Prenatal ß-catenin/Brn2/Tbr2 transcriptional cascade regulates adult social and stereotypic behaviors.

Social interaction is a fundamental behavior in all animal species, but the developmental timing of the social neural circuit formation and the cellular and molecular mechanisms governing its formation are poorly understood. We generated a mouse model with mutations in two Disheveled genes, Dvl1 and Dvl3, that displays adult social and repetitive behavioral abnormalities associated with transient embryonic brain enlargement during deep layer cortical neuron formation. These phenotypes were mediated by the embryonic expansion of basal neural progenitor cells (NPCs) via deregulation of a ß-catenin/Brn2/Tbr2 transcriptional cascade. Transient pharmacological activation of the canonical Wnt pathway during this period of early corticogenesis rescued the ß-catenin/Brn2/Tbr2 transcriptional cascade and the embryonic brain phenotypes. Remarkably, this embryonic treatment prevented adult behavioral deficits and partially rescued abnormal brain structure in Dvl mutant mice. Our findings define a mechanism that links fetal brain development and adult behavior, demonstrating a fetal origin for social and repetitive behavior deficits seen in disorders such as autism.

Ghrelin is expressed in a novel endocrine cell type in developing rat islets and inhibits insulin secretion from INS-1 (832/13) cells.

Ghrelin is produced mainly by endocrine cells in the stomach and is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). It also influences feeding behavior, metabolic regulation, and energy balance. It affects islet hormone secretion, and expression of ghrelin and GHS-R in the pancreas has been reported. In human islets, ghrelin expression is highest pre- and neonatally. We examined ghrelin and GHS-R in rat islets during development with immunocytochemistry and in situ hybridization. We also studied the effect of ghrelin on insulin secretion from INS-1 (832/13) cells and the expression of GHS-R in these cells. We found ghrelin expression in rat islet endocrine cells from mid-gestation to 1 month postnatally. Islet expression of GHS-R mRNA was detected from late fetal stages to adult. The onset of islet ghrelin expression preceded that of gastric ghrelin. Islet ghrelin cells constitute a separate and novel islet cell population throughout development. However, during a short perinatal period a minor subpopulation of the ghrelin cells co-expressed glucagon or pancreatic polypeptide. Markers for cell lineage, proliferation, and duct cells revealed that the ghrelin cells proliferate, originate from duct cells, and share lineage with glucagon cells. Ghrelin dose-dependently inhibited glucose-stimulated insulin secretion from INS-1 (832/13) cells, and GHS-R was detected in the cells. We conclude that ghrelin is expressed in a novel developmentally regulated endocrine islet cell type in the rat pancreas and that ghrelin inhibits glucose-stimulated insulin secretion via a direct effect on the beta-cell.

Combinatorial roles of the nuclear receptor corepressor in transcription and development.

Transcriptional repression plays crucial roles in diverse aspects of metazoan development, implying critical regulatory roles for corepressors such as N-CoR and SMRT. Altered patterns of transcription in tissues and cells derived from N-CoR gene-deleted mice and the resulting block at specific points in CNS, erythrocyte, and thymocyte development indicated that N-CoR was a required component of short-term active repression by nuclear receptors and MAD and of a subset of long-term repression events mediated by REST/NRSF. Unexpectedly, N-CoR and a specific deacetylase were also required for transcriptional activation of one class of retinoic acid response element. Together, these findings suggest that specific combinations of corepressors and histone deacetylases mediate the gene-specific actions of DNA-bound repressors in development of multiple organ systems.

A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system.

Axon pathfinding relies on the ability of the growth cone to detect and interpret guidance cues and to modulate cytoskeletal changes in response to these signals. We report that the murine POU domain transcription factor Brn-3.2 regulates pathfinding in retinal ganglion cell (RGC) axons at multiple points along their pathways and the establishment of topographic order in the superior colliculus. Using representational difference analysis, we identified Brn-3.2 gene targets likely to act on axon guidance at the levels of transcription, cell-cell interaction, and signal transduction, including the actin-binding LIM domain protein abLIM. We present evidence that abLIM plays a crucial role in RGC axon pathfinding, sharing functional similarity with its C. elegans homolog, UNC-115. Our findings provide insights into a Brn-3.2-directed hierarchical program linking signaling events to cytoskeletal changes required for axon pathfinding.

The role of POU domain proteins in the regulation of mammalian pituitary and nervous system development.

