Eric E. Turner

BRN-3.0 EXPRESSION IDENTIFIES EARLY POST-MITOTIC CNS NEURONS AND SENSORY NEURAL PRECURSORS

The mammalian POU-domain factor Brn-3.0 (Brn-3, Brn-3a) is a member of the POU-IV class of transcription factors which resemble the C. elegans factor unc-86 in structure, DNA-binding properties and expression in subsets of sensory neurons. Using specific antisera, we have explored the expression of Brn-3.0 in the early development of the mouse nervous system. Brn-3.0 expression begins at embryonic day 8.5 (E8.5) in a specific set of midbrain tectal neurons whose time and place of appearance are consistent with the earliest CNS neurons previously identified using non-specific markers of neural differentiation. By E9.5, Brn-3.0 immunoreactivity also identifies early CNS neurons in the hindbrain and spinal cord. In the peripheral sensory ganglia, Brn-3.0 expression is first observed at E9.0 in migrating precursors of the trigeminal ganglion, followed by the other sensory cranial and dorsal root ganglia, in a rostral to caudal sequence. Double-label immunofluorescence with Brn-3.0 and the markers of cell division PCNA and BrdU demonstrate that Brn-3.0 is restricted to the post-mitotic phase of CNS development. In the sensory cranial and dorsal root ganglia, however, Brn-3.0 is expressed in dividing neural precursors, suggesting that the nature or timing of developmental events controlled by Brn-3.0 are distinct in the CNS and peripheral neurons. Restriction of Brn-3.0 expression to post-mitotic CNS neurons demonstrates that Brn-3.0 is not required for neurogenesis or patterning of the neuroepithelium in the CNS, but suggests a role in specification of mature neuronal phenotypes.

Brn-3.2: A Brn-3-related transcription factor with distinctive central nervous system expression and regulation by retinoic acid

The identification and molecular characterization of Brn-3.2 has revealed a family of Brn-3-related mammalian POU proteins that share homology with the C. elegans developmental regulator Unc-86 and extended similarity with the Drosophila neurodevelopmental gene I-POU, which defines a novel POU-IV box. Brn-3.2 exhibits DNA binding properties similar to those of Brn-3.0, but its expression is uniquely regulated by retinoic acid in teratocarcinoma and neuroblastoma cells. In the developing PNS and retina, the expression pattern of Brn-3.2 is similar to that of Brn-3.0. In the caudal CNS (spinal cord, hindbrain, and midbrain) Brn-3.2 and Brn-3.0 are initially coexpressed, but diverge later in development. Rostral to the midbrain, Brn-3.2 and Brn-3.0 exhibit nonoverlapping patterns of expression, suggesting divergence of gene function in more recently evolved structures. Our analysis suggests that in the CNS Brn-3.2 is selectively expressed in postmitotic neurons, implying a role in specifying terminally differentiated neuronal phenotypes.

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.

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/β-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.

Lack of association between an RFLP near the D2 dopamine receptor gene and severe alcoholism

Blum et al (1990) have recently examined a restriction fragment length polymorphism (RFLP) detected by TaqI RFLP to the dopamine D2 receptor gene (DRD2) in deceased alcoholics and nonalcoholics, and reported an association between alcoholism and the A1 allele. Subsequent studies, however, by other investigators have failed to confirm this. We have examined the DRD2 TaqI RFLP in 47 living Caucasian males with severe alcoholism. All alcoholic subjects were thoroughly characterized by a structured interview, and met DSM-III-R criteria for alcohol dependence. Only 9/47 (19%) (1990) of these alcoholics had the AI allele compared to 14/22 (64%) reported by Blum et al. This rate was not significantly different from the rates reported in control populations by Blum et al (1990), CEPH, or Bolos et al (1990), and differed only slightly from those reported by Grandy et al (1990). Alcoholics selected for severe medical complications also displayed a similar rate. Our data do not support an association between alcoholism and the D2 dopamine receptor gene in this population.

