[show abstract][hide abstract] ABSTRACT: Androgens play key roles in the morphological specification of male type sex attractive and reproductive organs, while little is known about the developmental mechanisms of such secondary sex characters. Medaka offers a clue about sexual differentiation. They show a prominent masculine sexual character for appendage development, the formation of papillary processes in the anal fin, which has been induced in females by exogenous androgen exposure. This current study shows that the development of papillary processes is promoted by androgen-dependent augmentation of Bone morphogenic protein 7 (Bmp7) and Lymphoid enhancer-binding factor-1 (Lef1). Androgen receptor (AR) subtypes, ARα and ARβ, are expressed in the distal region of outgrowing bone nodules of developing papillary processes. Development of papillary processes concomitant with the induction of Bmp7 and Lef1 in the distal bone nodules by exposure to methyltestosterone (MT) was significantly suppressed by an anti-androgen, flutamide, in female medaka. When Bmp signaling was inhibited in MT-exposed females by its inhibitor, dorsomorphin, Lef1 expression was suppressed accompanied by reduced proliferation in the distal bone nodules and retarded bone deposition. These observations indicate that androgen-dependent expressions of Bmp7 and Lef1 are required for the bone nodule outgrowth leading to the formation of these secondary sex characteristics in medaka. The formation of androgen-induced papillary processes may provide insights into the mechanisms regulating the specification of sexual features in vertebrates.
[show abstract][hide abstract] ABSTRACT: The ubiquitous, freshwater microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have a well annotated, reference genome assembly that revealed an unusually high gene count highlighted by a large gene orphanage,-i.e., previously uncharacterized genes. Daphnia are capable of either clonal or sexual reproduction, making them ideally suited for genetic manipulation, but the establishment of gene manipulation techniques is needed to accurately define gene functions. Although previous investigations developed an RNA interference (RNAi) system for one congener D. magna, these methods are not appropriate for D. pulex because of the smaller size of their early embryos. In these studies, we develop RNAi techniques for D. pulex by first determining the optimum culture conditions of their isolated embryos and then applying these conditions to the development of microinjection techniques and proof-of-principle RNAi experiments.
We found that isolated embryos were best cultured on a 2% agar plate bathed in 60 mM sucrose dissolved in M4 media, providing optimal conditions for microinjections. Then, we injected double-stranded (ds)RNA specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for limb development in invertebrates and vertebrates. Injected embryos presented with defects in the second antenna and appendage development, and dsRNA induced the degradation of Dll mRNAs, indicating that this technique successfully inhibited transcription of the target gene.
We developed a microinjection system for RNAi studies in D. pulex. These techniques add to the growing genomic toolbox and enhance the genetic tractability of this important model for environmental, evolutionary, and developmental genomics.
[show abstract][hide abstract] ABSTRACT: Juvenile hormone is an essential regulator of major developmental and life history events in arthropods. Most of the insects use juvenile hormone III as the innate juvenile hormone ligand. By contrast, crustaceans use methyl farnesoate. Despite this difference that is tied to their deep evolutionary divergence, the process of this ligand transition is unknown. Here we show that a single amino-acid substitution in the receptor Methoprene-tolerant has an important role during evolution of the arthropod juvenile hormone pathway. Microcrustacea Daphnia pulex and D. magna share a juvenile hormone signal transduction pathway with insects, involving Methoprene-tolerant and steroid receptor coactivator proteins that form a heterodimer in response to various juvenoids. Juvenile hormone-binding pockets of the orthologous genes differ by only two amino acids, yet a single substitution within Daphnia Met enhances the receptor's responsiveness to juvenile hormone III. These results indicate that this mutation within an ancestral insect lineage contributed to the evolution of a juvenile hormone III receptor system.
