Mutations in the EXT1 gene are responsible for human hereditary multiple exostosis type 1. The Drosophila EXT1 homologue, tout-velu, regulates Hedgehog diffusion and signaling, which play an important role in tissue patterning during both invertebrate and vertebrate development. The EXT1 protein is also required for the biosynthesis of heparan sulfate glycosaminoglycans that bind Hedgehog. In this study, we generated EXT1-deficient mice by gene targeting. EXT1 homozygous mutants fail to gastrulate and generally lack organized mesoderm and extraembryonic tissues, resulting in smaller embryos compared to normal littermates. RT-PCR analysis of markers for visceral endoderm and mesoderm development indicates the delayed and abnormal development of both of these tissues. Immunohistochemical staining revealed a visceral endoderm pattern of Indian hedgehog (Ihh) in wild-type E6.5 embryos. However, in both EXT1-deficient embryos and wild-type embryos treated with heparitinase I, Ihh failed to associate with the cells. The effect of the EXT1 deletion on heparan sulfate formation was tested by HPLC and cellular glycosyltransferase activity assays. Heparan sulfate synthesis was abolished in EXT1 -/- ES cells and decreased to less than 50% in +/- cell lines. These results indicate that EXT1 is essential for both gastrulation and heparan sulfate biosynthesis in early embryonic development.

The application of plant growth promoting rhizobacteria (PGPR) to agro-ecosystems is considered to have the potential for improving plant growth in extreme environments featured by water shortage. Herein, we isolated bacterial strains from foxtail millet (Setaria italica L.), a drought-tolerant crop cultivated in semiarid regions in the northeast of China. Four isolates were initially selected for their ability to produce ACC deaminase as well as drought tolerance. The isolates were identified as Pseudomonas fluorescens, Enterobacter hormaechei, and Pseudomonas migulae on the basis of 16S rRNA sequence analysis. All of these drought-tolerant isolates were able to produce EPS (exopolysaccharide). Inoculation with these strains stimulated seed germination and seedling growth under drought stress. Pseudomonas fluorescens DR7 showed the highest level of ACC deaminase and EPS-producing activity. DR7 could efficiently colonize the root adhering soil, increased soil moisture, and enhance the root adhering soil/root tissue ratio. These results suggest drought tolerant PGPR from foxtail millet could enhance plant growth under drought stress conditions and serve as effective bioinoculants to sustain agricultural production in arid regions.

Gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) play critical roles in vertebrate reproduction. In the present study, we cloned and characterized zebrafish FSHbeta (fshb), LHbeta (lhb), and GTHalpha (cga) subunits. Compared with the molecules of other teleosts, the cysteine residues and potential glycosylation sites are fully conserved in zebrafish Lhb and Cga but not in Fshb, whose cysteines exhibit unique distribution. Interestingly, in addition to the pituitary, fshbeta, lhbeta, and cga were also expressed in some extrapituitary tissues, particularly the gonads and brain. In situ hybridization showed that zebrafish fshbeta and lhbeta were expressed in two distinct populations of gonadotrophs in the pituitary. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that all the three subunits increased expression before ovulation (0100-0400) when the germinal vesicles in the full-grown follicles were migrating toward the periphery, but the levels dropped at 0700, when ovulation occurred. Recombinant zebrafish FSH (zfFSH) and LH (zfLH) were produced in the Chinese hamster ovary (CHO) cells and their effects on the cognate receptors (zebrafish Fshr and Lhr) tested. Interestingly, zfFSH specifically activated zebrafish Fshr expressed together with a cAMP-responsive reporter gene in the CHO cells, whereas zfLH could stimulate both Fshr and Lhr. In conclusion, the present study systematically investigated gonadotropins in the zebrafish in terms of their structure, spatial-temporal expression patterns, and receptor specificity. These results, together with the availability of recombinant zfFSH and zfLH, provide a solid foundation for further studies on the physiological relevance of FSH and LH in the zebrafish, one of the top biological models in vertebrates.

Vertebrate reproduction is controlled by two gonadotropins (FSH and LH) from the pituitary. Despite numerous studies on FSH and LH in fish species, their functions in reproduction still remain poorly defined. This is partly due to the lack of powerful genetic approaches for functional studies in adult fish. This situation is now changing with the emergence of genome-editing technologies, especially Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). In this study, we deleted the hormone-specific β-genes of both FSH and LH in the zebrafish using TALEN. This was followed by a phenotype analysis for key reproductive events, including gonadal differentiation, puberty onset, gametogenesis, final maturation, and fertility. FSH-deficient zebrafish (fshb(-/-)) were surprisingly fertile in both sexes; however, the development of both the ovary and testis was significantly delayed. In contrast, LH-deficient zebrafish (lhb(-/-)) showed normal gonadal growth, but the females failed to spawn and were therefore infertile. Using previtellogenic follicles as the marker, we observed a significant delay of puberty onset in the fshb mutant but not the lhb mutant females. Interestingly, FSH seemed to play a role in maintaining the female status because we repeatedly observed sexual reversal in the fshb mutant. Neither the fshb nor lhb mutation alone seemed to affect gonadal differentiation; however, the double mutation of the two genes led to all males, although the development of the testis was significantly delayed. In summary, our data confirmed some well-known functions of FSH and LH in fish while also providing evidence for novel functions, which would be difficult to reveal using traditional biochemical and physiological approaches.

