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Agonistic encounters are powerful effectors of future behavior, and the ability to learn from this type of social challenge is an essential adaptive trait. We recently identified a conserved transcriptional program defining the response to social challenge across animal species, highly enriched in transcription factor (TF), energy metabolism, and developmental signaling genes. To understand the trajectory of this program and to uncover the most important regulatory influences controlling this response, we integrated gene expression data with the chromatin landscape in the hypothalamus, frontal cortex, and amygdala of socially challenged mice over time. The expression data revealed a complex spatiotemporal patterning of events starting with neural signaling molecules in the frontal cortex and ending in the modulation of developmental factors in the amygdala and hypothalamus, underpinned by a systems-wide shift in expression of energy metabolism-related genes. The transcriptional signals were correlated with significant shifts in chromatin accessibility and a network of challenge-associated TFs. Among these, the conserved metabolic and developmental regulator ESRRA was highlighted for an especially early and important regulatory role. Cell-type deconvolution analysis attributed the differential metabolic and developmental signals in this social context primarily to oligodendrocytes and neurons, respectively, and we show that ESRRA is expressed in both cell types. Localizing ESRRA binding sites in cortical chromatin, we show that this nuclear receptor binds both differentially expressed energy-related and neurodevelopmental TF genes. These data link metabolic and neurodevelopmental signaling to social challenge, and identify key regulatory drivers of this process with unprecedented tissue and temporal resolution.

Mammalian genomes contain hundreds of genes transcribed by RNA Polymerase III (Pol III), encoding noncoding RNAs and especially the tRNAs specialized to carry specific amino acids to the ribosome for protein synthesis. In addition to this well-known function, tRNAs and their genes (tDNAs) serve a variety of other critical cellular functions. For example, tRNAs and other Pol III transcripts can be cleaved to yield small RNAs with potent regulatory activities. Furthermore, from yeast to mammals, active tDNAs and related "extra-TFIIIC" (ETC) loci provide the DNA scaffolds for the most ancient known mechanism of three-dimensional chromatin architecture. Here we identify the ZSCAN5 TF family - including mammalian ZSCAN5B and its primate-specific paralogs - as proteins that occupy mammalian Pol III promoters and ETC sites. We show that ZSCAN5B binds with high specificity to a conserved subset of Pol III genes in human and mouse. Furthermore, primate-specific ZSCAN5A and ZSCAN5D also bind Pol III genes, although ZSCAN5D preferentially localizes to MIR SINE- and LINE2-associated ETC sites. ZSCAN5 genes are expressed in proliferating cell populations and are cell-cycle regulated, and siRNA knockdown experiments suggested a cooperative role in regulation of mitotic progression. Consistent with this prediction, ZSCAN5A knockdown led to increasing numbers of cells in mitosis and the appearance of cells. Together, these data implicate the role of ZSCAN5 genes in regulation of Pol III genes and nearby Pol II loci, ultimately influencing cell cycle progression and differentiation in a variety of tissues.

The T-box transcription factor TBX18 is essential to mesenchymal cell differentiation in several tissues and Tbx18 loss-of-function results in dramatic organ malformations and perinatal lethality. Here we demonstrate for the first time that Tbx18 is required for the normal development of periductal smooth muscle stromal cells in prostate, particularly in the anterior lobe, with a clear impact on prostate health in adult mice. Prostate abnormalities are only subtly apparent in Tbx18 mutants at birth; to examine postnatal prostate development we utilized a relatively long-lived hypomorphic mutant and a novel conditional Tbx18 allele. Similar to the ureter, cells that fail to express Tbx18 do not condense normally into smooth muscle cells of the periductal prostatic stroma. However, in contrast to ureter, the periductal stromal cells in mutant prostate assume a hypertrophic, myofibroblastic state and the adjacent epithelium becomes grossly disorganized. To identify molecular events preceding the onset of this pathology, we compared gene expression in the urogenital sinus (UGS), from which the prostate develops, in Tbx18-null and wild type littermates at two embryonic stages. Genes that regulate cell proliferation, smooth muscle differentiation, prostate epithelium development, and inflammatory response were significantly dysregulated in the mutant urogenital sinus around the time that Tbx18 is first expressed in the wild type UGS, suggesting a direct role in regulating those genes. Together, these results argue that Tbx18 is essential to the differentiation and maintenance of the prostate periurethral mesenchyme and that it indirectly regulates epithelial differentiation through control of stromal-epithelial signaling.

