We thank Dr. Clark Riley, Ms. Carol Davenport, and Ms. Jani ne Ptak for synthetic oligonucleotides; Dr. Keith Fry for the gift of mAb ABS; Ms. Cathy Blizzard for cat eyes; Dr. Mark Molliver for donating fixed monkey eyes; Drs. Masafumi Tanaka and Wi nshi p Herr for Oct.1 cDNA; Mr. Hao Zhou for the GST-Ott-1 POU domain protein; Dr. Y.-W. Peng for rabbit retinas and Dr. Hua-Shun Li for chicken retinas; Dr. Elio Ravi ol a for advice on histologic techniques; and Drs. Robert Rodieck, Jen-Chi h Hsieh, and King-wai Yau for hel pful comments on the manuscript.

The three members of the Brn-3 family of POU domain transcription factors are found in highly restricted sets of central nervous system neurons. Within the retina, these factors are present only within subsets of ganglion cells. We show here that in the developing mouse retina, Immunohistochemistry. Immunostaining of dorsal root and trigeminal ganglia, retinal sections, and retinal flat mounts was performed as previously described (6, 7). Antibodies to Brn3a, Brn-3b, and Brn-3c are described in refs. 6 and 7. Additional antibodies were obtained from the following sources: antiAbbreviations: ES, embryonic stem; mAb, monoclonal antibody; CNS, central nervous system; en, embryonic day n; DAPI, 4',6-diamidino-2-phenylindole; TUNEL, terminal dUTP nick end labeling.

During vertebrate retinogenesis, seven classes of cells are specified from multipotent progenitors. To date, the mechanisms underlying multipotent cell fate determination by retinal progenitors remain poorly understood. Here, we show that the Foxn4 winged helix/forkhead transcription factor is expressed in a subset of mitotic progenitors during mouse retinogenesis. Targeted disruption of Foxn4 largely eliminates amacrine neurons and completely abolishes horizontal cells, while overexpression of Foxn4 strongly promotes an amacrine cell fate. These results indicate that Foxn4 is both necessary and sufficient for commitment to the amacrine cell fate and is nonredundantly required for the genesis of horizontal cells. Furthermore, we provide evidence that Foxn4 controls the formation of amacrine and horizontal cells by activating the expression of the retinogenic factors Math3, NeuroD1, and Prox1. Our data suggest a model in which Foxn4 cooperates with other key retinogenic factors to mediate the multipotent differentiation of retinal progenitors.

The vertebrate neural retina comprises six classes of neurons and one class of glial cells, all derived from a population of multipotent progenitors. There is little information on the molecular mechanisms governing the specification of cell type identity from multipotent progenitors in the developing retina. We report that Ptf1a, a basic-helix-loop-helix (bHLH) transcription factor, is transiently expressed by post-mitotic precursors in the developing mouse retina. Recombination-based lineage tracing analysis in vivo revealed that Ptf1a expression marks retinal precursors with competence to exclusively produce horizontal and amacrine neurons. Inactivation of Ptf1a leads to a fate-switch in these precursors that causes them to adopt a ganglion cell fate. This misspecification of neurons results in a complete loss of horizontal cells, a profound decrease of amacrine cells and an increase in ganglion cells. Furthermore, we identify Ptf1a as a primary downstream target for Foxn4, a forkhead transcription factor involved in the genesis of horizontal and amacrine neurons. These data, together with the previous findings on Foxn4, provide a model in which the Foxn4-Ptf1a pathway plays a central role in directing the differentiation of retinal progenitors towards horizontal and amacrine cell fates.

The Brn-3 subfamily of POU-domain transcription factor genes consists of three highly homologous members-Brn-3a, Brn-3b, and Brn-3c-that are expressed in sensory neurons and in a small number of brainstem nuclei. This paper describes the role of Brn-3c in auditory and vestibular system development. In the inner ear, the Brn-3c protein is found only in auditory and vestibular hair cells, and the Brn-3a and Brn-3b proteins are found only in subsets of spiral and vestibular ganglion neurons. Mice carrying a targeted deletion of the Brn-3c gene are deaf and have impaired balance. These defects ref lect a complete loss of auditory and vestibular hair cells during the late embryonic and early postnatal period and a secondary loss of spiral and vestibular ganglion neurons. Together with earlier work demonstrating a loss of trigeminal ganglion neurons and retinal ganglion cells in mice carrying targeted disruptions in the Brn-3a and Brn-3b genes, respectively, the Brn-3c phenotype reported here demonstrates that each of the Brn-3 genes plays distinctive roles in the somatosensory, visual, and auditory͞ vestibular systems.A number of transcription factors have been implicated in decisions related to neuronal vs. nonneuronal cell fate, regional specification in the nervous system, or determination of the terminally differentiated phenotype. For example, bHLH factors such as neuroD and the achaete-scute family control neural vs. ectodermal cell fates (1, 2), Hox genes control regional specification along the neuraxis (3), and several POU-domain genes act at late stages to control the survival and final differentiated phenotype of particular neuronal subtypes (4). The POU-domain family was intially defined by the mammalian pituitary-specific transcription factor Pit-1͞ GHF-1, the octamer binding proteins Oct-1 and Oct-2, and the Caenorhabditis elegans gene Unc-86 (5). Genetic studies in mice and humans indicate that many POU-domain genes function in the terminal stages of central nervous system development. SCIP͞Tst-1͞Oct-6 controls the differentiation of Schwann cells (6-8), Pit-1͞GHF-1 is required for the normal development of the anterior pituitary (9), Brn-4͞RHS2͞ POU3F4 is required for the normal development of the middle ear (10), and Brn-2 is required for the specification of subsets of neurons in the hypothalamus (11,12).The class IV POU-domain group is defined by the Unc-86 gene (13), the Drosophila I-POU gene (14,15), and the three vertebrate Brn-3 genes (16-20). The Unc-86 protein is found exclusively within a subset of neurons and neuroblasts, and Unc-86 loss-of-function mutations affect some of these cells by causing a daughter cell to assume the fate of its mother or by altering cell phenotypes postmitotically (21-23). In mammals, the three highly homologous class IV POU-domain genes, Brn-3a, Brn-3b, and Brn-3c (also referred to as Brn-3.0, Brn-3.2, and Brn-3.1, respectively), are expressed in distinct but overlapping patterns in the developing and adult brainstem, retina, and dorsal root and trigemina...