POU domain proteins represent a subfamily of homeodomain-containing transcription factors that are expressed in many animal orders in a number of distinct regions in the developing and adult organism. In mammals, the expression profiles of these factors have suggested roles for class I, class III, and class IV POU domain proteins in the development, maintenance, and function of the endocrine and nervous systems. The genetic characterizations of the functions of these proteins during the generation, differentiation, and maturation of cells comprising these tissues have revealed a requirement for the individual actions of these transcription factors in the development of various elements of the anterior pituitary, the brain, and the somatosensory, vestibular/cochlear, and visual systems.

POU domain factors in neural development.

Transcription factors serve critical roles in the progressive development of general body plan, organ commitment, and finally, specific cell types. Comparison of the biological roles of a series of individual members within a family permits some generalizations to be made regarding the developmental events that are likely to be regulated by a particular class of transcription factors. Here, we evidence that the developmental functions of the family of transcription factors characterized by the POU DNA binding motif exerts roles in mammalian development. The POU domain family of transcription factors was defined following the observation that the products of three mammalian genes, Pit-1, Oct-1, and Oct-2, and the protein encoded by the C. elegans gene unc-86, shared a region of homology, known as the POU domain. The POU domain is a bipartite DNA binding domain, consisting of two highly conserved regions, tethered by a variable linker. The approximately 75 amino acid N-terminal region was called the POU-specific domain and the C-terminal 60 amino acid region, the POU-homeodomain. High-affinity site-specific DNA binding by POU domain transcription factors requires both the POU-specific and the POU-homeodomain. Resolution of the crystal structures of Oct-1 and Pit-1 POU domains bound to DNA as a monomer and homodimer, respectively, confirmed several of the in vitro findings regarding interactions of this bipartite DNA binding domain with DNA and has provided important information regarding the flexibility and versatility of POU domain proteins. Overall the crystal structure of a monomer of the Oct-1 POU domain bound to the octamer element was similar to that predicted by the NMR solution structures of the POU-specific domain and the POU-homeodomain in isolation, with the POU-specific domain consists of four alpha helices, with the second and third helices forming a structure similar to the helix-turn-helix motif of the lambda and 434 repressors; several of the DNA base contacts are also conserved. A homodimer of the Pit-1 POU domain was crystallized bound to a Pit-1 dimer DNA element that is closely related to a site in the proximal promoter of the prolactin gene. The structure of the Pit-1 POU domain on DNA is very similar to that of Oct-1, and the Pit-1 POU-homeodomain/DNA structure is strikingly similar to that of other homeodomains, including the Oct-1 POU-homeodomain. The DNA contacts made by the Pit-1 POU-specific domain are also similar to those of Oct-1 and conserved with many made by the prokaryotic repressors. In the Oct-1 crystal, the POU-specific domain recognizes a GCAT half-site, while the corresponding sequence recognized by the Pit-1 POU-specific domain, GTAT, is on the opposing strand. As a result, the orientation of the Pit-1 POU-specific domain relative to the POU-homeodomain is flipped, as compared to the Oct-1 crystal structure, indicating the remarkable flexibility of the POU-specific domain in adapting to variations in sequence within the site. Also in contrast to the Oct-1 monomer structure is the observation that the POU-specific and POU-homeodomain of each Pit-1 molecule make major groove contacts on the same face of the DNA, consistent with the constraints imposed by its 15 amino acid linker. As a result, the Pit-1 POU domain homodimer essentially surrounds its DNA binding site. In the Pit-1 POU domain homodimer the dimerization interface is formed between the C-terminal end of helix 3 of the POU-homeodomain of one Pit-1 molecule and the N-terminus of helix 1 and the loop between helices 3 and 4 of the POU-specific domain of the other Pit-1 molecule. In contrast to other homeodomain crystal structures, the C-terminus of helix 3 in the Pit-1 POU-homeo-domain has an extended structure. (ABSTRACT TRUNCATED)

Functions of the POU domain genes Skn-1a/i and Tst-1/Oct-6/SCIP in epidermal differentiation.

Here we report on investigation of the role of the POU domain genes Skin-1a/i (Skn-1a/i/Epoc/Oct-11) and Testes-1 (Tst-1/Oct-6/SCIP) in epidermis where proliferating basal keratinocytes withdraw from the cell cycle, migrate suprabasally, and terminally differentiate to form a multilayered, stratified epithelium. The expression of the Skn-1a/i and Tst-1 genes is linked to keratinocyte differentiation in vivo and in vitro, whereas the ubiquitous POU domain factor Oct-1 is expressed highly in both proliferating and post-mitotic keratinocytes. Analysis of Skn-1a/i gene-deleted mice reveals that the Skn-1a/i gene modulates the pattern of expression of the terminal differentiation marker loricrin and inhibits expression of genes encoding markers of the epidermal keratinocyte wounding response. Although epidermis from Tst-1 gene-deleted mice develops normally, epidermis from mice deleted for both Skn-1a/i and Tst-1 is hyperplastic and fails to suppress expression of K14 and Spr-1 in suprabasal cells when transplanted onto athymic mice. This suggests that Skn-1a/i and Tst-1 serve redundant functions in epidermis. Therefore, at least two POU domain genes, Skn-1a/i and Tst-1, serve both distinct and overlapping functions to regulate differentiation of epidermal keratinocytes during normal development and wound healing.