The ability to image the entire adult mouse heart at high resolution in 3-D would provide enormous advantages in the study of heart disease. However, a technique for imaging nuclear/cellular detail as well as the overall structure of the entire heart in 3-D with minimal effort is lacking. To solve this problem, we modified the benzyl alcohol:benzyl benzoate (BABB) clearing technique by labeling mouse hearts with periodic acid Schiff (PAS) stain. We then imaged the hearts with a combination of two-photon fluorescence microscopy and automated tile-scan imaging/stitching. Utilizing the differential spectral properties of PAS, we could identify muscle and nuclear compartments in the heart. We were also able to visualize the differences between a 3-month-old normal mouse heart and a mouse heart that had undergone heart failure due to the expression of cardiac myosin binding protein-C (cMyBP-C) gene mutation (t/t). Using 2-D and 3-D morphometric analysis, we found that the t/t heart had anomalous ventricular shape, volume, and wall thickness, as well as a disrupted sarcomere pattern. We further validated our approach using decellularized hearts that had been cultured with 3T3 fibroblasts, which were tracked using a nuclear label. We were able to detect the 3T3 cells inside the decellularized intact heart tissue, achieving nuclear/cellular resolution in 3-D. The combination of labeling, clearing, and two-photon microscopy together with tiling eliminates laborious and time-consuming physical sectioning, alignment, and 3-D reconstruction.

The vertebrate T-box transcription factor gene Tbx18 performs a vital role in development of multiple organ systems. Tbx18 insufficiency manifests as recessive phenotypes in the upper urinary system, cardiac venous pole, inner ear, and axial skeleton; homozygous null mutant animals die perinatally. Here, we report a new regulatory mutation of Tbx18, a reciprocal translocation breaking 78kbp downstream of the gene. 12Gso homozygotes present urinary and vertebral defects very similar to those associated with Tbx18-null mutations, but 12Gso is clearly not a global null allele since homozygotes survive into adulthood. We show that 12Gso down-regulates Tbx18 expression in a manner that is both spatially- and temporally-specific; combined with other data, the mutation points particularly to the presence of an essential urogenital enhancer located near the translocation breakpoint site. In support of this hypothesis, we identify a distal enhancer element, ECR1, which is active in developing urogenital and other tissues; we propose that disruption of this element leads to premature loss of Tbx18 function in 12Gso mutant mice. These data reveal a long-range regulatory architecture extending far downstream of Tbx18, identify a novel and likely essential urogenital enhancer, and introduce a new tool for dissecting postnatal phenotypes associated with dysregulation of Tbx18.

Certain complex phenotypes appear repeatedly across diverse species due to processes of evolutionary conservation and convergence. In some contexts like developmental body patterning, there is increased appreciation that common molecular mechanisms underlie common phenotypes; these molecular mechanisms include highly conserved genes and networks that may be modified by lineage-specific mutations. However, the existence of deeply conserved mechanisms for social behaviors has not yet been demonstrated. We used a comparative genomics approach to determine whether shared neuromolecular mechanisms could underlie behavioral response to territory intrusion across species spanning a broad phylogenetic range: house mouse (Mus musculus), stickleback fish (Gasterosteus aculeatus), and honey bee (Apis mellifera). Territory intrusion modulated similar brain functional processes in each species, including those associated with hormone-mediated signal transduction and neurodevelopment. Changes in chromosome organization and energy metabolism appear to be core, conserved processes involved in the response to territory intrusion. We also found that several homologous transcription factors that are typically associated with neural development were modulated across all three species, suggesting that shared neuronal effects may involve transcriptional cascades of evolutionarily conserved genes. Furthermore, immunohistochemical analyses of a subset of these transcription factors in mouse again implicated modulation of energy metabolism in the behavioral response. These results provide support for conserved genetic "toolkits" that are used in independent evolutions of the response to social challenge in diverse taxa.

