Biology subjects

Waters, P. D.

Publications and source records attributed to Waters, P. D..

12 recordsLinked to original sources

bioRxiv2026

DNA methylation regulates a key sex differentiation gene in Pogona vitticeps, a dragon lizard with sex reversal

During embryonic development bipotential gonads differentiate into either testes or ovaries under the direction of mutually exclusive gene networks. DNA methylation has been shown to regulate gene expression and to play a role in many developmental processes. This includes sex differentiation in which DNA methylation is linked to control of key sex genes. Whether DNA methylation regulates sexual differentiation in species where environmental influences such as temperature are involved is less clear. We conducted a genome-wide study in embryonic gonads of the central bearded dragon (Pogona vitticeps), a lizard with temperature induced sex reversal, and compared DNA methylation patterns to gene expression profiles at a stage of early sex differentiation. Overall, sex reversed ZZf females were found to have lower global methylation than both canonical sexes, ZZm males and ZWf females. We found that the expression of a key gene in sex differentiation, Amh, is regulated via DNA methylation. Amh is a driver of testis differentiation and is repressed in genetically determined females, as well as in temperature sex-reversed females, by hypermethylation around its transcription start site. Although the trigger of ovary determination is different in both groups of females, one by genetic complement and one by a temperature signal, downstream regulation of gene expression converges and seems to follow similar mechanisms.

genomics↗

bioRxiv2026

Characterising differential gene expression and alternative splicing in a sex reversing skink, Bassiana duperreyi

In some reptiles, genetic and environmental sex determination interact whereby extreme incubation temperatures override genetic sex determination (GSD) to produce sex-reversed individuals. In one lizard with temperature-influenced GSD, the central bearded dragon, intron retention in the histone-modifier genes Kdm6b and Jarid2 has been implicated as a candidate signal linking temperature to sex. Equivalent intron retention is also present in two species with temperature-dependent sex determination, the red-eared slider turtle and the American alligator. The eastern three-lined skink, Bassiana duperreyi, represents another lizard with temperature induced sex reversal. It has an XY sex determination system in which low temperature incubation causes sex reversal of XX embryos to produce phenotypic males. In this study, we performed splice-aware analysis of RNA sequencing from hatchling brains of the three-lined skink. We investigated differences in alternative splicing and gene expression between the three sex conditions: XY males (XYm), XX females (XXf), and sex-reversed XX males (XXm). Sex reversal specific intron retention was observed in the gene, Ttll7, which only occurred in XXm and not in XYm or XXf. Intron retention in Ttll7 could alter the function of the encoded protein, a tubulin polyglutamylase, but its effect on sex reversal here is unknown. In addition, intron retention in the histone-modifier genes Jarid2 and Kdm6b occurred in all conditions. The presence of Kdm6b and Jarid2 intron retention in all sex conditions suggests that the pattern of intron retention in sex reversal in the eastern three-lined skink is distinct compared to the bearded dragon. We conclude that a different molecular pathway for sex reversal is induced in the three-lined skink, the details of which remain elusive.

genomics↗

bioRxiv2025

DNA methylation reprogramming in marsupial embryos is restricted to the extraembryonic lineage

DNA methylation (5mC) is an epigenetic mark that plays a critical role in defining cell fate. Following fertilisation, DNA methylation inherited from gametes must be reprogrammed to establish totipotency and enable the parental-to-zygotic transition. To accomplish this, non-mammalian vertebrates such as zebrafish and medaka subtly reprogram maternal 5mC profiles while maintaining high methylation levels throughout embryogenesis. In contrast, eutherian mammals such as mouse and human undergo global 5mC erasure in both embryonic and extraembryonic lineages. However, while embryonic 5mC is rapidly re-established to high levels upon implantation, the trophectoderm, which gives rise to the placenta, displays sustained and conserved DNA hypomethylation, suggesting that this drastic 5mC erasure may be functionally linked to complex placentation in mammals. To clarify whether extensive post-fertilisation 5mC erasure co-evolved with placentation, we explored embryonic methylation dynamics in marsupials, a lineage of therian mammals with a short-lived placenta. We produced a near complete telomere-to-telomere (T2T) genome and generated detailed epigenome maps of embryonic development for an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata). We found the dunnart embryo exhibits genome wide DNA demethylation at the blastocyst stage, but these changes occur in the trophectoderm only, suggesting that 5mC erasure in the placenta is an ancestral state in therian mammals. Furthermore, the T2T-level dunnart genome assembly enabled identification of sex chromosomes, uncovering extensive hypomethylation of the paternally-inherited inactive X chromosome in females and revealing the previously unannotated master regulator of X chromosome inactivation, lncRNA Rsx. Our data indicate that while the use of genome-wide 5mC erasure differs between eutherian and marsupial lineages, 5mC erasure in extraembryonic tissue is ancestral to therian mammals and may be necessary to support placental development. HIGHLIGHTSO_LIFirst embryonic DNA methylation maps in an Australian marsupial C_LIO_LIExtensive global erasure of DNA methylation in the trophectoderm C_LIO_LIMaintenance of high DNA methylation in the embryonic lineage C_LIO_LIHypomethylated paternal X chromosome with methylated escapee genes C_LI