In the present study, we cloned and characterized zebrafish FSH receptor (Fshr) and LH receptor (Lhr). Both fshr and lhr were abundantly expressed in the zebrafish gonads; however, they could also be detected in the kidney and liver, respectively. When overexpressed in mammalian cell lines together with a cAMP-responsive reporter gene, zebrafish Fshr responded to goldfish pituitary extract but not hCG, whereas Lhr could be activated by both. It was further demonstrated that Fshr was specific to bFSH, while Lhr could be stimulated by both bovine FSH and LH. Low level of fshr expression could be detected in the immature ovary, but the level steadily increased during vitellogenesis of the first cohort of developing follicles. In contrast, the expression of lhr could barely be detected in the immature ovary, but it became detectable at the beginning of vitellogenesis and steadily increased afterward with the peak level reached at the full-grown stage. At the follicle level, the expression of fshr was very weak in the follicles of primary growth stage but significantly increased with the follicles entering vitellogenesis. However, after reaching the maximal level in the midvitellogenic follicles, the level of fshr expression dropped slightly but significantly at the full-grown stage. In comparison, the expression of lhr obviously lagged behind that of fshr. Its expression became detectable only when the follicles started to accumulate yolk granules, but the level rose steadily afterward and reached the peak at the full-grown stage before oocyte maturation. These results suggest differential roles for Fshr and Lhr in zebrafish ovarian follicle development.

Gonadotropins are primary hormones that control vertebrate reproduction. In a recent study, we analyzed the impacts of FSH and LH on zebrafish reproduction by disrupting FSH and LH-β genes (fshb and lhb) using transcription activator-like effector nuclease (TALEN) technology. Using the same approach, we successfully deleted FSH and LH receptor genes (fshr and lhcgr) in the present study. In contrast to the deficiency of its cognate ligand FSH, the fshr-deficient females showed a complete failure of follicle activation with all ovarian follicles arrested at the primary growth-previtellogenic transition, which is the marker for puberty onset in females. Interestingly, after blockade at the primary growth stage for varying times, all females reversed to males, and all these males were fertile. In fshr-deficient males, spermatogenesis was normal in adults, but the initiation of spermatogenesis in juveniles was retarded. In contrast to fshr, the deletion of the lhcgr gene alone caused no obvious phenotypes in both males and females; however, double mutation of fshr and lhcgr resulted in infertile males. In summary, our results in the present study showed that Fshr was indispensable to folliculogenesis and the disruption of the fshr gene resulted in a complete failure of follicle activation followed by masculinization into males. In contrast, lhcgr does not seem to be essential to zebrafish reproduction in both males and females. Neither Fshr nor Lhcgr deficiency could phenocopy the deficiency of their cognate ligands FSH and LH, which is likely due to the fact that Fshr can be activated by both FSH and LH in the zebrafish.

SignificanceIn vertebrates, steroid hormones regulate developmental transition from juveniles to adults. Insect steroid hormone, 20-hydroxyecdysone (20E), coordinates with juvenile hormone (JH) to regulate metamorphosis; however, the precise cross-talk mechanism is not well understood. Here, we report that JH and 20E antagonize each other’s biosynthesis in a major endocrine organ of Drosophila larvae: JH suppresses ecdysone biosynthesis and inhibits metamorphosis, whereas 20E suppresses JH biosynthesis and promotes metamorphosis. These data answer a long-standing question on how the mutual antagonism between the two major insect hormones regulates metamorphosis and may help to understand the hormonal regulation of developmental transition in mammals.

Sexual or gonadal differentiation is a complex event and its mechanism remains elusive in teleosts. Despite its complexity and plasticity, the process of ovarian differentiation is believed to involve gonadal aromatase (cyp19a1a) in nearly all species studied. However, most data concerning the role of aromatase have come from gene expression analysis or studies involving pharmacological approaches. There has been a lack of genetic evidence for the importance of aromatase in gonadal differentiation, especially the timing when the enzyme starts to exert its effect. This is due to the lack of appropriate loss-of-function approaches in fish models for studying gene functions. This situation has changed recently with the development of genome editing technologies, namely TALEN and CRISPR/Cas9. Using both TALEN and CRISPR/Cas9, we successfully established three mutant zebrafish lines lacking the ovarian aromatase. As expected, all mutant fish were males, supporting the view that aromatase plays a critical role in directing ovarian differentiation and development. Further analysis showed that the ovarian aromatase did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells; however, it was essential for further differentiation of the juvenile ovary into the true ovary.