Slow decrease in temperature produces readthrough transcripts in mammalian hibernation.

Accumulating evidence suggests that various cellular stresses interfere with the end processing of mRNA synthesis and lead to the production of abnormally long transcripts, known as readthrough transcripts (RTTs), which extend beyond the termination sites. Small mammalian hibernators repeatedly enter a state referred to as deep torpor (DT), where the metabolic rate, respiration rate, and core body temperature become extremely low, which produces various types of cellular stresses and therefore induces RTTs. However, the types of stresses and processes around the DT that cause RTTs are unclear. In the present study, we showed that RTTs are produced from different gene loci in the livers of Syrian hamsters under DT and summer-like conditions. Moreover, in vitro analysis using hamster primary hepatocytes revealed that DT-specific RTTs are induced by a slow decline in temperature, as seen in body temperature in the entrance phase of DT, but not by rapid cold treatment or hypoxia. In addition, it was observed that RTTs were not elongated under a significantly cold temperature (4 °C). These results indicate that DT-specific RTTs are produced during the entrance phase of torpor by a slow decrease in body temperature.

Estrogen mediates sex differences in preoptic neuropeptide and pituitary hormone production in medaka.

The preoptic area (POA) is one of the most evolutionarily conserved regions of the vertebrate brain and contains subsets of neuropeptide-expressing neurons. Here we found in the teleost medaka that two neuropeptides belonging to the secretin family, pituitary adenylate cyclase-activating polypeptide (Pacap) and vasoactive intestinal peptide (Vip), exhibit opposite patterns of sexually dimorphic expression in the same population of POA neurons that project to the anterior pituitary: Pacap is male-biased, whereas Vip is female-biased. Estrogen secreted by the ovary in adulthood was found to attenuate Pacap expression and, conversely, stimulate Vip expression in the female POA, thereby establishing and maintaining their opposite sexual dimorphism. Pituitary organ culture experiments demonstrated that both Pacap and Vip can markedly alter the expression of various anterior pituitary hormones. Collectively, these findings show that males and females use alternative preoptic neuropeptides to regulate anterior pituitary hormones as a result of their different estrogen milieu.

SOX2 regulates homeostasis of taste bud cells and lingual epithelial cells in posterior tongue.

Taste bud cells arise from local epithelial stem cells in the oral cavity and are continuously replaced by newborn cells throughout an animal's life. However, little is known about the molecular and cellular mechanisms of taste cell turnover. Recently, it has been demonstrated that SOX2, a transcription factor expressed in epithelial stem/progenitor cells of the oral cavity, regulates turnover of anterior tongue epithelium including gustatory and non-gustatory papillae. Yet, the role of SOX2 in regulating taste cell turnover in the posterior tongue is unclear. Prompted by the fact that there are regional differences in the cellular and molecular composition of taste buds and stem/progenitor cells in the anterior and posterior portions of tongue, which are derived from distinct embryonic origins, we set out to determine the role of SOX2 in epithelial tissue homeostasis in the posterior tongue. Here we report the differential requirement of SOX2 in the stem/progenitor cells for the normal turnover of lingual epithelial cells in the posterior tongue. Sox2 deletion in the stem/progenitor cells neither induced active caspase 3-mediated apoptotic cell death nor altered stem/progenitor cell population in the posterior tongue. Nevertheless, morphology and molecular feature of non-gustatory epithelial cells were impaired in the circumvallate papilla but not in the filiform papillae. Remarkably, taste buds became thinner, collapsed, and undetectable over time. Lineage tracing of Sox2-deleted stem/progenitor cells demonstrated an almost complete lack of newly generated basal precursor cells in the taste buds, suggesting mechanistically that Sox2 is involved in determining stem/progenitor cells to differentiate to gustatory lineage cells. Together, these results demonstrate that SOX2 plays key roles in regulating epithelial tissue homeostasis in the posterior tongue, similar but not identical to its function in the anterior tongue.

Male-predominant galanin mediates androgen-dependent aggressive chases in medaka.

