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In this study, we developed a method to identify core transcription factors (TFs) involved in differentiation using only comprehensive gene analysis. The theory of in silico screening using TFs regulatory network analysis (ISNA) required the following requirements: (1) estimating promoter regions, (2) constructing TFs regulatory network (TRN) relationships using the nucleotide sequence information in the promoters and score matrices derived from TF consensus sequences, and (3) identifying candidate core TFs by determining dissociation constants (K d values) within the relationships of TRN. ISNA demonstrated the ability to predict the core TFs involved in the endothelial-to-mesenchymal transition of human umbilical vein endothelial cell (HUVEC) and the differentiation of human embryonic stem cells into mesodermal cells. Using ISNA, we identified HMGA2 as a novel core TF in uterine epithelium. Notably, HMGA2 expression was predominantly detected in uterine epithelium, where it regulated cell proliferation in response to estrogen. These findings highlight ISNA’s potential to identify core TFs based on transcriptomic data.

Cereblon (CRBN) is a primary target of thalidomide and mediates its multiple pharmacological activities, including teratogenic and antimyeloma activities. CRBN functions as a substrate receptor of the E3 ubiquitin ligase CRL4, whose substrate specificity is modulated by thalidomide and its analogs. Although a number of CRL4 CRBN substrates have recently been identified, the substrate involved in thalidomide teratogenicity is unclear. Here we show that p63 isoforms are thalidomide-dependent CRL4 CRBN neosubstrates that are responsible, at least in part, for its teratogenic effects. The p53 family member p63 is associated with multiple developmental processes. ∆Np63α is essential for limb development, while TAp63α is important for cochlea development and hearing. Using a zebrafish model, we demonstrate that thalidomide exerts its teratogenic effects on pectoral fins and otic vesicles by inducing the degradation of ∆Np63α and TAp63α, respectively. These results may contribute to the invention of new thalidomide analogs lacking teratogenic activity. Zebrafish p63 isoforms were identified as thalidomide-dependent neosubstrates of the cereblon-containing E3 ligase complex. ∆Np63α and TAp63α are responsible for thalidomide-induced malformations of pectoral fins and otic vesicles, respectively.

DELLA protein is a key negative regulator of gibberellin (GA) signaling. Although how DELLA regulates downstream gene expression remains unclear, DELLA has been proposed to function as a transcriptional activator. However, because DELLA lacks a DNA-binding domain, intermediate protein(s) mediating the DELLA/DNA interaction are believed to be necessary for activating DELLA target genes. Here, using yeast hybrid screenings, we identified five members of INDETERMINATE DOMAIN (IDD) protein family which bind physically to both DELLA and the promoter sequence of the GA-positive regulator SCARECROW-LIKE 3 (SCL3), which previously was characterized as a DELLA direct target gene. Transient assays using Arabidopsis protoplasts demonstrated that a luciferase reporter controlled by the SCL3 promoter was additively transactivated by REPRESSOR of ga1-3 (RGA) and IDDs. Phenotypic analysis of transgenic plants expressing AtIDD3 (one of the 16 IDDs in the Arabidopsis genome) fused with the plant-specific repression domain (SRDX) supported the possibility that AtIDD3 is positively involved in GA signaling. In addition, we found that SCL3 protein also interacts with IDDs, resulting in the suppression of its target gene expression. In this context, DELLA and SCL3 interact competitively with IDD proteins to regulate downstream gene expression. These results suggest that the coregulators DELLA and SCL3, using IDDs as transcriptional scaffolds for DNA binding, antagonistically regulate the expression of their downstream targets to control the GA signaling pathway.