developmental biology↗

bioRxiv2025

A telomere to telomere phased genome assembly and annotation for the Australian central bearded dragon Pogona vitticeps

BackgroundThe central bearded dragon (Pogona vitticeps) is widely distributed in central eastern Australia and adapts readily to captivity. Among other attributes, it is distinctive because it undergoes sex reversal from ZZ genotypic males to phenotypic females at high incubation temperatures. Here, we report an annotated telomere to telomere phased assembly of the genome of a female ZW central bearded dragon. ResultsGenome assembly length is 1.75 Gbp with a scaffold N50 of 266.2 Mbp, N90 of 28.1 Mbp, 26 gaps and 42.2% GC content. Most (99.6%) of the reference assembly is scaffolded into 6 macrochromosomes and 10 microchromosomes, including the Z and W microchromosomes, corresponding to the karyotype. The genome assembly exceeds standard recommended by the Earth Biogenome Project (6CQ40): 0.003% collapsed sequence, 0.03% false expansions, 99.8% k-mer completeness, 97.9% complete single copy BUSCO genes and an average of 93.5% of transcriptome data mappable back to the genome assembly. The mitochondrial genome (16,731 bp) and the model rDNA repeat unit (length 9.5 Kbp) were assembled. Male vertebrate sex genes Amh and Amhr2 were discovered as copies in the small non-recombining region of the Z chromosome, absent from the W chromosome. This, coupled with the prior discovery of differential Z and W transcriptional isoform composition arising from pseudoautosomal sex gene Nr5a1, suggests that complex interactions between these genes, their autosomal copies and their resultant transcription factors and intermediaries, determines sex in the bearded dragon. ConclusionThis high-quality assembly will serve as a resource to enable and accelerate research into the unusual reproductive attributes of this species and for comparative studies across the Agamidae and reptiles more generally. Species TaxonomyEukaryota; Animalia; Chordata; Reptilia; Squamata; Iguania; Agamidae; Amphibolurinae; Pogona; Pogona vitticeps (Ahl, 1926) (NCBI:txid103695). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/651798v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1a9d5fforg.highwire.dtl.DTLVardef@1206061org.highwire.dtl.DTLVardef@97acf7org.highwire.dtl.DTLVardef@1dc939d_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

A genome assembly and annotation for the Australian alpine skink Bassiana duperreyi using long-read technologies

The eastern three-lined skink (Bassiana duperreyi) inhabits the Australian high country in the southwest of the continent including Tasmania. It is an oviparous species that is distinctive because it undergoes sex reversal (from XX genotypic females to phenotypic males) at low incubation temperatures. We present a chromosome-scale genome assembly of a Bassiana duperreyi XY male individual, constructed using a combination of PacBio HiFi and ONT long reads scaffolded using Illumina HiC data. The genome assembly length is 1.57 Gb with a scaffold N50 of 222 Mbp, N90 of 26 Mbp, 200 gaps and 43.10% GC content. Most (95%) of the assembly is scaffolded into 6 macrochromosomes, 8 microchromosomes and the X chromosome, corresponding to the karyotype. Fragmented Y chromosome scaffolds (n=11 > 1 Mbp) were identified using Y-specific contigs generated by genome subtraction. We identified two novel alpha-satellite repeats of 187 bp and 199 bp in the putative centromeres that did not form higher order repeats. The genome assembly exceeds the standard recommended by the Earth Biogenome Project; 0.02% false expansions, 99.63% kmer completeness, 94.66% complete single copy BUSCO genes and an average 98.42% of transcriptome data mappable to the genome assembly. The mitochondrial genome (17,506 bp) and the model rDNA repeat unit (15,154 bp) were assembled. The B. duperreyi genome assembly has one of the highest completeness levels for a skink and will provide a resource for research focused on sex determination and thermolabile sex reversal, as an oviparous foundation species for studies of the evolution of viviparity, and for other comparative genomics studies of the Scincidae. Species TaxonomyEukaryota; Animalia; Chordata; Reptilia; Squamata; Scincidae; Lygosominae; Eugongylini; Bassiana (=Acritoscincus); Bassiana duperreyi (Gray, 1838) (NCBI: txid316450).