[show abstract][hide abstract] ABSTRACT: BACKGROUND: The gene doublesex (dsx) is known as a key factor regulating genetic sex determination in many organisms. We previously identified two dsx genes (DapmaDsx1 and DapmaDsx2) from a freshwater branchiopod crustacean, Daphnia magna, which are expressed in males but not in females. D. magna produces males by parthenogenesis in response to environmental cues (environmental sex determination) and we showed that DapmaDsx1 expression during embryonic stages is responsible for the male trait development. The D. magna dsx genes are thought to have arisen by a cladoceran-specific duplication; therefore, to investigate evolutionary conservation of sex specific expression of dsx genes and to further assess their functions in the environmental sex determination, we searched for dsx homologs in four closely related cladoceran species. RESULTS: We identified homologs of both dsx genes from, D. pulex, D. galeata, and Ceriodaphnia dubia, yet only a single dsx gene was found from Moina macrocopa. The deduced amino acid sequences of all 9 dsx homologs contained the DM and oligomerization domains, which are characteristic for all arthropod DSX family members. Molecular phylogenetic analysis suggested that the dsx gene duplication likely occurred prior to the divergence of these cladoceran species, because that of the giant tiger prawn Penaeus monodon is rooted ancestrally to both DSX1 and DSX2 of cladocerans. Therefore, this result also suggested that M. macrocopa lost dsx2 gene secondarily. Furthermore, all dsx genes identified in this study showed male-biased expression levels, yet only half of the putative 5' upstream regulatory elements are preserved in D. magna and D. pulex. CONCLUSIONS: The all dsx genes of five cladoceran species examined had similar amino acid structure containing highly conserved DM and oligomerization domains, and exhibited sexually dimorphic expression patterns, suggesting that these genes may have similar functions for environmental sex determination in cladocerans.
[show abstract][hide abstract] ABSTRACT: The Eastern and Western mosquitofish (Gambusia holbrooki and G. affinis) are potential bioindicator organisms for endocrine disruptors. Male mosquitofish have an elongated anal fin (gonopodium) used for internal fertilization whose formation is driven by androgens. Normal female mosquitofish have a normal, rounded anal fin which undergoes elongation into a gonopodium structure when female mosquitofish are exposed to androgenic chemicals. Significant issues with using mosquitofish as a bioindicator include the lack of knowledge on how anal fin growth in females corresponds to endpoints relevant to biological integrity and the lack of information on the molecular pathways that regulate anal fin growth. The objectives of this study were to understand how androgen-induced anal fin elongation relates to changes in endpoints related to the female reproductive system and to understand how anal fin elongation occurs in androgen-exposed female mosquitofish. To achieve these objectives, adult female G. holbrooki were exposed to a vehicle control or one of three doses of the androgen 17β-trenbolone (TB) at nominal concentrations of 0.1, 1 or 10μg TB/L. Anal fin measurements were taken and livers were used for quantitative polymerase chain reaction analysis of vitellogenin (vtg) mRNA expression at multiple time points. 10μg TB/L induced anal fin elongation after 7 days of treatment (one-way ANOVA, p<0.05) as did 0.1 and 1μg TB/L at later time points (one-way ANOVA, p<0.05). 10μg TB/L significantly reduced hepatic vtg gene expression at all time points assessed (one-way ANOVA, p<0.05). There was no correlation between anal fin elongation levels and vtg gene expression (Spearman's ρ, p>0.05). In a separate experiment, female G. holbrooki and G. affinis were exposed to the vehicle control or 1μg TB/L. Anal fins were used for qualitative gene expression analysis of the genes sonic hedgehog (shh), muscle segment homeobox C (msxC), and fibroblast growth factor receptor 1 (fgfr1) by in situ hybridization. Shh was expressed in the distal tip of the gonopodium while msxC and fgfr1 were more widely expressed along the same anal fin rays during androgen exposure. These data provide insight into the molecular pathways involved in anal fin elongation and pave the way for future work toward developing the mosquitofish into a bioindicator organism for endocrine disruptors.
