School of BioSciences - Research Publications
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ItemThe role of mab-3 in spermatogenesis and ontogenesis of pinewood nematode, Bursaphelenchus xylophilus.(Wiley, 2021-01)BACKGROUND: Bursaphelenchus xylophilus is one of the most destructive invasive species, causing extensive economic losses worldwide. The sex ratio of female to male of B. xylophilus plays an important role in the nematode infestation. However, little is known about the processes of its sex determination. The double sex/mab-3-related family of transcription factors are highly conserved in animals, playing crucial roles in sex determination, spermatogenesis and ontogenesis. We therefore investigated its orthologue, Bxy-mab-3, in B. xylophilus. RESULTS: Bxy-mab-3 has two typical conserved DNA-binding domains. It was observed in J2 (the second-stage of juveniles), J3, J4 and male adults (specifically on the spicules), but not in eggs or female adults via mRNA in situ hybridization. RNA-Seq indicated significantly higher expression in males. RNAi showed that the body size and sperm size of male adults were markedly smaller than those of the controls. Meanwhile, almost all the RNAi-treated males failed to mate with the normal females, even 26.34% of interfered males did not produce sperm. However, RNAi of Bxy-mab-3 had no effect on the sex ratio of B. xylophilus. CONCLUSION: Bxy-mab-3 is indispensable for spermatogenesis, ontogenesis and mating behavior. It is a typical sex-determination gene with differential expression in males and females. However, knocking down Bxy-mab-3 expression could not alter the sex ratio as seen in other species. Our findings contribute towards a better understanding of the molecular events of Bxy-mab-3 in B. xylophilus, which provides promising hints for control of pine wilt disease by blocking ontogenesis and decreasing nematode fecundity.
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ItemRegulatory roles of Bxy-laf-1 in reproductive behaviour of Bursaphelenchus xylophilus.(Frontiers Media SA, 2022)Bursaphelenchus xylophilu is a worldwide quarantine nematode, causing huge economic losses and ecological disasters in many countries. The sex ratio of B. xylophilus plays an important role in the nematode infestation. The laf-1-related genes are highly conserved in animals, playing crucial roles in sex determination. Therefore, we investigated the expression pattern and biological function of its orthologue, Bxy-laf-1 in B. xylophilus. Bxy-laf-1 has two typical conserved DNA-binding domains, DEAD and Helicase C. The real-time quantitative PCR data revealed that Bxy-laf-1 expression was required throughout the entire life of B. xylophilus, with the maximum expression in the J2 stage and the lowest expression in the adult stage. mRNA in situ hybridization showed that Bxy-laf-1 is mainly located in the cephalopharynx and reproductive organs of B. xylophilus. RNA interference (RNAi) indicated that the head swing frequency was dramatically decreased. The RNA interference results displayed that a significant reduction in motility was observed in the hatched larvae. The female to male sex ratio was also decreased in the F0 and F1 generations, but recovered in the F2 generation. The tail of female adults with eggs in the belly appeared deformities. This phenomenon appeared in the F0 and F1 generations, but recovered in the F2 generation. Bxy-laf-1 is a typical sex-determination gene with distinct expression patterns in males and females. As demonstrated in other species, the sex ratio was altered after knocking down Bxy-laf-1 expression. The results of this study contribute to our understanding of the molecular processes of Bxy-laf-1 in B. xylophilus, which may provide clues about how to control pine wilt disease by inhibiting ontogenic growth and reducing nematode fertility.
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Itemmadd-4 plays a critical role in light against Bursaphelenchus xylophilus.(Springer Science and Business Media LLC, 2022-08-30)Bursaphelenchus xylophilus is a notorious invasive species, causing extensive losses to pine ecosystems globally. Previous studies had shown that the development of B. xylophilus was seriously suppressed by light. However, the mechanism involved in the inhibition is unknown. Here, it is the first report that Bxy-madd-4 is a light-regulated gene, plays a potential role in B. xylophilus in responding to the blue light. Transcriptome sequencing revealed that the expression level of Bxy-madd-4 declined by 86.39% under blue light. The reverse transcription quantitative real-time PCR results were in accord with the transcriptome sequencing, confirming the expression level of Bxy-madd-4 was suppressed by blue light. Bxy-madd-4 promoter::mCherry reporter constructed in Caenorhabditis elegans were utilized to mimic the spatiotemporal expression patterns of Bxy-madd-4. Bxy-madd-4A promoter activity had a strong continuity throughout all development stages in C. elegans. Further RNA interference indicated that only 36.8% of the Bxy-madd-4 dsRNA treated embryos were hatched. Moreover, 71.6% of the hatched nematodes were abnormal, such as particles on the body surface and concave tissues. Our findings contribute towards a better understanding of the mechanism of light against the destructive invasive nematode, providing a promising hint for control of the destructive invasive nematode.
