PBX1 acts as terminal selector for olfactory bulb dopaminergic neurons.

Neuronal specification is a protracted process that begins with the commitment of progenitor cells and culminates with the generation of mature neurons. Many transcription factors are continuously expressed during this process but it is presently unclear how these factors modify their targets as cells transition through different stages of specification. In olfactory bulb adult neurogenesis, the transcription factor PBX1 controls neurogenesis in progenitor cells and the survival of migrating neuroblasts. Here, we show that, at later differentiation stages, PBX1 also acts as a terminal selector for the dopaminergic neuron fate. PBX1 is also required for the morphological maturation of dopaminergic neurons and to repress alternative interneuron fates, findings that expand the known repertoire of terminal-selector actions. Finally, we reveal that the temporal diversification of PBX1 functions in neuronal specification is achieved, at least in part, through the dynamic regulation of alternative splicing. In Caenorhabditis elegans, PBX/CEH-20 also acts as a dopaminergic neuron terminal selector, which suggests an ancient role for PBX factors in the regulation of terminal differentiation of dopaminergic neurons.

A transcription factor collective defines the HSN serotonergic neuron regulatory landscape.

Cell differentiation is controlled by individual transcription factors (TFs) that together activate a selection of enhancers in specific cell types. How these combinations of TFs identify and activate their target sequences remains poorly understood. Here, we identify the cis-regulatory transcriptional code that controls the differentiation of serotonergic HSN neurons in Caenorhabditis elegans. Activation of the HSN transcriptome is directly orchestrated by a collective of six TFs. Binding site clusters for this TF collective form a regulatory signature that is sufficient for de novo identification of HSN neuron functional enhancers. Among C. elegans neurons, the HSN transcriptome most closely resembles that of mouse serotonergic neurons. Mouse orthologs of the HSN TF collective also regulate serotonergic differentiation and can functionally substitute for their worm counterparts which suggests deep homology. Our results identify rules governing the regulatory landscape of a critically important neuronal type in two species separated by over 700 million years.

Cyclin-Dependent Kinase 4 Regulates Adult Neural Stem Cell Proliferation and Differentiation in Response to Insulin.

Insulin is one of the standard components used to culture primary neurospheres. Although it stimulates growth of different types of cells, the effects of insulin on adult neural stem cells (NSCs) have not been well characterized. Here, we reveal that insulin stimulates proliferation, but not survival or self-renewal, of adult NSCs. This effect is mediated by insulin receptor substrate 2 (IRS2) and subsequent activation of the protein kinase B (or Akt), leading to increased activity of the G1-phase cyclin-dependent kinase 4 (Cdk4) and cell cycle progression. Neurospheres isolated from Irs2-deficient mice are reduced in size and fail to expand in culture and this impaired proliferation is rescued by introduction of a constitutively active Cdk4 (Cdk4R24C/R24C ). More interestingly, activation of the IRS2/Akt/Cdk4 signaling pathway by insulin is also necessary for the generation in vitro of neurons and oligodendrocytes from NSCs. Furthermore, the IRS2/Cdk4 pathway is also required for neuritogenesis, an aspect of neuronal maturation that has not been previously linked to regulation of the cell cycle. Differentiation of NSCs usually follows exit from the cell cycle due to increased levels of CDK-inhibitors which prevent activation of CDKs. In contrast, our data indicate that IRS2-mediated Cdk4 activity in response to a mitogen such as insulin promotes terminal differentiation of adult NSCs. Stem Cells 2017;35:2403-2416.

IRS2 signalling is required for the development of a subset of sensory spinal neurons.

Insulin and insulin-like growth factor-I play important roles in the development and maintenance of neurons and glial cells of the nervous system. Both factors activate tyrosine kinase receptors, which signal through adapter proteins of the insulin receptor substrate (IRS) family. Although insulin and insulin-like growth factor-I receptors are expressed in dorsal root ganglia (DRG), the function of IRS-mediated signalling in these structures has not been studied. Here we address the role of IRS2-mediated signalling in murine DRG. Studies in cultured DRG neurons from different embryonic stages indicated that a subset of nerve growth factor-responsive neurons is also dependent on insulin for survival at very early time points. Consistent with this, increased apoptosis during gangliogenesis resulted in a partial loss of trkA-positive neurons in DRG of Irs2 mutant embryos. Analyses in adult Irs2(-/-) mice revealed that unmyelinated fibre afferents, which express calcitonin gene-related peptide/substance P and isolectin B4, as well as some myelinated afferents to the skin were affected by the mutation. The diminished innervation of glabrous skin in adult Irs2(-/-) mice correlated with longer paw withdrawal latencies in the hot-plate assay. Collectively, these findings indicate that IRS2 signalling is required for the proper development of spinal sensory neurons involved in the perception of pain.

Satb2 regulates callosal projection neuron identity in the developing cerebral cortex.

Satb2 is a DNA-binding protein that regulates chromatin organization and gene expression. In the developing brain, Satb2 is expressed in cortical neurons that extend axons across the corpus callosum. To assess the role of Satb2 in neurons, we analyzed mice in which the Satb2 locus was disrupted by insertion of a LacZ gene. In mutant mice, beta-galactosidase-labeled axons are absent from the corpus callosum and instead descend along the corticospinal tract. Satb2 mutant neurons acquire expression of Ctip2, a transcription factor that is necessary and sufficient for the extension of subcortical projections by cortical neurons. Conversely, ectopic expression of Satb2 in neural stem cells markedly decreases Ctip2 expression. Finally, we find that Satb2 binds directly to regulatory regions of Ctip2 and induces changes in chromatin structure. These data suggest that Satb2 functions as a repressor of Ctip2 and regulatory determinant of corticocortical connections in the developing cerebral cortex.

SATB2 is a multifunctional determinant of craniofacial patterning and osteoblast differentiation.

Vertebrate skeletogenesis involves two processes, skeletal patterning and osteoblast differentiation. Here, we show that Satb2, encoding a nuclear matrix protein, is expressed in branchial arches and in cells of the osteoblast lineage. Satb2-/- mice exhibit both craniofacial abnormalities that resemble those observed in humans carrying a translocation in SATB2 and defects in osteoblast differentiation and function. Multiple osteoblast-specific genes were identified as targets positively regulated by SATB2. In addition, SATB2 was found to repress the expression of several Hox genes including Hoxa2, an inhibitor of bone formation and regulator of branchial arch patterning. Molecular analysis revealed that SATB2 directly interacts with and enhances the activity of both Runx2 and ATF4, transcription factors that regulate osteoblast differentiation. This synergy was genetically confirmed by bone formation defects in Satb2/Runx2 and Satb2/Atf4 double heterozygous mice. Thus, SATB2 acts as a molecular node in a transcriptional network regulating skeletal development and osteoblast differentiation.