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.

A central role for Islet1 in sensory neuron development linking sensory and spinal gene regulatory programs.

We used conditional knockout strategies in mice to determine the developmental events and gene expression program regulated by the LIM-homeodomain factor Islet1 in developing sensory neurons. Early development of the trigeminal and dorsal root ganglia was grossly normal in the absence of Islet1. From E12.5 onward, however, Isl1 mutant embryos showed a loss of the nociceptive markers TrkA and Runx1 and a near absence of cutaneous innervation. Proprioceptive neurons characterized by the expression of TrkC, Runx3 and Etv1 were relatively spared. Microarray analysis of Isl1 mutant ganglia revealed prolonged expression of developmental regulators that are normally restricted to early sensory neurogenesis and ectopic expression of transcription factors that are normally found in the CNS, but not in sensory ganglia. Later excision of Isl1 did not reactivate early genes, but resulted in decreased expression of transcripts related to specific sensory functions. Together these results establish a central role for Islet1 in the transition from sensory neurogenesis to subtype specification.

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.

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.

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.

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.

Brn3a regulation of TrkA/NGF receptor expression in developing sensory neurons.

The TrkA/NGF receptor is essential for the survival and differentiation of sensory neurons. The molecular mechanisms regulating tissue and stage-specific expression of TrkA are largely unknown. The Brn3a POU-domain transcription factor has been implicated in the development of the PNS and proposed as a transcription regulator for TrkA. The molecular mechanisms underlying the regulation of TrkA by Brn3a is unclear. In this study, we provide genetic, transgenic and biochemical evidence that Brn3a binds to novel, specific sites in the 457 bp enhancer that regulates TrkA expression in embryonic sensory neurons. We employ Bax-knockout mice, in which sensory neurons no longer require neurotrophins for survival, to uncouple TrkA-dependent cell death from downregulation of TrkA expression. In addition, when mutagenized, the novel Brn3a-binding sites identified fail to drive appropriate reporter transgene expression in sensory neurons. Thus, TrkA, a gene that is crucial for the differentiation and survival of sensory nociceptive neurons, requires Brn3a to maintain normal transcriptional activity.

Unaltered expression of Bcl-2 and TAG-1/axonin-1 precedes sensory apoptosis in Brn3a knockout mice.

Mice lacking the POU-domain transcription factor Brn3a exhibit growth defects in trigeminal axons, undergo extensive sensory cell death in late gestation, and die at birth. Based on tissue culture studies, the mediator of apoptosis Bcl-2 has been suggested as a target of Brn3a regulation which could affect sensory viability in these mice. In addition, Bcl-2 and the neural cell adhesion molecule TAG-1/axonin-1 have both been implicated in sensory axon guidance. In this study we examined wild-type and Brn3a knockout embryos for alterations in the expression of these genes. Trigeminal ganglia were harvested from embryonic day 13.5 mouse embryos, and Bcl-2 and TAG-1 expression were measured by RT-PCR. TAG-1 expression was also examined in the embryonic trigeminal and dorsal root ganglia by immunohistochemistry. The developing trigeminal ganglia of Brn3a knockout mice exhibit similar levels of Bcl-2 and TAG-1 mRNA expression. Immunohistochemical staining of TAG-1 also appeared to be quantitatively similar in the sensory axons of wild-type and knockout embryos. It is unlikely that Bcl-2 is a regulatory target of Brn3a, or that either of these factors mediates the defects in axon guidance and neuronal survival observed in the sensory ganglia of Brn3a knockout mice.

Direct autoregulation and gene dosage compensation by POU-domain transcription factor Brn3a.

Brn3a is a POU-domain transcription factor expressed in peripheral sensory neurons and in specific interneurons of the caudal CNS. Sensory expression of Brn3a is regulated by a specific upstream enhancer, the activity of which is greatly increased in Brn3a knockout mice, implying that Brn3a negatively regulates its own expression. Brn3a binds to highly conserved sites within this enhancer, and alteration of these sites abolishes Brn3a regulation of reporter transgenes. Furthermore, endogenous Brn3a expression levels in the sensory ganglia of Brn3a(+/+) and Brn3a(+/-) mice are similar, demonstrating that autoregulation can compensate for the loss of one allele by increasing transcription of the remaining gene copy. Conversely, transgenic overexpression of Brn3a in the trigeminal ganglion suppresses the expression of the endogenous gene. These findings demonstrate that the Brn3a locus functions as a self-regulating unit to maintain a constant expression level of this key regulator of neural development.