Requirement for Brn-3.0 in differentiation and survival of sensory and motor neurons.

Specific families of transcription factors mediate events in the sequential maturation of distinct neuronal phenotypes. Members of one such family, the class IV POU domain transcription factor Brn-3.0, and two highly related factors Brn-3.1 and Brn-3.2, are differentially expressed in the developing and mature mammalian nervous system. The expression pattern of Brn-3.0 suggested that it has an important role in the development of sensory ganglia, as well as red nucleus, inferior olive, and nucleus ambiguus. Analysis of mice null for the Brn-3.0 locus shows that Brn-3.0 is required for the survival of subpopulations of proprioceptive, mechanoreceptive and nociceptive sensory neurons, where deletion of the gene affects neurotrophin and neurotrophin-receptor gene expression. Deletion of Brn-3.0 also alters either differentiation, migration or survival of specific central neuronal populations.

Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development.

The neurally expressed genes Brn-3.1 and Brn-3.2 (refs 1-6) are mammalian orthologues of the Caenorhabditis elegans unc-86 gene that constitute, with Brn-3.0 (refs 1-3,8,9), the class IV POU-domain transcription factors. Brn-3.1 and Brn-3.2 provide a means of exploring the potentially distinct biological functions of expanded gene families in neural development. The highly related members of the Brn-3 family have similar DNA-binding preferences and overlapping expression patterns in the sensory nervous system, midbrain and hindbrain, suggesting functional redundancy. Here we report that Brn-3.1 and Brn-3.2 critically modulate the terminal differentiation of distinct sensorineural cells in which they exhibit selective spatial and temporal expression patterns. Deletion of the Brn-3.2 gene causes the loss of most retinal ganglion cells, defining distinct ganglion cell populations. Mutation of Brn-3.1 results in complete deafness, owing to a failure of hair cells to appear in the inner ear, with subsequent loss of cochlear and vestibular ganglia.

Development and survival of the endocrine hypothalamus and posterior pituitary gland requires the neuronal POU domain factor Brn-2.

Neurons comprising the endocrine hypothalamus are disposed in several nuclei that develop in tandem with their ultimate target the pituitary gland, and arise from a primordium in which three related class III POU domain factors, Brn-2, Brn-4, and Brn-1, are initially coexpressed. Subsequently, these factors exhibit stratified patterns of ontogenic expression, correlating with the appearance of distinct neuropeptides that define three major endocrine hypothalamic cell types. Strikingly, deletion of the Brn-2 genomic locus results in loss of endocrine hypothalamic nuclei and the posterior pituitary gland. Lack of Brn-2 does not affect initial hypothalamic developmental events, but instead results in a failure of differentiation to mature neurosecretory neurons of the paraventricular and supraoptic nuclei, characterized by an inability to activate genes encoding regulatory neuropeptides or to make correct axonal projections, with subsequent loss of these neurons. Thus, both neuronal and endocrine components of the hypothalamic-pituitary axis are critically dependent on the action of specific POU domain factors at a penultimate step in the sequential events that underlie the appearance of mature cellular phenotypes.

Brn-3.0: a POU-domain protein expressed in the sensory, immune, and endocrine systems that functions on elements distinct from known octamer motifs.

Characterization of Brn-3.0 and identification of a highly related member (Brn-3.1) of the class IV POU-domain family suggest potential roles of Brn-3.0 in the development of retinal ganglion cells and sensory neurons, as well as potential roles in the pituitary gland and the immune system. Brn-3.0 is expressed in the pituitary gland and in a corticotroph cell line. A functional DNA response element has been identified in the proopiomelanocortin promoter. In contrast to previously described mammalian POU-domain proteins, Brn-3.0 binds relatively ineffectively to known octamer DNA motifs, but instead binds with high affinity to a distinct set of DNA elements, functioning as a transcriptional activator. Brn-3.0, Brn-3.1, and the Drosophila tI-POU share an N-terminal region of homology, referred to as the "POU-IV box," which is similar to a conserved functional domain in the c-myc gene family.