While many vertebrate transcription factor (TF) families are conserved, the C2H2 zinc finger (ZNF) family stands out as a notable exception. In particular, novel ZNF gene types have arisen, duplicated, and diverged independently throughout evolution to yield many lineage-specific TF genes. This evolutionary dynamic not only raises many intriguing questions but also severely complicates identification of those ZNF genes that remain functionally conserved. To address this problem, we searched for vertebrate "DNA binding orthologs" by mining ZNF loci from eight sequenced genomes and then aligning the patterns of DNA-binding amino acids, or "fingerprints," extracted from the encoded ZNF motifs. Using this approach, we found hundreds of lineage-specific genes in each species and also hundreds of orthologous groups. Most groups of orthologs displayed some degree of fingerprint divergence between species, but 174 groups showed fingerprint patterns that have been very rigidly conserved. Focusing on the dynamic KRAB-ZNF subfamily--including nearly 400 human genes thought to possess potent KRAB-mediated epigenetic silencing activities--we found only three genes conserved between mammals and nonmammalian groups. These three genes, members of an ancient familial cluster, encode an unusual KRAB domain that functions as a transcriptional activator. Evolutionary analysis confirms the ancient provenance of this activating KRAB and reveals the independent expansion of KRAB-ZNFs in every vertebrate lineage. Most human ZNF genes, from the most deeply conserved to the primate-specific genes, are highly expressed in immune and reproductive tissues, indicating that they have been enlisted to regulate evolutionarily divergent biological traits.

Pax6 encodes a transcription factor with key roles in the development of the pancreas, central nervous system, and eye. Gene expression is orchestrated by several alternative promoters and enhancer elements that are distributed over several hundred kilobases. Here, we describe a reciprocal translocation, called 1Gso, which disrupts the integrity of transcripts arising from the 5′‐most promoter, P0, and separates downstream promoters from enhancers active in pancreas and eye. Despite this fact, 1Gso animals exhibit none of the dominant Pax6 phenotypes, and the translocation complements recessive brain and craniofacial phenotypes. However, 1Gso fails to complement Pax6 recessive effects in lacrimal gland, conjunctiva, lens, and pancreas. The 1Gso animals also express a corneal phenotype that is related to but distinct from that expressed by Pax6 null mutants, and an abnormal density and organization of retinal ganglion cell axons; these phenotypes may be related to a modest upregulation of Pax6 expression from downstream promoters that we observed during development. Our investigation maps the activities of Pax6 alternative promoters including a novel one in developing tissues, confirms the phenotypic consequences of upstream enhancer disruption, and limits the likely effects of the P0 transcript null mutation to recessive abnormalities in the pancreas and specific structures of the eye. genesis 51:630–646. © 2013 Wiley Periodicals, Inc.

We previously showed that the expression of troponin T1 (Tnnt 1) was induced in the central nervous system (CNS) of adult mice 30min after treatment with ketamine, a glutamate N-methyl-d-aspartic acid (NMDA) receptor antagonist. We hypothesized that Tnnt 1 expression may be an early molecular biomarker of stress response in the CNS of mice. To further evaluate this hypothesis, we investigated the regional expression of Tnnt 1 in the mouse brain using RNA in situ hybridization 4h after systemic exposure to interferon-α (IFN-α) and gamma ionizing radiation, both of which have be associated with wide ranges of neuropsychiatric complications. Adult B6C3F1 male mice were treated with either human IFN-α (a single i.p. injection at 1×105IU/kg) or whole body gamma-radiation (10cGy or 2Gy). Patterns of Tnnt 1 transcript expression were compared in various CNS regions after IFN-α, radiation and ketamine treatments (previous study). Tnnt 1 expression was consistently induced in pyramidal neurons of cerebral cortex and hippocampus after all treatment regimens including 10cGy of ionizing radiation. Regional expression of Tnnt 1 was induced in Purkinje cells of cerebellum after ionizing radiation and ketamine treatment; but not after IFN-α treatment. None of the three treatments induced Tnnt 1 expression in glial cells. The patterns of Tnnt 1 expression in pyramidal neurons of cerebral cortex and hippocampus, which are both known to play important roles in cognitive function, memory and emotion, suggest that the expression of Tnnt 1 may be an early molecular biomarker of induced CNS stress.