genomics↗

bioRxiv2023

Functional coherence of the Xist and RSX protein interactomes: X chromosome inactivation marsupials

Long range epigenetic silencing is epitomised by X chromosome inactivation (XCI) in mammals. It is mediated by independently evolved, non-homologous long noncoding RNAs in eutherian and marsupial mammals (XIST and RSX). The Xist interactome, comprising proteins that mediate the silencing process, is well documented in mouse studies. Here we interrogate proteins that interact with RSX using chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) in a marsupial representative, Monodelphis domestica. We identify 135 proteins that interact with RSX, of which 56 have orthologues in the Xist interactome. Remarkably, nearly 90% of the combined Xist and RSX interactomes are within the same protein-protein association network. This network clustered into three major groups with distinctive functional enrichments, including RNA splicing and processing, regulation of translation and ribosomal biogenesis, and epigenetic transcriptional silencing. The proteins of the RSX interactome were enriched for regions of intrinsic disorder in common with the Xist interactome, identifying this as a feature of ribonucleoprotein complexes associated with XCI. We also show that RNAi knockdown of representative RSX interactors, HNRNPK and CKAP4, led to reactivation of transcription from the inactive X chromosome, indicating a role for each in marsupial XCI. Thus, despite the absence of linear sequence homology between Xist and RSX, they exhibit extraordinary functional coherence that indicates potential for post-transcriptional regulation, a feature not previously associated with the molecular machinery of XCI.

genetics↗

bioRxiv2023

Both phenotypic and genotypic sex influence sex chromosome dosage compensation in a sex reversing lizard

BackgroundLizards have sex determination systems that can differ between even closely related species. These include XY and ZW systems, and thermolabile systems where genes and temperature interact to determine sex. The eastern three-lined skink (Bassiana duperreyi) has a differentiated XY sex determination system, in which low temperature incubation during development can cause female to male sex reversal, producing XX males. This provides a unique opportunity to investigate how genotype and sexual phenotype affect dosage compensation. ResultsHere, we present a draft genome assembly of the Eastern three-lined skink generated from nanopore sequencing. We also generated transcriptomes from brain and heart tissue of normal adult males and females, along with brain tissue of sex-reversed XX males. We observed partial dosage compensation between XX females and XY males in both brain and heart, with median gene expression from the X in normal males being 0.7 times that of normal females. In brain of sex reversed XX males the median X chromosome output matched that of the normal XX female level, and not that of normal XY males. ConclusionsPartial dosage compensation in the Eastern three-lined skink is similar to several other species of lizard. However, here for the first time we describe dosage compensation in a lizard with natural sex reversal, and show that in sex reversed individuals dosage compensation of the X chromosome follows genotypic sex and not phenotypic sex.

genomics↗

bioRxiv2023

Incomplete transcriptional dosage compensation of vertebrate sex chromosomes is balanced by post-transcriptional compensation.

Heteromorphic sex chromosomes (XY or ZW) present problems of gene dosage imbalance between the sexes, and with the autosomes. Mammalian X chromosome inactivation was long thought to imply a critical need for dosage compensation in vertebrates. However, the universal importance of sex chromosome dosage compensation was questioned by mRNA abundance measurements that demonstrated sex chromosome transcripts are neither balanced between the sexes or with autosomes in monotreme mammals or birds. Here, we demonstrate unbalanced mRNA levels of X genes in platypus males and females that correlate with differential loading of histone modifications, and confirm that transcripts of Z genes are unbalanced between males and females also in chicken. However, we found that in both species, median male to female protein abundance ratios were 1:1, implying an additional level of post-transcriptional control. We conclude that parity of sex chromosome output is achieved in birds, as well as all mammal groups, by a combination of transcriptional and post-transcriptional control, consistent with an essential role for sex chromosome dosage compensation in vertebrates.

evolutionary biology↗

bioRxiv2023

Divergent patterns of meiotic double strand breaks and synapsis initiation dynamics suggest an evolutionary shift in the meiosis program between American and Australian marsupials