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ItemMolecular characterization and functional analysis of akt-1 in pinewood nematode, Bursaphelenchus xylophilus(WILEY, 2021-02)Abstract Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which leads to the devastation of millions of hectares of pine trees worldwide. Protein kinase B (AKT) is a structurally conserved serine/threonine kinase with a pleckstrin homology domain and a kinase domain, and it plays versatile biological roles across different animal kingdoms. We, therefore, investigated the molecular characteristics and biological function of akt‐1 in B. xylophilus (Bxy‐akt‐1). In propagative stages, reverse transcription quantitative real‐time PCR revealed that Bxy‐akt‐1 expression gradually increased during embryogenesis, reaching the highest level at the second‐stage juvenile (J2), and then steadily decreased from J3 to adults. Intriguingly, the expression of Bxy‐akt‐1 was significantly higher in males than in females. Moreover, the expression level of Bxy‐akt‐1 in dispersal third‐stage dauer larvae (DL3) was higher than that in J2, but it decreased to normal level in DL4. Bxy‐akt‐1 promoter::gfp reporter constructed in Caenorhabditis elegans was utilized to mimic the spatio‐temporal expression pattern of Bxy‐akt‐1. The results showed that Bxy‐akt‐1 was broadly expressed in the hypodermis around the head during embryogenesis and was mainly expressed in the muscle cells of pharynx, vulva, tail and nerve systems in post‐embryonic stages. Abnormal embryos were observed, and the hatching rate was significantly decreased after Bxy‐akt‐1 was knocked down in B. xylophilus, but no constitutive developmental arrest of dauer larvae was observed. The results indicated that Bxy‐akt‐1 is indispensable for embryogenesis, but might not play a significant role in controlling dauer entry, as in C. elegans.
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ItemEvolution of coding and non-coding genes in HOX clusters of a marsupial(BMC, 2012-06-18)BACKGROUND: The HOX gene clusters are thought to be highly conserved amongst mammals and other vertebrates, but the long non-coding RNAs have only been studied in detail in human and mouse. The sequencing of the kangaroo genome provides an opportunity to use comparative analyses to compare the HOX clusters of a mammal with a distinct body plan to those of other mammals. RESULTS: Here we report a comparative analysis of HOX gene clusters between an Australian marsupial of the kangaroo family and the eutherians. There was a strikingly high level of conservation of HOX gene sequence and structure and non-protein coding genes including the microRNAs miR-196a, miR-196b, miR-10a and miR-10b and the long non-coding RNAs HOTAIR, HOTAIRM1 and HOXA11AS that play critical roles in regulating gene expression and controlling development. By microRNA deep sequencing and comparative genomic analyses, two conserved microRNAs (miR-10a and miR-10b) were identified and one new candidate microRNA with typical hairpin precursor structure that is expressed in both fibroblasts and testes was found. The prediction of microRNA target analysis showed that several known microRNA targets, such as miR-10, miR-414 and miR-464, were found in the tammar HOX clusters. In addition, several novel and putative miRNAs were identified that originated from elsewhere in the tammar genome and that target the tammar HOXB and HOXD clusters. CONCLUSIONS: This study confirms that the emergence of known long non-coding RNAs in the HOX clusters clearly predate the marsupial-eutherian divergence 160 Ma ago. It also identified a new potentially functional microRNA as well as conserved miRNAs. These non-coding RNAs may participate in the regulation of HOX genes to influence the body plan of this marsupial.
