Brn3a regulates neuronal subtype specification in the trigeminal ganglion by promoting Runx expression during sensory differentiation.

The transcription factor Brn3a, product of the pou4f1 gene, is expressed in most sensory neurons throughout embryogenesis. Prior work has demonstrated a role for Brn3a in the repression of early neurogenic genes; here we describe a second major role for Brn3a in the specification of sensory subtypes in the trigeminal ganglion (TG). Sensory neurons initially co-express multiple Trk-family neurotrophin receptors, but are later marked by the unique expression of TrkA, TrkB or TrkC. Maturation of these sensory subtypes is known to depend on the expression of Runx transcription factors. Newborn Brn3a knockout mice fail to express TrkC, which is associated in the TG with mechanoreceptors, plus a set of functional genes associated with nociceptor subtypes. In embryonic Brn3a-/- ganglia, the normal expression of Runx3 is never initiated in TrkC+ neurons, and Runx1 expression is greatly attenuated in TrkA+ nociceptors. These changes are accompanied by expanded expression of TrkB in neurons that abnormally express multiple Trks, followed by the loss of TrkC and TrkA expression. In transgenic embryos expressing a Brn3a-VP16 dominant transactivator, Runx3 mRNA expression is increased, suggesting that it is a direct regulatory target of Brn3a. Chromatin immunoprecipitation confirms that Brn3a binds in vivo to a conserved upstream enhancer element within histone H3-acetylated chromatin in the Runx3 locus. Together these data show that Brn3a acts upstream of the Runx factors, which then repress TrkB expression to allow establishment of the non-overlapping Trk receptor profiles and correct terminally differentiated phenotypes.

Brn3a regulates the transition from neurogenesis to terminal differentiation and represses non-neural gene expression in the trigeminal ganglion.

The POU-domain transcription factor Brn3a is expressed in developing sensory neurons at all levels of the neural axis, including the trigeminal ganglion, hindbrain sensory ganglia, and dorsal root ganglia. Changes in global gene expression in the trigeminal ganglion from E11.5 to E13.5 reflect the repression of early neurogenic genes, exit from the cell cycle, and initiation of the expression of definitive markers of sensory function. A majority of these developmental changes are perturbed in the trigeminal ganglia of Brn3a knockout mice. At E13.5, Brn3a(-/-) trigeminal neurons fail to repress a battery of developmental regulators that are highly expressed at E11.5 and are normally down-regulated as development progresses, and also fail to appropriately activate a set of definitive sensory genes. Remarkably, developing Brn3a(-/-) trigeminal neurons also ectopically express multiple regulatory genes associated with cardiac and/or cranial mesoderm development, although definitive myogenic programs are not activated. The majority of these genes are not ectopically expressed in the dorsal root ganglia of Brn3a null mice, perhaps due to redundant mechanisms of repression at spinal levels. These results underscore the importance of gene repression in regulating neuronal development, and the need for unbiased screens in the determination of developmental gene regulatory programs.

Brn3a target gene recognition in embryonic sensory neurons.

Numerous transcription factors have been identified which have profound effects on developing neurons. A fundamental problem is to identify genes downstream of these factors and order them in developmental pathways. We have previously identified 85 genes with changed expression in the trigeminal ganglia of mice lacking Brn3a, a transcription factor encoded by the Pou4f1 gene. Here we use locus-wide chromatin immunoprecipitation in embryonic trigeminal neurons to show that Brn3a is a direct repressor of two of these downstream genes, NeuroD1 and NeuroD4, and also directly modulates its own expression. Comparison of Brn3a binding to the Pou4f1 locus in vitro and in vivo reveals that not all high affinity sites are occupied, and several Brn3a binding sites identified in the promoters of genes that are silent in sensory ganglia are also not occupied in vivo. Site occupancy by Brn3a can be correlated with evolutionary conservation of the genomic regions containing the recognition sites and also with histone modifications found in regions of chromatin active in transcription and gene regulation, suggesting that Brn3a binding is highly context dependent.

POU-domain factor Brn3a regulates both distinct and common programs of gene expression in the spinal and trigeminal sensory ganglia.