Recent studies in mice demonstrate that a subset of neurons in the medial preoptic area (MPOA) that express galanin play crucial roles in regulating parental behavior in both sexes. However, little information is available on the function of galanin in social behaviors in other species. Here, we report that, in medaka, a subset of MPOA galanin neurons occurred nearly exclusively in males, resulting from testicular androgen stimulation. Galanin-deficient medaka showed a greatly reduced incidence of male-male aggressive chases. Furthermore, while treatment of female medaka with androgen induced male-typical aggressive acts, galanin deficiency in these females attenuated the effect of androgen on chases. Given their male-biased and androgen-dependent nature, the subset of MPOA galanin neurons most likely mediate androgen-dependent male-male chases. Histological studies further suggested that variability in the projection targets of the MPOA galanin neurons may account for the species-dependent functional differences in these evolutionarily conserved neural substrates.

Neuropeptide B mediates female sexual receptivity in medaka fish, acting in a female-specific but reversible manner.

Male and female animals display innate sex-specific mating behaviors. In teleost fish, altering the adult sex steroid milieu can effectively reverse sex-typical mating behaviors, suggesting remarkable sexual lability of their brains as adults. In the teleost medaka, neuropeptide B (NPB) is expressed female-specifically in the brain nuclei implicated in mating behavior. Here, we demonstrate that NPB is a direct mediator of estrogen action on female mating behavior, acting in a female-specific but reversible manner. Analysis of regulatory mechanisms revealed that the female-specific expression of NPB is dependent on direct transcriptional activation by estrogen via an estrogen-responsive element and is reversed in response to changes in the adult sex steroid milieu. Behavioral studies of NPB knockouts revealed that female-specific NBP mediates female receptivity to male courtship. The female-specific NPB signaling identified herein is presumably a critical element of the neural circuitry underlying sexual dimorphism and lability of mating behaviors in teleosts.

Localization of three forms of gonadotropin-releasing hormone in the brain and pituitary of the self-fertilizing fish, Kryptolebias marmoratus.

The localization of gonadotropin-releasing hormone (GnRH) in the brain and pituitary of the self-fertilizing mangrove killifish Kryptolebias marmoratus was examined by immunohistochemistry and in situ hybridization to understand its neuroendocrine system. The genome assembly of K. marmoratus did not have any sequence encoding GnRH1, but sequences encoding GnRH2 (chicken GnRH-II) and GnRH3 (salmon GnRH) were found. Therefore, GnRH1 was identified by in silico cloning. The deduced amino acid sequence of the K. marmoratus GnRH1 (mature peptide) was identical to that of the medaka GnRH. GnRH1 neurons were detected in the ventral part of the preoptic nucleus by immunohistochemistry and in situ hybridization, and GnRH1-immunoreactive (ir) fibers were observed throughout the brain. GnRH1-ir fibers were in close contact with luteinizing hormone (LH)-ir cells in the pituitary using double immunohistochemistry. GnRH2 neurons were detected in the midbrain tegmentum by immunohistochemistry and in situ hybridization. Although GnRH2-ir fibers were observed throughout the brain, they were not detected in the pituitary. GnRH3 neurons were detected in the lateral part of the ventral telencephalic area by both methods. GnRH3-ir fibers were observed throughout the brain, and a few GnRH3-ir fibers were in close contact with LH-ir cells in the pituitary. These results indicate that GnRH1 and possibly GnRH3 are responsible for gonadal maturation through LH secretion and that all three forms of GnRH function as neurotransmitters or neuromodulators in the brain of K. marmoratus.

Skn-1a/Pou2f3 functions as a master regulator to generate Trpm5-expressing chemosensory cells in mice.