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ItemHOXA13 and HOXD13 expression during development of the syndactylous digits in the marsupial Macropus eugenii(BMC, 2012-01-11)BACKGROUND: Kangaroos and wallabies have specialised limbs that allow for their hopping mode of locomotion. The hindlimbs differentiate much later in development but become much larger than the forelimbs. The hindlimb autopod has only four digits, the fourth of which is greatly elongated, while digits two and three are syndactylous. We investigated the expression of two genes, HOXA13 and HOXD13, that are crucial for digit patterning in mice during formation of the limbs of the tammar wallaby. RESULTS: We describe the development of the tammar limbs at key stages before birth. There was marked heterochrony and the hindlimb developed more slowly than the forelimb. Both tammar HOXA13 and HOXD13 have two exons as in humans, mice and chickens. HOXA13 had an early and distal mRNA distribution in the tammar limb bud as in the mouse, but forelimb expression preceded that in the hindlimb. HOXD13 mRNA was expressed earlier in the forelimb than the hindlimb and was predominantly detected in the interdigital tissues of the forelimb. In contrast, the hindlimb had a more restricted expression pattern that appeared to be expressed at discrete points at both posterior and anterior margins of the limb bud, and was unlike expression seen in the mouse and the chicken. CONCLUSIONS: This is the first examination of HOXA and HOXD gene expression in a marsupial. The gene structure and predicted proteins were highly conserved with their eutherian orthologues. Interestingly, despite the morphological differences in hindlimb patterning, there were no modifications to the polyalanine tract of either HOXA13 or HOXD13 when compared to those of the mouse and bat but there was a marked difference between the tammar and the other mammals in the region of the first polyserine tract of HOXD13. There were also altered expression domains for both genes in the developing tammar limbs compared to the chicken and mouse. Together these findings suggest that the timing of HOX gene expression may contribute to the heterochrony of the forelimb and hindlimb and that alteration to HOX domains may influence phenotypic differences that lead to the development of marsupial syndactylous digits.
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ItemDifferential roles of TGIF family genes in mammalian reproduction(BIOMED CENTRAL LTD, 2011-09-29)BACKGROUND: TG-interacting factors (TGIFs) belong to a family of TALE-homeodomain proteins including TGIF1, TGIF2 and TGIFLX/Y in human. Both TGIF1 and TGIF2 act as transcription factors repressing TGF-β signalling. Human TGIFLX and its orthologue, Tex1 in the mouse, are X-linked genes that are only expressed in the adult testis. TGIF2 arose from TGIF1 by duplication, whereas TGIFLX arose by retrotransposition to the X-chromosome. These genes have not been characterised in any non-eutherian mammals. We therefore studied the TGIF family in the tammar wallaby (a marsupial mammal) to investigate their roles in reproduction and how and when these genes may have evolved their functions and chromosomal locations. RESULTS: Both TGIF1 and TGIF2 were present in the tammar genome on autosomes but TGIFLX was absent. Tammar TGIF1 shared a similar expression pattern during embryogenesis, sexual differentiation and in adult tissues to that of TGIF1 in eutherian mammals, suggesting it has been functionally conserved. Tammar TGIF2 was ubiquitously expressed throughout early development as in the human and mouse, but in the adult, it was expressed only in the gonads and spleen, more like the expression pattern of human TGIFLX and mouse Tex1. Tammar TGIF2 mRNA was specifically detected in round and elongated spermatids. There was no mRNA detected in mature spermatozoa. TGIF2 protein was specifically located in the cytoplasm of spermatids, and in the residual body and the mid-piece of the mature sperm tail. These data suggest that tammar TGIF2 may participate in spermiogenesis, like TGIFLX does in eutherians. TGIF2 was detected for the first time in the ovary with mRNA produced in the granulosa and theca cells, suggesting it may also play a role in folliculogenesis. CONCLUSIONS: The restricted and very similar expression of tammar TGIF2 to X-linked paralogues in eutherians suggests that the evolution of TGIF1, TGIF2 and TGIFLX in eutherians was accompanied by a change from ubiquitous to tissue-specific expression. The distribution and localization of TGIF2 in tammar adult gonads suggest that there has been an ultra-conserved function for the TGIF family in fertility and that TGIF2 already functioned in spermatogenesis and potentially folliculogenesis long before its retrotransposition to the X-chromosome of eutherian mammals. These results also provide further evidence that the eutherian X-chromosome has actively recruited sex and reproductive-related genes during mammalian evolution.