The Müllerian duct develops into the oviduct, uterus, and vagina, all of which are quite distinct in their morphology and function. The epithelial fate of these female reproductive organs in developing mice is determined by factors secreted from the stroma; however, how stromal differentiation occurs in the female reproductive organs derived from the Müllerian duct is still unclear. In the present study, roles of retinoic acid (RA) signaling in developing female reproductive tracts were investigated. Retinol dehydrogenase 10 (RDH10) and aldehyde dehydrogenase family 1 subfamily A2 (ALDH1A2) mRNAs and proteins and transactivation activity of endogenous RA were found in the stroma of proximal Müllerian ducts and gradually decreased from the proximal to caudal regions in fetal mice. In organcultured Müllerian ducts, retinaldehyde or RA treatment induced uterine epithelial differentiation, defined as a layer of columnar epithelial cells negative for oviductal and vaginal epithelial markers. In contrast, inhibition of RA receptor (RAR) signaling induced vaginal epithelial differentiation, characterized as vaginal epithelial marker genes–positive stratified epithelium. Grafting experiments of the organ-cultured Müllerian duct revealed irreversible epithelial fate determination. Although RAR did not directly bind to the homeobox A10 (Hoxa10) promoter region, RA–RAR signaling stimulated Hoxa10 expression. Thus, RA–RAR signaling in the Müllerian duct determines the fate of stroma to form the future uterus and vagina.

Nosocomial infections or hospital-acquired infections (HAIs) are common health problems affecting patients in human and animal hospitals. Herein, we hypothesised that HAIs could be spread through human and animal movement, contact with veterinary medical supplies, equipment, or instruments. We used a combination of social network analysis and genotyping techniques to find key players (or key nodes) and spread patterns using Escherichia coli as a marker. This study was implemented in the critical care unit, outpatient department, operation room, and ward of a small animal hospital. We conducted an observational study used for key player determination (or key node identification), then observed the selected key nodes twice with a one-month interval. Next, surface swabs of key nodes and their connecting nodes were analysed using bacterial identification, matrix-assisted laser desorption/ionisation-time of flight mass spectrometry, and pulsed-field gel electrophoresis. Altogether, our results showed that veterinarians were key players in this contact network in all departments. We found two predominant similarity clusters; dendrogram results suggested E. coli isolates from different time points and places to be closely related, providing evidence of HAI circulation within and across hospital departments. This study could aid in limiting the spread of HAIs in veterinary and human hospitals.

Mechanisms underlying sex determination and differentiation in animals are known to encompass a diverse array of molecular clues. Recent innovations in high-throughput sequencing and mass spectrometry technologies have been widely applied in non-model organisms without reference genomes. Crustaceans are no exception. They are particularly diverse among the Arthropoda and contain a wide variety of commercially important fishery species such as shrimps, lobsters and crabs (Order Decapoda), and keystone species of aquatic ecosystems such as water fleas (Order Branchiopoda). In terms of decapod sex determination and differentiation, previous approaches have attempted to elucidate their molecular components, to establish mono-sex breeding technology. Here, we overview reports describing the physiological functions of sex hormones regulating masculinization and feminization, and gene discovery by transcriptomics in decapod species. Moreover, this review summarizes the recent progresses of studies on the juvenile hormone-driven sex determination system of the branchiopod genus Daphnia, and then compares sex determination and endocrine systems between decapods and branchiopods. This review provides not only substantial insights for aquaculture research, but also the opportunity to re-organize the current and future trends of this field.

The anterior (DA) and posterior parts of the deltoid (DP) show alternating contraction during shoulder flexion and extension movements. It is expected that an inhibitory spinal reflex between the DA and DP exists. In this study, spinal reflexes between the DA and DP were examined in healthy human subjects using post-stimulus time histogram (PSTH) and electromyogram averaging (EMG-A). Electrical conditioning stimulation was delivered to the axillary nerve branch that innervates the DA (DA nerve) and DP (DP nerve) with the intensity below the motor threshold. In the PSTH study, the stimulation to the DA and DP nerves inhibited (decrease in the firing probability) 31 of 54 DA motor units and 31 of 51 DP motor units. The inhibition was not provoked by cutaneous stimulation. The central synaptic delay of the inhibition between the DA and DP nerves was 1.5 ± 0.5 ms and 1.4 ± 0.4 ms (mean ± SD) longer than those of the homonymous facilitation of the DA and DP, respectively. In the EMG-A study, conditioning stimulation to the DA and DP nerves inhibited the rectified and averaged EMG of the DP and DA, respectively. The inhibition diminished with tonic vibration stimulation to the DA and DP and recovered 20–30 min after vibration removal. These findings suggest that oligo(di or tri)-synaptic inhibition mediated by group Ia afferents between the DA and DP exists in humans.