[show abstract][hide abstract] ABSTRACT: The Wnt/β-catenin signaling is essential for various organogenesis and is often implicated during tumorigenesis. Dysregulated β-catenin signaling is associated with the formation of endometrial adenocarcinomas (EACs), which is considered as the common form of endometrial cancer in women. In the current study, we investigate the downstream target of Wnt/β-catenin signaling in the uterine epithelia and the mechanism leading to the formation of endometrial hyperplasia. We report that conditional ablation and activation of β-catenin in the uterine epithelia lead to aberrant epithelial structures and endometrial hyperplasia formation, respectively. We demonstrate that β-catenin regulates Foxa2 with its candidate upstream region for the uterine epithelia. Furthermore, knockdown of Foxa2 leads to defects in cell cycle regulation, suggesting a possible function of Foxa2 in the control of cell proliferation. We also observe that β-catenin and Foxa2 expression levels are augmented in the human specimens of complex atypical endometrial hyperplasia, which is considered to have a greater risk of progression to EACs. Thus, our study indicates that β-catenin regulates Foxa2 expression, and this interaction is possibly essential to control cell cycle progression during endometrial hyperplasia formation. Altogether, the augmented expression levels of β-catenin and Foxa2 are essential features during the formation of endometrial hyperplasia.Oncogene advance online publication, 3 September 2012; doi:10.1038/onc.2012.376.
[show abstract][hide abstract] ABSTRACT: Exposure to estrogenic chemicals discharged into the aquatic environment has been shown to induce feminization in wild freshwater fish and although fish species have been reported to differ in their susceptibility for these effects, empirical studies that directly address this hypothesis are lacking. In this study, in vitro ERα activation assays were applied in a range of fish species used widely in chemical testing (including, zebrafish, fathead minnow, medaka) and/or as environmental monitoring species (including, roach, stickleback, carp) to assess their comparative responsiveness to natural (estrone, estradiol, estriol) and synthetic (17α-ethinylestradiol (EE2), diethylstilbestrol (DES)) estrogens. In vivo exposures to EE2 via the water (nominal 2 and 10 ng/L for 7 days) were also conducted for seven fish species to compare their responsiveness for hepatic vitellogenin (VTG) mRNA induction (an ER mediated response). Of the fish species tested, zebrafish ERα was found to be the most responsive and carp and stickleback ERα the least responsive to natural steroid estrogens. This was also the case for exposure to EE2 with an ERα-mediated response sensitivity order of zebrafish > medaka > roach > fathead minnow > carp > stickleback. For VTG mRNA induction in vivo, the order of species responsiveness was: rainbow trout (not tested in the ERα activation assays) > zebrafish > fathead minnow > medaka > roach > stickleback > carp. Overall, the responses to steroid estrogens in vitro via ERα compared well with those seen in vivo (VTG induction for exposure to EE2) showing in vitro screening of chemicals using fish ERα-mediated responses indicative of estrogenic responses (VTG induction) in vivo.
[show abstract][hide abstract] ABSTRACT: The occurrence of oocytes in the testis (testis-ova) of several fish species is often associated with exposure of estrogenic chemicals. However, induction mechanisms of the testis-ova remain to be elucidated. To develop marker genes for detecting testis-ova in the testis, adult male medaka were exposed to nominal concentration of 100 ng L(-1) of 17α-ethinylestradiol (EE2) for 3-5 weeks, and 800 ng estradiol benzoate (EB) for 3 weeks (experiment I), and a measured concentration of 20 ng L(-1) EE2 for 1-6 weeks (experiment II). Histological analysis was performed for the testis, and microarray analyses were performed for the testis, liver and brain. Microarray analysis in the estrogen-exposed medaka liver showed vitellogenin and choriogenin as estrogen responsive genes. Testis-ova were induced in the testis after 4 weeks of exposure to 100 ng L(-1) EE2, 3 weeks of exposure to 800 ng EB, and 6 weeks of exposure to 20 ng L(-1) EE2. Microarray analysis of estrogen-exposed testes revealed up-regulation of genes related to zona pellucida (ZP) and the oocytes marker gene, 42Sp50. Using quantitative RT-PCR we confirmed that Zpc5 gene can be used as a marker for the detection of testis-ova in male medaka.
