5), Sip1, expressed strongly by cortical plate neurons, represses NT3 expression, which in turn ensures generation of deep layer rather than upper layer neurons (a). In Sip1 conditional mutants, lack of Sip1 leads to premature overexpression of NT3, which signals back to VZ progenitors to induce a fate switch from DL to UL neuron production (b). At later stages (E17.5), Sip1 represses Fgf9 expression in CP neurons, and feedback signaling from CP to VZ ensures a precisely regulated end of neurogenesis and onset of gliogenesis (c). In Sip1 conditional mutants, absence of Sip1 in the CP leads to precocious upregulation of Fgf9, which signals back to VZ precursors to firstly, induce

premature neurogenic to gliogenic fate switch, and secondly, to promote proliferation of progenitors committed to an astrocytic lineage (d).

own designated time. However, in case of Brn2+ upper layer neurons, which are indeed detected precociously in the mutant cortex, even the differentiation of committed progenitors seems to have shifted to at least one day earlier. This, however, may not be completely true, since Brn2 is known to be expressed in migrating neurons as well, before they fully differentiate into mature upper layer neurons. Since our claim is based mostly on BrdU pulse- chase experiments, there could be an alternative explanation to our observations. The possibility that upper layer neurons are born in their usual temporal sequence but that it is the deep layer neurons that undergo a postmitotic cell fate shift to an upper layer neuron by early postnatal stages, cannot be precluded. On the other hand, if we were to apply the same logic to the neuron to glial fate switch at later stages, the likelihood of a neuron transdifferentiating into an astrocyte seems very low. Also, using the Fgf9 coated beads on slice cultures, we have shown that the increased number of Olig2+ cells is confined to the region close to the VZ (Fig15). This implies that Fgf9 expression in the cortical plate signals back to the VZ/SVZ and induces proliferation of glial precursors.

Moreover, it is highly likely that Sip1 acts via the same conceptual mechanism at both early and late stages, though the intermediate signaling pathways might be different.

Therefore, we strongly suspect Sip1 to non- cell autonomously influence progenitor commitment rather than be involved in maintenance of the differentiated state of a neuron.

So far, several instances of premature astrocyte production in the cortex have been reported. These include, 1. Exogenous application of growth factors such as LIF, CNTF, CT-1, Fgf2, CNTF and BMPs to neural stem cell cultures; 2. lack of function of neurogenic bHLH genes such as Ngn2,Mash1 and Ngn1 (Nieto et al., 2001; Sun et al., 2001); 3. Overexpression of EGF receptor (Burrows et al., 1997; Sun et al., 2005); 4.

Genetic ablation of certain growth factors, receptors, signaling molecules and transcription factors. Some examples include, neuregulin receptors ErbB2 and ErbB4 (Sardi et al., 2006;

Schmid et al., 2003), SHP-2, a growth factor regulated phosphatase known to modulate MEK-ERK and gp130-JAK-STAT pathways (Miller and Gauthier, 2007) and N-CoR (Hermanson et al., 2002) (other reports reviewed in (Miller and Gauthier, 2007). Our studies report for the first time, precocious astrocytogenesis as a consequence of deletion

of a postmitotic transcription factor leading to gliogenic feedback signaling from the cortical plate to the VZ/SVZ.

The only instances of premature cortical neurogenesis that have been reported so far involve a shift in the peak of the entire neurogenesis and not just of some cell types, as we have shown in our conditional mutants. Examples of the former include, knockout of Id4 (Yun et al., 2004) and several Notch pathway related mutants (reviewed in (Yoon and Gaiano, 2005).