The circadian clock controls many physiological parameters including immune response to infectious agents, which is mediated by activation of the transcription factor NF-κB. It is widely accepted that circadian regulation is based on periodic changes in gene expression that are triggered by transcriptional activity of the CLOCK/BMAL1 complex. Through the use of a mouse model system we show that daily variations in the intensity of the NF-κB response to a variety of immunomodulators are mediated by core circadian protein CLOCK, which can up-regulate NF-κB-mediated transcription in the absence of BMAL1; moreover, BMAL1 counteracts the CLOCK-dependent increase in the activation of NF-κB-responsive genes. Consistent with its regulatory function, CLOCK is found in protein complexes with the p65 subunit of NF-κB, and its overexpression correlates with an increase in specific phosphorylated and acetylated transcriptionally active forms of p65. In addition, activation of NF-κB in response to immunostimuli in mouse embryonic fibroblasts and primary hepatocytes isolated from Clock-deficient mice is significantly reduced compared with WT cells, whereas Clock-Δ19 mutation, which reduces the transactivation capacity of CLOCK on E-box-containing circadian promoters, has no effect on the ability of CLOCK to up-regulate NF-κB-responsive promoters. These findings establish a molecular link between two essential determinants of the circadian and immune mechanisms, the transcription factors CLOCK and NF-κB, respectively.

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.

Brn3a/Brn-3.0 is a POU-domain transcription factor expressed in primary sensory neurons of the cranial and dorsal root ganglia and in specific neurons in the caudal CNS. Mice lacking Brn3a undergo extensive sensory neural death late in gestation and die at birth. To further examine Brn3a expression and the abnormalities that accompany its absence, we constructed a transgene containing 11 kb of Brn3a upstream regulatory sequence linked to a LacZ reporter. Here we show that these regulatory sequences direct transgene expression specifically to Brn3a peripheral sensory neurons of the cranial and dorsal root ganglia. Furthermore, expression of the 11 kb/LacZ reporter in the sensory neurons of the mesencephalic trigeminal, but not other Brn3a midbrain neurons, demonstrates that cellspecific transgene expression is targeted to a functional class of neurons rather than to an anatomical region. We then interbred the 11 kb/LacZ reporter strain with mice carrying a null mutant allele of Brn3a to generate 11 kb/LacZ, Brn3a knockout mice. ␤-Galactosidase expression in these mice reveals significant axonal growth defects, including excessive and premature branching of the major divisions of the trigeminal nerve and a failure to correctly innervate whisker follicles, all of which precede sensory neural death in these mice. These defects in Brn3a Ϫ/Ϫ mice resemble strongly those seen in mice lacking the mediators of sensory pathfinding semaphorin 3A and neuropilin-1. Here we show, however, that sensory neurons are able to express neuropilin-1 in the absence of Brn3a. Key words: POU-domain; homeodomain; Brn3; TrkC; trigeminal ganglion; sensory ganglion; axon guidanceThe vertebrate nervous system contains a large number of specific cell types, and how this neuronal diversity is generated is a central question in the study of brain development. Many neurons in the developing brain can be recognized by the expression of specific "neural identity" genes that are first detectable at approximately the time of exit from cell division and may persist throughout embryogenesis. Most such genes discovered to date have been transcription factors, often but not always containing a homeodomain motif (Rubenstein and Puelles, 1994). A specific neural identity gene may be expressed in a recognizable class of neurons, such as the motor neurons of the brainstem and spinal cord, or it may characterize a group of cells that have no other known features in common. Accordingly, they are good candidates for establishing the neuronal phenotypes characterized by, among others, the cellspecific expression of neurotransmitters and their receptors, axonal guidance to selected targets, and synaptic specificity. However, the neuronal properties that are actually regulated by these factors, and how this regulation is accomplished, remain primarily undiscovered.The "Brn3" or POU-IV class of transcription factors is comprised of three members in vertebrates that share very similar DNA recognition properties to their invertebrate counterparts (Gruber et al., 199...

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.