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.