Chromosomal organization of mammalian POU domain factors.

We present the chromosomal locations in mouse of eight new members of the mammalian POU domain family of transcriptional regulators. Chromosomal assignments were made for Brn-1 (Chr 1), Brn-2 (Chr 4), Brn-4 (Chr X), Brn-3.0 (Chr 14), Brn-3.1 (Chr 18), Brn-5.0 (Chr 15), Skn-1a/i (Chr 9), and Sprm-1 (Chr 13) in addition to the previously reported Pit-1 (Chr 16), Tst-1 (Chr 4), Oct-3/4 (Chr 17), Oct-1 (Chr 1), and Oct-2 (Chr 7) genes. Several conclusions have emerged from this analysis. First, among the most highly related family members (Brn-1, Brn-2, Brn-4, and Tst-1; Brn-3.0 and Brn-3.1; Oct-1, Oct-2, and Skn-1a/i) no chromosomal linkage is noted. Second, no clusters of genes are observed, irrespective of homology. Finally, no obvious linkages to genes for known additional regulatory factors with a specific origin of cell type are apparent. Thus, members of this large gene family, presumably arising as duplication events from common ancestral genes, apparently function in distinct chromosomal milieu under independent regulation. Some of these newly localized genes map in close proximity to existing mouse mutations.

Patterns of cytokine gene expression by CD4+ T cells from young and old mice.

We have analyzed the patterns of induced cytokine gene expression and cell cycle activity by CD4+ cells from mice, and have examined how these response patterns change during the aging process. CD4+ cells were isolated from spleens of young adult and old C57BL/6NNia mice and were stimulated in vitro with plate-bound anti-CD3 epsilon mAb. The cells were then assessed over time for the capacity to accumulate transcripts for IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IFN-gamma, TNF-alpha, and TNF-beta; to secrete IL-2, IL-3, IL-4, IL-5, IL-6, and IFN-gamma; and to progress through S phase. Before the first major cell division in culture (< 32 h), stimulated CD4+ cells of the old group contained similar peak levels of IL-2, TNF-alpha, and TNF-beta transcripts relative to young adult controls, whereas IL-3, IL-4, IL-5, and IFN-gamma transcripts accumulated to significantly higher peak levels in the old group. These findings were consistent with the patterns of cytokine secretion later in culture (24 to 72 h): the peak IL-2 levels were similar between age groups, but the old group exhibited an enhanced capacity to release IL-3, IL-4, IL-5, and IFN-gamma. In contrast, CD4+ cells of the young group were superior in the hyper-expression of the housekeeping gene, rpL32, before cell division and in the levels of S phase activity throughout 3-day cultures. Similar analyses of CD4+ cells from mice of intermediate ages showed that the alterations in cytokine profiles occurred gradually from young adulthood to old age, whereas the reductions in proliferative capacity were late life changes. Consistent with previous reports, we found that the splenic CD4+ cell group also underwent a progressive, age-dependent increase in the proportions of cells expressing high levels of membrane CD44 (a phenotype associated with memory or effector cells). Moreover, the analysis of IL-3, IL-5, and IFN-gamma production by isolated CD4+CD44lo and CD4+CD44hi cells revealed that the capacity to produce these cytokines segregated predominantly with the CD44hi subset, regardless of donor age. Taken together, our data suggest that gradual age-associated shifts in the subset composition of the splenic CD4+ cell pool underlie progressive changes in the patterns of cytokine gene expression by this cell group.

Interleukin-6 production by murine B cells and B cell lines.

We have analyzed interleukin (IL)-6 gene transcription and IL-6 secretion by murine B cells in vitro. Mitogenic doses of lipopolysaccharide (LPS) or LPS in combination with F(ab')2 goat anti-mouse IgM antibodies (GAMmu), but not GAMmu alone, induced B cells to synthesize and release IL-6. In time course experiments, the accumulation of IL-6 mRNA was first detectable at 24-36 hr of culture and the levels were maintained through 60 hr; these kinetics correlated well with increases in supernatant IL-6 levels and were coincident with vigorous cell cycle activity. We also analyzed constitutive and LPS-induced IL-6 gene expression by the murine B cell lines: 70Z/3, 38C-13, WEHI-231, X16C, WEHI-279, and BCL1. Only the WEHI-279 and BCL1 lines produced detectable IL-6 constitutively, and the BCL1 cells could be further induced by treatment with LPS. Of the remaining cell lines, only WEHI-231 and X16C could be stimulated with LPS to produce IL-6. To evaluate whether IL-6 could influence proliferation and Ig secretion by the cell lines, low cell density cultures were established in the presence of various doses of human rIL-6 and were assessed over time for levels of [3H]thymidine uptake and supernatant Ig. Under these conditions, IL-6 had no effect on either cell function.