POU domain factors of the Brn-3 class recognize functional DNA elements which are distinctive, symmetrical, and highly conserved in evolution

To better understand the diversity of function within the POU domain class of transcriptional regulators, we have determined the optimal DNA recognition site of several proteins of the POU-IV (Brn-3) subclass by random oligonucleotide selection. The consensus recognition element derived in this study, ATAATTAAT, is clearly distinct from octamer sites described for the POU factor Oct-1. The optimal POU-IV site determined here also binds Brn-3.0 with significantly higher affinity than consensus recognition sites previously proposed for this POU subclass. The binding affinity of Brn-3.0 on its optimal site, several variants of this site, and several naturally occurring POU recognition elements is highly correlated with the activation of reporter gene expression by Brn-3.0 in transfection assays. The preferred DNA recognition site of Brn-3.0 resembles strongly the optimal sites of another mammalian POU-IV class protein, Brn-3.2, and of the Caenorhabditis elegans Brn-3.0 homolog Unc-86, demonstrating that the site-specific DNA recognition properties of these factors are highly conserved between widely divergent species.

Twin of I-POU: A Two Amino Acid Difference in the I-POU Homeodomain Distinguishes an Activator from an Inhibitor of Transcription

I-POU, a POU domain nuclear protein that lacks two conserved basic amino acids of the POU homeodomain is coexpressed in the developing Drosophila nervous system with a second POU domain transcription factor, Cf1-a. I-POU does not bind to DNA but forms a POU domain-mediated, high affinity heterodimer with Cf1-a, inhibiting its ability to bind and activate the dopa decarboxylase gene. The I-POU/Cf1-a dimerization interface encompasses only the N-terminal basic region and helices 1 and 2 of the POU homeodomains with precise amino acid and alpha-helical requirements. twin of I-POU, an alternatively spliced transcript of the I-POU gene, encodes a protein containing the two basic amino acid residues absent in I-POU. Twin of I-POU is incapable of dimerizing with Cf1-a, but can act as a positive transcription factor on targets distinct from those regulated by Cf1-a. These findings suggest that the I-POU genomic locus simultaneously generates both a specific activator and inhibitor of gene transcription, capable of modulating two distinct regulatory programs during neural development.

Ovothiol replaces glutathione peroxidase as a hydrogen peroxide scavenger in sea urchin eggs

Despite its potential toxicity, H2O2 is used as an extracellular oxidant by Stronglylocentrotus purpuratus eggs to cross-link their fertilization envelopes. These eggs contain 5 mM 1-methyl-N alpha,N alpha-dimethyl-4-mercaptohistidine (ovothiol C), which reacts with H2O2. In consuming H2O2 and being reduced by glutathione, ovothiol acts as a glutathione peroxidase and replaces the function of the enzyme in eggs. The ovothiol system is more effective than egg catalase in destroying H2O2 at concentrations produced during fertilization and constitutes a principal mechanism for preventing oxidative damage at fertilization.

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 and Nurr1 mediate a gene regulatory pathway for habenula development

The habenula is a dorsal diencephalic structure consisting of medial and lateral subnuclei and a principal output tract, the fasciculus retroflexus, which together form a link between the limbic forebrain and ventral midbrain. Here, we have used microarray and bioinformatic approaches in the mouse to show that the habenula is a distinctive molecular territory of the CNS, with a unique profile of neurotransmitter, ion channel, and regulatory factor expression. Neurons of the medial habenula and part of the lateral habenula express the transcription factor Brn3a/Pou4f1, and Brn3a-expressing habenular neurons project exclusively to the interpeduncular nucleus in the ventral midbrain. In Brn3a mutant embryos, the fasciculus retroflexus is directed appropriately, but habenular neurons fail to innervate their targets. Microarray analysis of Brn3a null embryos shows that this factor regulates an extensive program of habenula-enriched genes, but not generic neural properties. The orphan nuclear receptor Nurr1/Nr4a2 is coexpressed with Brn3a in the developing habenula, is downstream of Brn3a, and mediates expression of a subset of Brn3a-regulated transcripts. Together, these findings begin to define a gene regulatory pathway for habenula development in mammals.