Figure 3. Prdm16 is required for survival, cell cycle regulation, and self-renewal in neural stem cells.

a) The brains of neonatal Prdm16^LacZ/LacZ mice were significantly smaller than those of Prdm16^+/+ or Prdm16^LacZ/+ mice (the number of mice of each genotype is shown under each bar). b–c) Hematoxylin and eosin staining of coronal sections showed that Prdm16^LacZ/LacZ brains (c) were smaller and that morphology was disrupted relative to control brains (b). Brackets show reduced cortical thickness, arrowheads show narrower lateral ventricle, and arrows point to the lack of a corpus callosum in the Prdm16^LacZ/LacZ brain. d–e) Agenesis of the corpus callosum was confirmed by staining with the neuronal marker TuJ1 which revealed axon tracts that crossed the midline in wild-type (arrow, d) but Probst bundles that did not cross the midline in Prdm16^LacZ/LacZ brains (*, e). f–j) Some primary neurospheres of all genotypes underwent multilineage differentiation, forming neurons (TuJ1+), astrocytes (GFAP+), and oligodendrocytes (O4+); however, a significantly lower percentage of VZ cells from newborn Prdm16^LacZ/LacZ mice formed multipotent neurospheres compared to littermate controls (h) and the diameter (i) and self-renewal potential (j) of Prdm16^LacZ/LacZ neurospheres was significantly less than control neurospheres (the number of mice per genotype is indicated in each panel; panels h, i, and j reflect 6, 4, and 3 independent experiments). Self-renewal was quantified as the number of multipotent secondary neurospheres generated upon the subcloning of individual primary neurospheres. k–o) Significantly fewer dividing cells were observed in the VZ of newborn Prdm16^LacZ/LacZ mice compared to littermate controls based on phospho-histone3 (pH3) staining (3 mice per genotype and 4 sections per mouse). p–r) Significantly more cells underwent cell death in the lateral ventricle VZ of newborn Prdm16^LacZ/LacZ mice compared to littermate controls (* marks lateral ventricle) in sections (p–q), and by flow cytometry (6 mice/genotype from 3 independent experiments). (*, P<0.05; **, P<0.01; ***, P<0.001, error bars always represent SD).