Translocations have provided invaluable tools for identifying both cancer-linked genes and loci associated with heritable human diseases, but heritable human translocations are rare and few mouse models exist. Here we report progress on analysis of a collection of heritable translocations generated by treatment of mice with specific chemicals or radiation during late spermatogenic stages. The translocation mutants exhibit a range of visible phenotypes reflecting the disruption of coding sequences or the separation of genes from essential regulatory elements. The breakpoints of both radiation-induced and chemically induced mutations in these mice are remarkably clean, with very short deletions, duplications, or inversions in some cases, and ligation mediated by microhomology, suggesting nonhomologous end joining as the major path of repair. These mutations provide new tools for the discovery of novel genes and regulatory elements linked to human developmental disorders and new clues to the molecular basis of human genetic disease.

The glutamatergic system has been implicated in neuropsychiatric disorders, such as schizophrenia, bipolar disorder and Alzheimer's disease, which also have a high prevalence of metabolic syndrome. Treatment with ketamine, a non-competitive glutamate N-methyl-d-aspartic acid (NMDA) receptor antagonist, is known to have paradoxical effects of neuroprotection and neurotoxicity. We investigated gene expression in brain tissue of adult mice treated with ketamine to characterize the expression profiles and to identify the affected metabolic pathways. Adult male mice were treated by a single intraperitoneal (i.p.) injection of either s(+)ketamine (80 mg/kg) or distilled water (as the control). Fifty genes were differentially expressed in ketamine-treated mouse brains compared with control mice using oligonucleotide microarray analysis, and the expression of Troponin T1 (Tnnt1) gene was consistently elevated (2- to 4-fold) (p<0.001). Ketamine-induced Tnnt1 expression was confirmed and characterized using RNA in situ hybridization techniques in paraffin embedded brain tissue sections. Tnnt1 expression was induced in the granule layer of the hippocampus, amygdala, hypothalamus, Purkinje cells of cerebellum (p<0.0001), and cerebral cortex. Tnnt1 gene is known to interact directly with FoxO1, which is involved in multiple peripheral metabolic pathways and central energy homeostasis. Our findings suggest that the induction of Tnnt1 gene expression in adult mouse brains by ketamine may illustrate the genes involved in the metabolic syndromes observed in neuropsychiatric disorders.

YY2 was originally identified due to its unusual similarity to the evolutionarily well-conserved zinc finger gene YY1. In this study, we have determined the evolutionary origin and conservation of YY2 using comparative genomic approaches. Our results indicate that YY2 is a retroposed copy of YY1 that has been inserted into another gene locus named Mbtps2 (membrane-bound transcription factor protease site 2). This retroposition is estimated to have occurred after the divergence of placental mammals from other vertebrates based on the detection of YY2 only in the placental mammals. The N- and C-terminal regions of YY2 have evolved under different selection pressures. The N-terminal region has evolved at a very fast pace with very limited functional constraints, whereas the DNA-binding, C-terminal region still maintains a sequence structure very similar to that of YY1 and is also well conserved among placental mammals. In situ hybridizations using different adult mouse tissues indicate that mouse YY2 is expressed at relatively low levels in Purkinje and granular cells of cerebellum and in neuronal cells of cerebrum, but at very high levels in testis. The expression levels of YY2 are much lower than those of YY1, but the overall spatial expression patterns are similar to those of Mbtps2, suggesting a possible shared transcriptional control between YY2 and Mbtps2. Taken together, the formation and evolution of YY2 represent a very unusual case where a transcription factor was first retroposed into another gene locus encoding a protease and survived with different selection schemes and expression patterns.