Transient receptor potential channel M5 (Trpm5)-expressing cells, such as sweet, umami, and bitter taste cells in the oropharyngeal epithelium, solitary chemosensory cells in the nasal respiratory epithelium, and tuft cells in the small intestine, that express taste-related genes function as chemosensory cells. Previous studies demonstrated that Skn-1a/Pou2f3, a POU homeodomain transcription factor is expressed in these Trpm5-expressing chemosensory cells, and is necessary for their generation. Trpm5-expressing cells have recently been found in trachea, auditory tube, urethra, thymus, pancreatic duct, stomach, and large intestine. They are considered to be involved in protective responses to potential hazardous compounds as Skn-1a-dependent bitter taste cells, respiratory solitary chemosensory cells, and intestinal tuft cells are. In this study, we examined the expression and function of Skn-1a/Pou2f3 in Trpm5-expressing cells in trachea, auditory tube, urethra, thymus, pancreatic duct, stomach, and large intestine. Skn-1a/Pou2f3 is expressed in a majority of Trpm5-expressing cells in all tissues examined. In Skn-1a/Pou2f3-deficient mice, the expression of Trpm5 as well as marker genes for Trpm5-expressing cells were absent in all tested tissues. Immunohistochemical analyses demonstrated that two types of microvillous cells exist in trachea, urethra, and thymus, Trpm5-positive and Trpm5-negative cells. In Skn-1a/Pou2f3-deficient mice, a considerable proportion of Trpm5-negative and villin-positive microvillous cells remained present in these tissues. Thus, we propose that Skn-1a/Pou2f3 is the master regulator for the generation of the Trpm5-expressing microvillous cells in multiple tissues.

Mechanistic Studies on the Generation and Properties of Superelectrophilic Singlet Carbenes from Bis(perfluoroalkanesulfonyl)bromonium Ylides.

Pyrolysis of bis(perfluoroalkanesulfonyl)bromonium ylides in various olefins results in highly stereospecific formation of cyclopropanes via unimolecular decomposition. Product analysis, kinetic study, substituent effects, and theoretical study revealed the generation of singlet bis(perfluoroalkanesulfonyl)carbenes stabilized by intramolecular coordination of sulfonyl oxygen.

Glucocorticoid receptor exhibits sexually dimorphic expression in the medaka brain.

The differential impact of stress on brain functions of males and females has been widely observed in vertebrates. Recent evidence suggests that stress-induced glucocorticoid signaling affects sexual differentiation and sex changes in teleost fish. These facts led us to postulate that there were sex differences in glucocorticoid signaling in the teleost brain that underlie some sex differences in their physiological and behavioral traits. Here we found sexually dimorphic expression of a glucocorticoid receptor gene (gr1) in the brain of medaka fish (Oryzias latipes), with females having greater expression in several preoptic and thalamic nuclei. Further, gr1 exhibits female-biased expression in neurons of the anterior parvocellular preoptic nucleus that produce the neuropeptides vasotocin and gonadotropin-releasing hormone 1 (these neuropeptides have been implicated in the regulation of neuroendocrine and behavioral functions). These findings suggest that glucocorticoids have a greater influence on physiology and behavior mediated by these neuropeptides in females than in males, which may contribute to sex differences in the brain's response to stress.

Disturbed biopterin and folate metabolism in the Qdpr-deficient mouse.

Quinonoid dihydropteridine reductase (QDPR) catalyzes the regeneration of tetrahydrobiopterin (BH4), a cofactor for monoamine synthesis, phenylalanine hydroxylation and nitric oxide production. Here, we produced and analyzed a transgenic Qdpr(-/-) mouse model. Unexpectedly, the BH4 contents in the Qdpr(-/-) mice were not decreased and even increased in some tissues, whereas those of the oxidized form dihydrobiopterin (BH2) were significantly increased. We demonstrated that unlike the wild-type mice, dihydrofolate reductase regenerated BH4 from BH2 in the mutants. Furthermore, we revealed wide alterations in folate-associated metabolism in the Qdpr(-/-) mice, which suggests an interconnection between folate and biopterin metabolism in the transgenic mouse model.

Sexually dimorphic expression of the sex chromosome-linked genes cntfa and pdlim3a in the medaka brain.

