The POU domain transcription factor Brn-3a protects cortical neurons from apoptosis.

We have demonstrated previously that exogenously expressed Brn-3a is capable of protecting neurons of the peripheral nervous system against apoptosis. In these previous studies Brn-3a showed a degree of neuronal sub-type specificity, in that while it could promote survival in NGF-dependent sensory neurons, no effect was observed in NGF-dependent neurons of the sympathetic nervous system. In this report, we show that Brn-3a delivered using a herpes simplex virus is capable of protecting cultures of rat cerebrocortical neurons of the central nervous system against two types of cell death stimuli, including glutamate neurotoxicity. Hence the protective effect of Brn-3a is not confined to neurons of the peripheral nervous system but can also occur in neurons of the CNS.     

Slit1b-Robo3 signaling and N-cadherin regulate apical process retraction in developing retinal ganglion cells

When neurons exit the cell cycle after their terminal mitosis, they detach from the apical surface of the neuroepithelium. Despite the fact that this detachment is crucial for further neurogenesis and neuronal migration, the underlying mechanisms are still not understood. Here, taking advantage of the genetics and imaging possibilities of the zebrafish retina as a model system, we show by knockdown experiments that the guidance molecule Slit1b and its receptor Robo3 are required for apical retraction of retinal ganglion cells (RGCs). In contrast, N-cadherin seems to be responsible for maintenance of apical attachment, as expression of dominant-negative N-cadherin causes RGCs to lose apical attachments prematurely and rescues retraction in slit1b morphants. These results suggest that Slit-Robo signaling downregulates N-cadherin activity to allow apical retraction in newly generated RGCs.     

Brn-3a deficiency increases tyrosine hydroxylase-immunoreactive neurons in the dorsal root ganglion

Immunohistochemistry for tyrosine hydroxylase (TH) was performed on the dorsal root ganglia (DRG) in wild-type, heterozygous and Brn-3a knockout mice at embryonic day 18.5. TH-immunoreactive (-IR) neurons were detected in the DRG of wild-type and heterozygous mice, but their proportion was greatly increased by the loss of Brn-3a function (wild-type and heterozygot, 8.4%; knockout, 20.9%). IR neurons were of various sizes in wild-type (mean+/-S.D.=118.1+/-55.4 microm2, range=26.6-306.3 microm2) and heterozygous mice. In the knockout mice, however, TH-IR neurons were mostly small (mean+/-S.D.=68.2+/-34.3 microm2, range=11.8-166.8 microm2). The present study suggests that Brn-3a may normally suppress TH expression in many small DRG neurons but activate TH expression in large DRG neurons.     

Effect of Brn-3a deficiency on CGRP-immunoreactivity in the dorsal root ganglion

Immunohistochemistry for calcitonin gene-related peptide (CGRP) was performed on the dorsal root ganglion (DRG) and spinal cord in wildtype and knockout mice for Brn-3a. CGRP-immunoreactive (-IR) neurons were abundant in the DRG of wildtype, heterozygous and knockout mice. Cell size analysis revealed that CGRP-IR neurons were of various sizes in wildtype and heterozygous mice. In the knockout mice, however, most of CGRP-IR neurons were small. In the spinal cord of knockout mice, the number of CGRP-IR fibers increased in the dorsal column but decreased in the deep part of the dorsal horn. The loss of Brn-3a may have different effects on CGRP-IR expression in small and large DRG neurons.     

Dynamic expression of ganglion cell markers in retinal progenitors during the terminal cell cycle

The vertebrate neural retina contains seven major cell types, which arise from a common multipotent progenitor pool. During neurogenesis, these cells stop cycling, commit to a single fate, and differentiate. The mechanism and order of these steps remain unclear. The first-born type of retinal neurons, ganglion cells (RGCs), develop through the actions of Math5 (Atoh7), Brn3b (Pou4f2) and Islet1 (Isl1) factors, whereas inhibitory amacrine and horizontal precursors require Ptf1a for differentiation. We have examined the link between these markers, and the timing of their expression during the terminal cell cycle, by nucleoside pulse-chase analysis in the mouse retina. We show that G2 phase lasts 1-2 h at embryonic (E) 13.5 and E15.5 stages. Surprisingly, we found that cells expressing Brn3b and/or Isl1 were frequently co-labeled with EdU after a short chase (<1 h) in early embryos (E15), Brn3b and Isl1 were exclusively expressed in post-mitotic cells, even as new RGCs are still generated. In contrast, Ptf1a and amacrine marker AP2α were detected only after terminal mitosis, at all developmental stages. Using a retroviral tracer in embryonic retinal explants (E12-E13), we identified two-cell clones containing paired ganglion cells, consistent with RGC fate commitment prior to terminal mitosis. Thus, although cell cycle exit and fate determination are temporally correlated during retinal neurogenesis, the order of these events varies according to developmental stage and final cell type.