Tolerance-related V beta clonal deletions in normal CD4-8-, TCR-alpha/beta + and abnormal lpr and gld cell populations.

We have analyzed tolerance-related clonal deletion of Mls-and I-E-reactive thymocytes at the RNA level using a multi-V beta probe RNAse protection assay, and used this phenomenon to identify the maturation stage of the abnormally expanded CD4-8-, TCR-alpha/beta + subset in lpr and gld homozygous mice, and of the phenotypically similar minor thymocyte subset found in normal mice. Essentially complete V beta clonal deletions were detected in lpr and gld cells of all appropriate background strains. Substantial, but not complete, V beta clonal deletions were also detected in the CD4-8- TCR-alpha/beta + subset of normal mice. Since expression of CD4/CD8 is required for V beta clonal deletions to occur, we conclude that lpr and gld cells, and at least a portion of CD4-8- TCR-alpha/beta + thymocytes in normal mice, are derived by secondary loss of CD4/CD8 accessory molecules from more mature CD4+8+ precursors. One possible interpretation of these findings is that such CD4/CD8 loss may affect a class of self-reactive thymocytes that have escaped direct clonal deletion. Exportation and expansion of such cells in the periphery may be an important contributory factor in the induction of systemic autoimmunity.

T-cell receptor alpha-chain variable-region haplotypes of normal and autoimmune laboratory mouse strains.

We used Southern blotting and mRNA analysis to characterize allelic polymorphisms among genes of the T-cell antigen receptor (TCR) alpha-chain variable-region (V alpha) locus in a large panel of normal and autoimmune-susceptible or autoimmune-contributing strains of laboratory mice. Four major V alpha haplotypes were defined on the basis of multiple restriction fragment length polymorphisms for each of nine V alpha subfamily probes used. Southern blotting also revealed haplotype-specific loss of bands within some V alpha subfamilies, consistent with the deletion of particular V alpha genes or sets of genes from haplotype to haplotype. In contrast to the situation in the V beta locus, however, deletion of entire V alpha subfamilies was not observed. The nature of V alpha allelic variability was further explored by using an RNase protection assay to analyze expressed V alpha mRNA sequences in thymocyte RNA. Such analysis revealed both shared and unique patterns of V alpha mRNA expression among the different haplotypes and supported the conclusion that haplotype differences sometimes involve V alpha gene deletions. Interestingly, a disproportionate number of, but not all, autoimmune-susceptible strains, including NZB, SJL, SWR, PL/J, and NOD, share a common V alpha haplotype. The identification of murine TCR V alpha haplotypes should provide a basis for understanding the role of TCR diversity in normal immunoregulatory and immune-response phenomena, as well as autoimmune-disease predisposition.

Clonal diversity and T-cell receptor beta-chain variable gene expression in enlarged lymph nodes of MRL-lpr/lpr lupus mice.

The autosomal recessive lpr gene accelerates a systemic lupus erythematosus-like disease in genetically predisposed mice and induces autoantibodies in mice of normal genetic background. The molecular mode(s) of action of the lpr gene and its chromosomal location remain unknown, but it is primarily expressed as a massive T-cell proliferation manifested only in the presence of a thymus. To define the clonal diversity and maturational stage of the abnormally proliferating T cells found in enlarged lymph nodes of MRL-lpr/lpr mice, and their possible role in autoreactive B-cell activation, we analyzed their T-cell receptor beta-chain variable region (V beta) gene sequences. Twenty-five VDJ-containing beta-chain cDNA sequences were examined, each of which was found to derive from a distinct rearrangement in the correct reading frame, yielding translatable beta-chain mRNAs. An additional 10 clones were derived from truncated nonfunctional mRNAs. D beta 1 and D beta 2 elements were used equally in the sequenced clones, and 10 of the possible 12 mouse J beta elements were represented. Remarkably, 60% of the functional beta-chain mRNAs expressed V beta 8.2 or V beta 8.3 genes, whereas the equally homologous V beta 8.1 gene was not represented at all. Other V beta genes were found at lower frequencies in the library, including one previously unidentified V beta gene. The results indicate that the clonal makeup of the abnormally proliferating lymph node T cells in MRL-lpr/lpr mice is heterogeneous, but V beta gene expression is significantly skewed in favor of V beta 8.2/8.3 genes. The preferential representation of V beta 8 genes might be caused by lpr gene-induced modification of T-cell thymic processing and relate to the lpr gene-associated autoimmunity.