Genetic and developmental data supporting a spatial to temporal patterning switch in the evolution of the mammalian neocortex
A hallmark of the evolution of the mammalian neocortex is the emergence of a SVZ in the DP (Martínez-Cerdeño et al., 2006; Cheung et al., 2010), and interestingly the intermediate progenitors (IPc) that populate this germinative layer are mainly involved in the generation of UL neurons (Tarabykin et al., 2001; Martínez-Cerdeño et al., 2006; Kowalczyk et al., 2009). Although, an SVZ is not always evident in sauropsids, studies in turtle and chick showed that putative IP like cells are present in late developmental phases of the LP and VP of turtle and chick (Martínez-Cerdeño et al., 2006; Cheung et al., 2007). The acquisition by DP progenitors of a character (the IPc) that pre-existed in LP/VP progenitors is consistent with our hypothesis. Nonetheless, the IPc step is a common feature in stem cell systems and it has been described for multiple neuronal progenitors populations in both vertebrate and invertebrate brains (Brand and Livesey, 2011). Mammalian DP progenitors may have independently increased the generation of IP to amplify neuron production. Future studies defining the role of the SVZ during pallial development will be necessary to understand the role of this germinative layer in the emergence of the neocortex. While deciphering the developmental program set up by pallial progenitors is a fundamental issue, recent studies also tried to extend previous inter-species comparisons of pallial neuronal types with more modern molecular techniques. The comparison of the chick and mouse transcriptomes of telencephalic regions with either disputed or undisputed homology (Belgard et al., 2013) revealed significant similarities for the hippocampus but failed to identify specific relationships between any other pallial region. The only exception was a weak correlation between the neocortical layer IV and a thalamorecipient field of the nidopallium (a VP derivative). Along with our hypothesis for the evolution of layer II/III it would be interesting to evaluate whether the appearance of stellate cells in layer IV was due to the co-option of the developmental program of the thalamo-recipient VP cells. Unfortunately the olfactory cortex was not analyzed in this study, probably because it is highly reduced in chick. These transcriptomes comparisons supported the view that DP and VP derivatives underwent dramatic changes in morphology and function during amniote evolution (Montiel and Molnár, 2013). At the same time, although such analyses can make a strong case for homology, negative results are more difficult to interpret. Huge differences in the transcriptome do not rule out the occurrence of homologous cell types that greatly changed their relative proportions or mixed with novel cell types. This further indicates the importance of defining the evolutionary history of individual pallial cell types (the so called cell type homology or deep-homology; Arendt, 2008; Shubin et al., 2009) to understand the divergence of DP derivatives in amniotes.
In this perspective, in the last years different authors have analyzed the pattern of expression of the sauropsid orthologs of genes expressed in specific neocortical layers (Nomura et al., 2008, 2013; Dugas-Ford et al., 2012; Suzuki et al., 2012; Chen et al., 2013; Suzuki and Hirata, 2014). The drawback of this approach is that the few individual genes that have been analyzed are expressed by multiple cell types not only in the neocortex but also in other brain regions (Medina et al., 2013). Moreover, the layer specificity of some of the markers of upper layer cells have been disputed (Dugas-Ford et al., 2012). Nonetheless, from these studies a general pattern emerged in which the orthologs of DL markers tend to be expressed more medially than those of the UL. These latter genes are mostly expressed in LP derivatives such as the mesopallium/pallial thickening or the olfactory cortex (Dugas-Ford et al., 2012; Suzuki and Hirata, 2013; Nomura et al., 2014). Since clonal analyses in chick indicate that pallial neurons expressing the orthologs of DL and UL markers are produced by spatially segregated progenitors (Suzuki et al., 2012), these observations are consistent with the hypothesis that the evolution of the mammalian neocortex involved a spatial to temporal patterning switch (Dugas-Ford et al., 2012; Suzuki and Hirata, 2013; Nomura et al., 2014).
Surprisingly, early dorso-medial and dorso-lateral progenitors of the chick pallium were able to sequentially produce cells expressing DL and UL markers in vitro (Suzuki et al., 2012). Caution should be made in the interpretation of these data, first because the authors did not verified the purity of the explanted regions and second because the expression of few marker is a very weak evidence that chick and neocortical progenitors generates the same cell types.
Nonetheless, these results introduce the intriguing possibility that an intrinsic temporal patterning mechanism specifying pallio-fugal, thalamo-recipient, and pallio-pallial neuronal types was present in pallial progenitors of the common ancestor of all amniotes or even vertebrates. This idea would be consistent with the fact that temporal patterning of primary progenitors is a major mechanism for generating neuronal diversity in Drosophila (Li et al., 2013a,b; Eroglu et al., 2014). At some point in vertebrate evolution, spatial patterning cues may have differentially repressed specific parts of this program along medio-lateral and anterior-posterior axes. The molecular mechanism that led to the evolution of the six-layered neocortex could thus be a de-repression of the ancestral developmental program in DP progenitors or a subpopulation of them. A similar idea has also been proposed by Luis Puelles to explain the stratified birth dates of VP derived neurons migrating to the neocortex (Puelles, 2011): “One wonders whether this implies a normally repressed, cryptic 6-layer potency existing throughout the pallium, which is simply de-repressed and thus allowed to emerge at the neocortex.” Interestingly, the transcription factor zbtb20 has been recently shown to play a general repressive activity over the specification of neocortical cell types of both UL and DL (Nielsen et al., 2014). In the mammalian pallium, this transcription factor is expressed in MP, LP, and VP but not DP regions and gain and loss of functions have been shown to shift the neocortical limit, at least medially (Nielsen et al., 2007, 2014; Rosenthal et al., 2012). Detailed comparative analyses will be necessary to understand if down-regulation of zbtb20 or other transcriptional repressors in DP progenitors may have played a role in the evolution of the neocortex.
In conclusion, our understanding of the genetic logic of cell type specification in the neocortex and other pallial regions of amniotes is constantly growing and this will likely enable to test current theories of the evolution of the mammalian pallium. These analyses would be greatly helped by the comparison of the genetic fingerprint of more restricted cell populations and the layer II DCX+/Tbr1+ cells represent an attractive candidate for such analyses.

Conflict of interest statement
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.