BACKGROUND: General somatic sensation is conveyed to the central nervous system at cranial levels by the trigeminal ganglion (TG), and at spinal levels by the dorsal root ganglia (DRG). Although these ganglia have similar functions, they have distinct embryological origins, in that both contain neurons originating from the neural crest, while only the TG includes cells derived from the placodal ectoderm. RESULTS: Here we use microarray analysis of E13.5 embryos to demonstrate that the developing DRG and TG have very similar overall patterns of gene expression. In mice lacking the POU-domain transcription factor Brn3a, the DRG and TG exhibit many common changes in gene expression, but a subset of Brn3a target genes show increased expression only in the TG. In the wild-type TG these Brn3a-repressed genes are silent, yet their promoter regions exhibit histone H3-acetylation levels similar to constitutively transcribed gene loci. This increased H3-acetylation is not observed in the DRG, suggesting that chromatin modifications play a role in cell-specific target gene regulation by Brn3a. CONCLUSION: These results demonstrate that one developmental role of Brn3a is to repress potential differences in gene expression between sensory neurons generated at different axial levels, and to regulate a convergent program of developmental gene expression, in which functionally similar populations of neurons are generated from different embryological substrates.

Regulation of FGF10 by POU transcription factor Brn3a in the developing trigeminal ganglion.

The POU-domain transcription factor Brn3a is expressed in specific neurons of the caudal CNS and peripheral sensory nervous system. The sensory neurons of mice lacking Brn3a exhibit marked defects in axon growth and extensive apoptosis in late gestation. Here we show that expression of the developmental regulator FGF10 is approximately 35-fold increased in the developing trigeminal ganglia of Brn3a-null mice. In order to determine whether FGF10 regulates other changes in gene expression observed in Brn3a knock-out ganglia, we have used a sensory-specific enhancer to over-express FGF10 in transgenic mice. Microarray analysis of trigeminal ganglia from individual transgenic founders effectively excludes the cell-autonomous activity of FGF10 as a mechanism for mediating the downstream effects of the loss of Brn3a, probably because developing sensory neurons lack the appropriate type of FGF receptor.

Brn3a-expressing retinal ganglion cells project specifically to thalamocortical and collicular visual pathways.

Retinal ganglion cells (RGCs) innervate several specific CNS targets serving cortical and subcortical visual pathways and the entrainment of circadian rhythms. Recent studies have shown that retinal ganglion cells express specific combinations of POU- and LIM-domain transcription factors, but how these factors relate to the subsequent development of the retinofugal pathways and the functional identity of RGCs is mostly unknown. Here, we use targeted expression of an genetic axonal tracer, tau/beta-galactosidase, to examine target innervation by retinal ganglion cells expressing the POU-domain factor Brn3a. Brn3a is expressed in RGCs innervating the principal retinothalamic/retinocollicular pathway mediating cortical vision but is not expressed in RGCs of the accessory optic, pretectal, and hypothalamic pathways serving subcortical visuomotor and circadian functions. In the thalamus, Brn3a ganglion cell fibers are primarily restricted to the outer shell of the dorsal lateral geniculate, providing new evidence for the regionalization of this nucleus in rodents. Brn3a RGC axons have a relative preference for the contralateral hemisphere, but known mediators of the laterality of RGC axons are not repatterned in the absence of Brn3a. Brn3a is coexpressed extensively with the closely related factor Brn3b in the embryonic retina, and the effects of the loss of Brn3a in retinal development are not severe, suggesting partial redundancy of function in this gene class.

Coordinated regulation of gene expression by Brn3a in developing sensory ganglia.

Mice lacking the POU-domain transcription factor Brn3a exhibit marked defects in sensory axon growth and abnormal sensory apoptosis. We have determined the regulatory targets of Brn3a in the developing trigeminal ganglion using microarray analysis of Brn3a mutant mice. These results show that Brn3 mediates the coordinated expression of neurotransmitter systems, ion channels, structural components of axons and inter- and intracellular signaling systems. Loss of Brn3a also results in the ectopic expression of transcription factors normally detected in earlier developmental stages and in other areas of the nervous system. Target gene expression is normal in heterozygous mice, consistent with prior work showing that autoregulation by Brn3a results in gene dosage compensation. Detailed examination of the expression of several of these downstream genes reveals that the regulatory role of Brn3a in the trigeminal ganglion appears to be conserved in more posterior sensory ganglia but not in the CNS neurons that express this factor.