[show abstract][hide abstract] ABSTRACT: The epididymis is a male accessory organ and functions for sperm maturation and storage under the control of androgen. The development of the epididymis is also androgen dependent. The Wolffian duct (WD), anlagen of the epididymis, is formed in both male and female embryos; however, it is stabilized only in male embryos by testicular androgen. Androgen drives subsequent differentiation of the WD into the epididymis. Although the essential roles of androgen in WD masculinization and epididymal function have been established, little is known about cellular events regulated precisely by androgen signaling during these processes. It is also unclear whether androgen signaling, especially in the epithelia, has further function for epididymal epithelial cell differentiation. In this study we examined the cellular death and proliferation controlled by androgen signaling via the androgen receptor (AR) in WD stabilization. Analyses using AR knockout mice revealed that androgen signaling inhibits epithelial cell death in this process. Analysis of AP2α-Cre;AR(flox/Y) mice, in which AR function is deleted in the WD epithelium, revealed that epithelial AR is not required for the WD stabilization but is required for epithelial cell differentiation in the epididymis. Specifically, loss of epithelial AR significantly reduced expression of p63 that is essential for differentiation of basal cells in the epididymal epithelium. We also interrogated the possibility of regulation of the p63 gene (Trp63) by AR in vitro and found that p63 is a likely direct target of AR regulation.
[show abstract][hide abstract] ABSTRACT: Vertebrates show diverse sexual characters in sexually attractive and reproductive organs, which are regulated by steroid hormones, particularly androgens. However, the evolutionary history of androgen receptor (AR) gene remains largely unknown on the basis of phylogenic and functional analyses. To elucidate the evolutionary history and functional diversification of AR genes in vertebrates, we cloned the AR cDNAs from a shark, basal ray-finned fishes (Actinopterygii), namely bichir and sturgeon (Acipenseriformes), and teleosts including a basal teleost, arowana (Osteoglossiformes). Molecular phylogenetic analysis revealed that the gene duplication event that gave rise to two different teleost ARs (alpha and beta) likely occurred in the actinopterygian lineage leading to teleosts after the divergence of Acipenseriformes but before the split of Osteoglossiformes, which is compatible with the phylogenetic timing of teleost-specific genome duplication. Searching for AR genes in the medaka genome indicated that the teleost AR gene duplication has been associated with the duplication between chromosomes 10 and 14. Our functional analysis revealed that the shark AR activates the target gene via androgen response element by classical androgens. The teleost ARalpha showed the unique intracellular localization with a significantly higher transactivating capacity than that by teleost ARbeta. These findings indicate that the most ancient type of AR, as activated by the classical androgens as ligands, emerged before the Chondrichthyes-Osteichthyes split, and the AR gene was duplicated during the teleost-specific genome duplication event. We report here for the first time the accurate evolutionary history of AR gene and functional characterization of AR duplicates in teleost lineage.
[show abstract][hide abstract] ABSTRACT: Recent, molecular biology techniques have accomplished a large contribution to developmental biology. Especially, gene transduction techniques are indispensable to study the roles of regulatory genes underlying embryogenesis.
Electroporation and Sonoporation in Developmental Biology. 01/2009;
[show abstract][hide abstract] ABSTRACT: Androgen plays a crucial role in initiating and maintaining the expression of male sexual characteristics in mammals. In humans and mice, any defects along the pathway of androgen functions result in congenital urogenital abnormalities. The genital tubercle (GT), an anlage of the external genitalia, differentiates into a penis in males and a clitoris in females. Although masculinization of the external genitalia is androgen-dependent, the molecular pathway of its potential downstream genes is largely unclear. To identify the genes involved in mouse GT masculinization, we performed gene expression analyses, such as real-time quantitative polymerase chain reaction and section in situ hybridization analysis. From our studies we have identified candidate genes, Cyp1b1, Fkbp51 and MafB as potential androgen targets during mouse GT masculinization.