The aryl hydrocarbon receptor (AHR) belongs to the PAS (PER-ARNT-SIM) family transcription factors and mediates broad responses to numerous environmental pollutants and cellular metabolites, modulating diverse biological processes from adaptive metabolism, acute toxicity, to normal physiology of vascular and immune systems. The AHR forms a transcriptionally active heterodimer with ARNT (AHR nuclear translocator), which recognizes the dioxin response element (DRE) in the promoter of downstream genes. We determined the crystal structure of the mammalian AHR-ARNT heterodimer in complex with the DRE, in which ARNT curls around AHR into a highly intertwined asymmetric architecture, with extensive heterodimerization interfaces and AHR interdomain interactions. Specific recognition of the DRE is determined locally by the DNA-binding residues, which discriminates it from the closely related hypoxia response element (HRE), and is globally affected by the dimerization interfaces and interdomain interactions. Changes at the interdomain interactions caused either AHR constitutive nuclear localization or failure to translocate to nucleus, underlying an allosteric structural pathway for mediating ligand-induced exposure of nuclear localization signal. These observations, together with the global higher flexibility of the AHR PAS-A and its loosely packed structural elements, suggest a dynamic structural hierarchy for complex scenarios of AHR activation induced by its diverse ligands.he aryl hydrocarbon receptor (AHR) belongs to the PER-ARNT-SIM (PAS) family transcription factor that mediates broad responses to cellular and environmental cues. The AHR has been shown to be activated by diverse environmental toxicants and endogenous ligands, and play an important role in adaptive metabolism, dioxin toxicity, and normal vascular and immune development (1, 2), ever since it was identified four decades ago for mediating metabolic responses to aryl hydrocarbon toxicants (3, 4) and the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (5). The AHR was more recently found to mediate diverse cellular and physiological responses and likely respond to unknown endogenous AHR ligands (1, 2, 6-8). Developmentally, the AHR plays a role in the normal development and function of both the vascular and immune systems (9-12), and has close links to cancer, metabolic, immune, and cardiovascular diseases (13-18).Intense efforts in the past four decades have yielded important insights into the molecular processes governing AHR signaling. Newly synthesized AHR is located in cytosol and associated with the chaperones Hsp90 (19), P23 (20, 21), and AHR associated protein 9 (ARA9, also known as XAP2 or AIP) (22-24). Binding of ligands induces conformational changes in the AHR that lead to exposure of nuclear localization sequences (NLS) (25, 26). Following nuclear translocation, the AHR exchanges chaperones for a transcription partner, ARNT (1) and the AHR-ARNT heterodimer binds near the promoters of target genes at dioxin-response element (DRE...

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In the developing central nervous system, cellular diversity depends in part on organising signals that establish regionally restricted progenitor domains, each of which produces distinct types of differentiated neurons. However, the mechanisms of neuronal subtype specification within each progenitor domain remain poorly understood. The p2 progenitor domain in the ventral spinal cord gives rise to two interneuron (IN) subtypes, V2a and V2b, which integrate into local neuronal networks that control motor activity and locomotion. Foxn4, a forkhead transcription factor, is expressed in the common progenitors of V2a and V2b INs and is required directly for V2b but not for V2a development. We show here in experiments conducted using mouse and chick that Foxn4 induces expression of delta-like 4 (Dll4) and Mash1 (Ascl1). Dll4 then signals through Notch1 to subdivide the p2 progenitor pool. Foxn4, Mash1 and activated Notch1 trigger the genetic cascade leading to V2b INs, whereas the complementary set of progenitors, without active Notch1, generates V2a INs. Thus, Foxn4 plays a dual role in V2 IN development: (1) by initiating Notch-Delta signalling, it introduces the asymmetry required for development of V2a and V2b INs from their common progenitors; (2) it simultaneously activates the V2b genetic programme.