Increased susceptibility to gastric cancer has been associated with a wide range of host genetic and environmental factors, including Helicobacter pylori infection. Helicobacter pylori infection is postulated to initiate a progression through atrophic gastritis, metaplasia and dysplasia to cancer, and has been associated with reduction of acid output and dysregulation of stomach mucins. Here, we present the characterization of two mouse lines carrying mutant alleles of the gene encoding the Kcnq1 potassium channel, which very rapidly establish chronic gastritis in a pathogen-exposed environment. These mice develop gastric hyperplasia, hypochlorhydria and mucin dysregulation independent of infection. Metaplasia, dysplasia and pre-malignant adenomatous hyperplasia of the stomach have been observed in these Kcnq1 mutant mice, also independent of infection. The data presented here suggest that Kcnq1 mutant mice can be used both as an efficient model for the development of atrophic gastritis after infection and to determine the processes during the later stages of progression to gastric cancer independent of infection. Thus, Kcnq1 mutant mice are a powerful new tool for investigating the connection between acid balance, Helicobacter infection and mucin disruption in the progression to gastric cancer.

We have isolated Kruppel-type (C2H2) zinc-finger genes, ZIM3 (zinc-finger gene 3 from imprinted domain) and ZNF264, located downstream of human and mouse USP29 genes (encoding ubiquitin-specific processing protease 29). In human, both ZIM3 and ZNF264 encode zinc-finger proteins with Kruppel-associated box (KRAB) A and B domains at the amino-terminal regions of the predicted proteins. In contrast, mouse Zim3 and Zfp264 seem to have lost protein-coding capability based on the lack of open reading frames (ORFs) in their cDNA sequences. In particular, the 3' end of the Zim3 transcript overlaps with the coding region of the adjacent gene Usp29 in an antisense orientation, indicating the conversion of mouse Zim3 into an antisense transcript gene for Usp29. The expression patterns of ZIM3 and ZNF264 have been largely conserved between human and mouse, with testis-specific expression of ZIM3 and ubiquitous expression of ZNF264, but high expression levels in adult testes in both species. Our studies also demonstrate that both mouse genes are imprinted with maternal expression of Zim3 in adult testes and paternal expression of Zfp264 in neonatal and adult brain. The reciprocal imprinting of two neighboring mouse genes, Zim3 and Zfp264, is consistent with a pattern observed frequently in other imprinted domains, and suggests that the imprinting of these two genes might be coregulated.

In analysis of a conserved region of proximal mouse chromosome 7 and human chromosome 19q, we have isolated a novel mouse gene, Zim1 (imprinted zinc-finger gene 1), encoding a typical Kruppel-type (C2H2) zinc-finger protein, located within 30 kb of a known imprinted gene, Peg3 (paternally expressed gene 3). Our studies demonstrate that Zim1 is also imprinted; the gene is expressed mainly from the maternal allele and at high levels only during embryonic and neonatal stages. In contrast to most tissues, Zim1 is expressed biallelically in neonatal and adult brain with slightly more input from the maternal allele. Zim1 produces multiple transcripts that range in size from 7.5 to 15 kb. The 7.5 kb transcript is expressed at highest levels and appears to be embryo specific. Whole mount in situ hybridization analysis indicates that Zim1 is expressed at significant levels in the apical ectodermal ridge of the limb buds during embryogenesis, suggesting a potential role of Zim1 in limb formation. We have identified the potential human ortholog of Zim1 near PEG3 in a conserved, gene-rich region of human chromosome 19q13.4. The close juxtaposition of reciprocally imprinted genes has also been seen in other imprinted regions, such as human 11p15.5/Mmu7 ( H19 / Igf2 ) and suggests that the two genes may be co-regulated. These and other data suggest the presence of an unexplored, conserved imprinted domain in human chromosome 19q13.4 and proximal Mmu7.