Pax6 limits the competence of developing cerebral cortical cells to respond to inductive intercellular signals

Abstract

The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here, we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single-cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors’ competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6’s main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side effects of morphogens engaged contemporaneously in other essential functions.

Developmental disruption and restoration of brain synaptome architecture in the Pax6 neurodevelopmental disease model

“…It is likely, therefore, that the abnormalities in synaptome architecture reported here could arise from both the reduction in Pax6 expression at postnatal ages as well as from residual defects in neurons whose lineages no longer express Pax6. During a critical period of early development, Pax6 protects early progenitors from erroneous specification by inductive signals and thereby controls the identity of neurons long after Pax6 has ceased to be expressed 56 . Neurons that would be expected to have cell-autonomous defects in Pax6 +/− mice project their axons to neurons that do not express Pax6, such as midbrain neurons, and these postsynaptic neurons might alter their PSD95 and SAP102 expression as a result of the mutation-dependent changes in neuronal activity in the input neurons.…”

Pax6 mutant cerebral organoids partially recapitulate phenotypes of Pax6 mutant mouse strains

“…Interestingly, we saw a much larger variation in the amount of β-tubulin+ neurons produced in Pax6 -/- organoids than in controls. One possible explanation for this is that Pax6 acts to protect cells from the influence of signalling molecules in the extracellular environment as has recently been shown in mice [ 23 ]. It is possible that the variation in the extent of precocious neural differentiation in Pax6 -/- organoids is due to variability in the presence of environmental signals that promote cell cycle exit.…”

“…shown in mice [23]. It is possible that the variation in the extent of precocious neural differentiation in Pax6 -/organoids is due to variability in the presence of environmental signals that promote cell cycle exit.…”

Loss of PAX6 alters the excitatory/inhibitory neuronal ratio in human cerebral organoids

“…Acute deletion of Pax6 in mouse cortical progenitors causes a subset of these cells to adopt a Gad1-positive inhibitory identity and segregate from excitatory-lineage cells in the cortical plate, forming an ectopic mass (Manuel et al, 2022). To examine the distribution of ectopic inhibitory cells in PAX6 -/organoids, we immunostained cryosections of day 60 organoids for TBR2 (EOMES) to label cortical IP cells ('dorsal' identity) and DLX2 to identify inhibitory cell precursors ('ventral' identity) (Fig 3A).…”