Theca cells produce androgen by 17α-hydroxylase-17,20-lyase encoded by Cyp17a1, and conversion of androgen to estrogen in granulosa cells is regulated by gonadotropins. Women with polycystic ovarian syndrome (PCOS) exhibit elevated levels of androgens due to high Cyp17a1 expression and alterations in gene expression in granulosa cells. The aim of this study was to examine the interaction between theca and granulosa cells in PCOS-model mice. To produce PCOS-model mice, neonatal mice were injected with 1 μg TP for 3 days from the day of birth. Gonadotropins were injected according to the superovulation protocol to 3-month-old control mice and PCOS-model mice. Histological changes and expression of genes involved in steroidogenesis, ovulation and luteinization were investigated by immunohistochemistry and real-time RT-PCR, respectively. Pregnant mare serum gonadotropin (PMSG) induced the expression of genes involved in steroidogenesis in control prepubertal mice, whereas human chorionic gonadotropin (hCG) reduced Cyp17a1 expression and induced phospho-ERK1/2 in granulosa cells. Cyp17a1 was reduced in PMSG-primed PCOS-model mice regardless of hCG injection, and PMSG induced phosphorylation of ERK1/2 in granulosa cells. Phospho-ERK1/2 in granulosa cells can be correlated with reduced Cyp17a1 expression in theca cells, and the interaction between granulosa and theca cells may be impaired in PCOS-model mice.

Teleost fishes exhibit complex sexual characteristics in response to androgens, such as fin enlargement and courtship display. However, the molecular mechanisms underlying their evolutionary acquisition remain largely unknown. To address this question, we analyse medaka ( Oryzias latipes ) mutants deficient in teleost-specific androgen receptor ohnologs ( ara and arb ). We discovered that neither ar ohnolog was required for spermatogenesis, whilst they appear to be functionally redundant for the courtship display in males. However, both were required for reproductive success: ara for tooth enlargement and the reproductive behaviour eliciting female receptivity, arb for male-specific fin morphogenesis and sexual motivation. We further showed that differences between the two ar ohnologs in their transcription, cellular localisation of their encoded proteins, and their downstream genetic programmes could be responsible for the phenotypic diversity between the ara and arb mutants. These findings suggest that the ar ohnologs have diverged in two ways: first, through the loss of their roles in spermatogenesis and second, through gene duplication followed by functional differentiation that has likely resolved the pleiotropic roles derived from their ancestral gene. Thus, our results provide insights into how genome duplication impacts the massive diversification of sexual characteristics in the teleost lineage. How has the genome duplication impacted the diversification of sexual characteristics in the teleost lineage? This study shows that androgen receptor ohnologs in medaka appear to have diverged in their roles for regulating morphological and behavioural sexual characteristics after loss from an ancestral role in spermatogenesis.

In the mouse ovary, interactions between oocytes and somatic cells are essential for folliculogenesis and subsequent follicle development. The polyovular follicle (PF), which contains more than two oocytes in a follicle, can be induced in the neonatal mouse ovary when interactions between oocytes and somatic cells are disrupted by agents such as the potent synthetic estrogen diethylstilbestrol (DES) acting through estrogen receptor (ER) β. Hedgehog signaling is known to regulate granulosa cell proliferation, thecal cell differentiation, and follicle growth. To investigate the role of hedgehog signaling in the early folliculogenesis and in PF induction by DES, neonatal mouse ovaries were cultured with or without 10 μM cyclopamine (CPA), an inhibitor of hedgehog signaling, and grafted under the kidney capsule of adult ovariectomized host mice. The number and the incidence of PFs were significantly increased in organ-cultured ovaries post-grafting. Expression of procollagen type IV, alpha 1 ( Col4a1 ) in organ-cultured ovaries was significantly reduced by CPA, but not by DES. The expression of two hedgehog ligands, Desert hedgehog (Dhh) and Indian hedgehog (Ihh), and a target gene, Hedgehog interacting protein (Hhip), was significantly increased by DES both in WT and ERβ KO mice. Therefore, we infer that DES can affect expression of those genes through ERα but not via suppression of hedgehog signaling. Thus, PFs are induced by DES or CPA, but the induction mechanism is different. Our results revealed an important role of hedgehog signaling in basement membrane remodeling during folliculogenesis even before thecal cell differentiation.