In vertebrates, sex differences in the brain have been attributed to differences in gonadal hormone secretion; however, recent evidence in mammals and birds shows that sex chromosome-linked genes, independent of gonadal hormones, also mediate sex differences in the brain. In this study, we searched for genes that were differentially expressed between the sexes in the brain of a teleost fish, medaka (Oryzias latipes), and identified two sex chromosome genes with male-biased expression, cntfa (encoding ciliary neurotrophic factor a) and pdlim3a (encoding PDZ and LIM domain 3 a). These genes were found to be located 3-4 Mb from and on opposite sides of the Y chromosome-specific region containing the sex-determining gene (the medaka X and Y chromosomes are genetically identical, differing only in this region). The male-biased expression of both genes was evident prior to the onset of sexual maturity. Sex-reversed XY females, as well as wild-type XY males, had more pronounced expression of these genes than XX males and XX females, indicating that the Y allele confers higher expression than the X allele for both genes. In addition, their expression was affected to some extent by sex steroid hormones, thereby possibly serving as focal points of the crosstalk between the genetic and hormonal pathways underlying brain sex differences. Given that sex chromosomes of lower vertebrates, including teleost fish, have evolved independently in different genera or species, sex chromosome genes with sexually dimorphic expression in the brain may contribute to genus- or species-specific sex differences in a variety of traits.

Skn-1a/Pou2f3 is required for the generation of Trpm5-expressing microvillous cells in the mouse main olfactory epithelium.

BACKGROUND: The main olfactory epithelium (MOE) in mammals is a specialized organ to detect odorous molecules in the external environment. The MOE consists of four types of cells: olfactory sensory neurons, supporting cells, basal cells, and microvillous cells. Among these, development and function of microvillous cells remain largely unknown. Recent studies have shown that a population of microvillous cells expresses the monovalent cation channel Trpm5 (transient receptor potential channel M5). To examine functional differentiation of Trpm5-expressing microvillous cells in the MOE, we investigated the expression and function of Skn-1a, a POU (Pit-Oct-Unc) transcription factor required for functional differentiation of Trpm5-expressing sweet, umami, and bitter taste bud cells in oropharyngeal epithelium and solitary chemosensory cells in nasal respiratory epithelium. RESULTS: Skn-1a is expressed in a subset of basal cells and apical non-neuronal cells in the MOE of embryonic and adult mice. Two-color in situ hybridization revealed that a small population of Skn-1a-expressing cells was co-labeled with Mash1/Ascl1 and that most Skn-1a-expressing cells coexpress Trpm5. To investigate whether Skn-1a has an irreplaceable role in the MOE, we analyzed Skn-1a-deficient mice. In the absence of Skn-1a, olfactory sensory neurons differentiate normally except for a limited defect in terminal differentiation in ectoturbinate 2 of some of MOEs examined. In contrast, the impact of Skn-1a deficiency on Trpm5-expressing microvillous cells is much more striking: Trpm5, villin, and choline acetyltransferase, cell markers previously shown to identify Trpm5-expressing microvillous cells, were no longer detectable in Skn-1a-deficient mice. In addition, quantitative analysis demonstrated that the density of superficial microvillous cells was significantly decreased in Skn-1a-deficient mice. CONCLUSION: Skn-1a is expressed in a minority of Mash1-positive olfactory progenitor cells and a majority of Trpm5-expressing microvillous cells in the main olfactory epithelium. Loss-of-function mutation of Skn-1a resulted in complete loss of Trpm5-expressing microvillous cells, whereas most of olfactory sensory neurons differentiated normally. Thus, Skn-1a is a critical regulator for the generation of Trpm5-expressing microvillous cells in the main olfactory epithelium in mice.

Pou2f3/Skn-1a is necessary for the generation or differentiation of solitary chemosensory cells in the anterior nasal cavity.

Solitary chemosensory cells in the non-neuronal epithelium of the anterior nasal cavity have bitter taste cell-like molecular characteristics and are involved in the detection of noxious substances. Here, we demonstrate that Pou2f3/Skn-1a, which is necessary for generation of sweet, umami, and bitter taste cells, is also necessary for the generation or differentiation of solitary chemosensory cells.