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ItemComparative analysis of the mammalian WNT4 promoter(BMC, 2009-09-06)BACKGROUND: WNT4 is a critical signalling molecule in embryogenesis and homeostasis, but the elements that control its transcriptional regulation are largely unknown. This study uses comparative cross species sequence and functional analyses between humans and a marsupial (the tammar wallaby,Macropus eugenii) to refine the mammalian Wnt4 promoter. RESULTS: We have defined a highly conserved 89 bp minimal promoter region in human WNT4 by comparative analysis with the tammar wallaby. There are many conserved transcription factor binding sites in the proximal promoter region, including SP1, MyoD, NFkappaB and AP2, as well as highly conserved CpG islands within the human, mouse and marsupial promoters, suggesting that DNA methylation may play an important role in WNT4 transcriptional regulation. CONCLUSION: Using a marsupial model, we have been able to provide new information on the transcriptional regulators in the promoter of this essential mammalian developmental gene, WNT4. These transcription factor binding sites and CpG islands are highly conserved in two disparate mammals, and are likely key controlling elements in the regulation of this essential developmental gene.
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ItemAndrogen and Oestrogen Affect the Expression of Long Non-Coding RNAs During Phallus Development in a Marsupial(MDPI, 2019-03)There is increasing evidence that long non-coding RNAs (lncRNAs) are important for normal reproductive development, yet very few lncRNAs have been identified in phalluses so far. Unlike eutherians, phallus development in the marsupial tammar wallaby occurs post-natally, enabling manipulation not possible in eutherians in which differentiation occurs in utero. We treated with sex steroids to determine the effects of androgen and oestrogen on lncRNA expression during phallus development. Hormonal manipulations altered the coding and non-coding gene expression profile of phalluses. We identified several predicted co-regulatory lncRNAs that appear to be co-expressed with the hormone-responsive candidate genes regulating urethral closure and phallus growth, namely IGF1, AR and ESR1. Interestingly, more than 50% of AR-associated coding genes and lncRNAs were also associated with ESR1. In addition, we identified and validated three novel co-regulatory and hormone-responsive lncRNAs: lnc-BMP5, lnc-ZBTB16 and lncRSPO4. Lnc-BMP5 was detected in the urethral epithelium of male phalluses and was downregulated by oestrogen in males. Lnc-ZBTB16 was downregulated by oestrogen treatment in male phalluses at day 50 post-partum (pp). LncRSPO4 was downregulated by adiol treatment in female phalluses but increased in male phalluses after castration. Thus, the expression pattern and hormone responsiveness of these lncRNAs suggests a physiological role in the development of the phallus.
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ItemDifferential expression of WNT4 in testicular and ovarian development in a marsupial(BMC, 2006-10-03)BACKGROUND: WNT4 is a key regulator of gonadal differentiation in humans and mice, playing a pivotal role in early embryogenesis. Using a marsupial, the tammar wallaby, in which most gonadal differentiation occurs after birth whilst the young is in the pouch, we show by quantitative PCR during early testicular and ovarian development that WNT4 is differentially expressed in gonads. RESULTS: Before birth, WNT4 mRNA expression was similar in indifferent gonads of both sexes. After birth, in females WNT4 mRNA dramatically increased during ovarian differentiation, reaching a peak by day 9-13 post partum (pp) when the ovarian cortex and medulla are first distinguishable. WNT4 protein was localised in the ovarian cortex and at the medullary boundary. WNT4 mRNA then steadily decreased to day 49, by which time all the female germ cells have entered meiotic arrest. In males, WNT4 mRNA was down-regulated in testes immediately after birth, coincident with the time that seminiferous cords normally form, and rose gradually after day 8. By day 49, when testicular androgen production normally declines, WNT4 protein was restricted to the Leydig cells. CONCLUSION: This is the first localisation of WNT4 protein in developing gonads and is consistent with a role for WNT4 in steroidogenesis. Our data provide strong support for the suggestion that WNT4 not only functions as an anti-testis gene during early development, but is also necessary for later ovarian and testicular function.