[show abstract][hide abstract] ABSTRACT: Split hand/foot malformation (SHFM) is syndromic ectrodactyly often associated with mental retardation and/or craniofacial defects. Several clinical reports previously described urogenital dysplasia such as micropenis, hypospadias, and small testis in SHFM patients. Genetic lesions in the Dlx5 and Dlx6 (Dlx5/6) locus are associated with the human genetic disorder SHFM type 1. Although Dlx5/6 are expressed in the testis, their possible function of Dlx5/6 during testis differentiation has not been described. In this study, we show that Dlx5/6 are expressed in the fetal Leydig cells during testis development. We examined the effect of Dlx5 expression on the promoter activation of the steroidogenic acute regulatory protein (StAR) gene, which is essential for gonadal and adrenal steroidogenesis, in a Leydig cell line. Dlx5 efficiently activates the StAR promoter when GATA-4, another transcription factor essential for testicular steroidogenesis, was coexpressed. The transcriptional activation required the GATA-4-recognition element in the StAR promoter region and Dlx5 can physically interact with GATA-4. Furthermore, we herein show that the double inactivation of Dlx5 and Dlx6 in the mouse leads to decreased testosterone level and abnormal masculinization phenotype. These results suggest that Dlx5 and Dlx6 participate in the control of steroidogenesis during testis development. The findings of this study may open the way to analyze human congenital birth defects.
[show abstract][hide abstract] ABSTRACT: Gene transduction technologies are essential tools for understanding of gene functions or gene cascades underlying embryogenesis. In this review, we introduce a gene transduction method using microbubble and ultrasound (hereafter referred to as sonoporation). Sonoporation is carried out with relatively simple procedures and easily transduces genes into mesenchymal cells without significant damage to target tissues. Therefore, sonoporation is effective for gene transduction to study the molecular mechanisms of morphogenesis.
[show abstract][hide abstract] ABSTRACT: We cloned a full-length androgen receptor (AR) cDNA from chicken (Gallus gallus) gonads. The cDNA sequence has an open reading frame of 2109 bp encoding 703 amino acids. The chicken AR (cAR) shares high homology with ARs from other species in its amino acid sequences, in particular DNA binding domain (DBD) and ligand binding domain (LBD). RT-PCR analysis revealed that cAR mRNA is expressed in several embryonic tissues of both sexes, and relatively higher expression was observed in left ovary compared with testis. The immunoreactive signal of AR was co-localized within the ovarian cell nucleus, while such nuclear localization was not detected in those of testis. To get insight on the possible role of androgen-AR signaling during gonadal development, non-steroidal AR antagonist, flutamide, was administrated in ovo. The treatment induced the disorganization of sex cords in ovarian cortex at day 12 of incubation. The effect was restored by testosterone co-treatment, implying the possibility that AR mediated signaling may be involved in ovarian morphogenesis. Furthermore, co-treatment of flutamide with estradiol-17beta (E2) also restored the phenotype, suggesting androgen-AR signaling might activate aromatase expression that is necessary for estrogen synthesis. These findings suggest androgen-AR signaling might contribute to chicken embryonic ovarian development.
[show abstract][hide abstract] ABSTRACT: The process of fetal external genitalia development might be divided into two processes. The first process accomplishes the initial outgrowth of the anlage, genital tubercle (GT). Previous analysis suggests that the distal urethral epithelium (DUE) of the GT, the Fgf8-expressing region, regulates the outgrowth of the GT. The second process eventually generates the sexually dimorphic development of the external genitalia, which is dependent on the action of steroid hormones. Several key genes, for example, RARs, RXRs, RALDH2, and CYP26, were dynamically expressed during GT development. The teratogenic dose of RA at 9.0 d.p.c. induced a drastic malformation of the urethral plate during GT formation, but did not show gross abnormalities in its outgrowth. In RA-treated embryos, Fgf8 expression was still detected in the distal GT regions. Possible regulatory roles of the FGF and RA signaling systems in external genitalia formation are discussed.
Annals of the New York Academy of Sciences 01/2006; 948(1):13 - 31. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Msx1 and Msx2 genes encode the homeodomain transcription factors. Several gene knockout mice and expression studies suggest that they possess functionally redundant roles in embryogenesis. In this study, we revealed that Msx1 and Msx2 were expressed during ventral body wall formation in an overlapping manner. Msx1/Msx2 double-mutant mice displayed embryonic abdominal wall defects with disorganized muscle layers and connective tissues. These findings indicate that Msx1 and Msx2 play roles in concert during embryonic ventral abdominal wall formation.
The Anatomical Record Part A Discoveries in Molecular Cellular and Evolutionary Biology 06/2005; 284(1):424-30.