In eutherian mammals, hundreds of programmed DNA double-strand breaks (DSBs) are generated at the onset of meiosis. The DNA damage response is then triggered. Although the dynamics of this response is well studied in eutherian mammals, recent findings have revealed different patterns of DNA damage signaling and repair in marsupial mammals. To better characterize these differences, here we analyzed synapsis and the chromosomal distribution of meiotic DSBs markers in three different marsupial species (Thylamys elegans, Dromiciops gliorides, and Macropus eugenii) that represent South American and Australian Orders. Our results revealed inter-specific differences in the chromosomal distribution of DNA damage and repair proteins, which were associated with differing synapsis patterns. In the American species T. elegans and D. gliroides, synapsis progressed exclusively from the chromosomal ends towards interstitial regions. This was accompanied by sparse H2AX phosphorylation, mainly accumulating at chromosomal ends, which appeared conspicuously polarized in a bouquet configuration at early stages of prophase I. Accordingly, RAD51 and RPA were mainly localized at chromosomal ends throughout prophaseI in both American marsupials, likely resulting in reduced recombination rates at interstitial positions. In sharp contrast, synapsis initiated at both interstitial and distal chromosomal regions in the Australian representative M. eugenii, {gamma}H2AX had a broad nuclear distribution, and RAD51 and RPA foci displayed an even chromosomal distribution. Given the basal evolutionary position of T. elegans, it is likely that the meiotic features reported in this species represent an ancestral pattern in marsupials and that a shift in the meiotic program occurred after the split of D. gliroides and the Australian marsupial clade. Our results open intriguing questions about the regulation and homeostasis of meiotic DSBs in marsupials. The low recombination rates observed at the interstitial chromosomal regions in American marsupials can result in the formation of large linkage groups, thus having an impact in the evolution of their genomes.

genetics↗

bioRxiv2021

Microchromosomes are building blocks of bird, reptile and mammal chromosomes

Microchromosomes, once considered unimportant shreds of the chicken genome, are gene rich elements with a high GC content and few transposable elements. Their origin has been debated for decades. We used cytological and whole genome sequence comparisons, and chromosome conformation capture, to trace their origin and fate in genomes of reptiles, birds and mammals. We find that microchromosomes as well as macrochromosomes are highly conserved across birds, and share synteny with single small chromosomes of the chordate amphioxus, attesting to their origin as elements of an ancient animal genome. Turtles and squamates (snakes and lizards) share different subsets of ancestral microchromosomes, having independently lost microchromosomes by fusion with other microchromosomes or macrochromosomes. Patterns of fusions were quite different in different lineages. Cytological observations show that microchromosomes in all lineages are spatially separated into a central compartment at interphase and during mitosis and meiosis. This reflects higher interaction between microchromosomes than with macrochromosomes, as observed by chromosome conformation capture, and suggests some functional coherence. In highly rearranged genomes fused microchromosomes retain most ancestral characteristics, but these may erode over evolutionary time; surprisingly de novo microchromosomes have rapidly adopted high interaction. Some chromosomes of early branching monotreme mammals align to several bird microchromosomes, suggesting multiple microchromosome fusions in a mammalian ancestor. Subsequently multiple rearrangements fueled the extraordinary karyotypic diversity of therian mammals. Thus microchromosomes, far from being aberrant genetic elements, represent fundamental building blocks of amniote chromosomes, and it is mammals, rather than reptiles, that are atypical. Significance StatementGenomes of birds and reptiles, but not mammals, consist of a few large chromosomes and many tiny microchromosomes. Once considered unimportant shreds of the genome, microchromosomes are gene rich and highly conserved among bird and reptiles, and share homology with one or more of the tiny chromosomes of an invertebrate that diverged from the vertebrate lineage 684 million years ago. Microchromosomes interact strongly and crowd together at the centre of cells, suggesting functional coherence. Many microchromosomes have been lost independently in turtles, snakes and lizards as they have fused with each other, or with larger chromosomes. In mammals they have completely disappeared, yet some chromosomes of the basal platypus line up with several microchromosomes, suggesting that they are the building blocks of the atypically variable chromosomes of mammals.

genomics↗

bioRxiv2019

A natural antisense to brain-derived neurotrophic factor impairs extinction of drug seeking

BACKGROUNDBrain derived neurotrophic factor (BDNF) is critical for the extinction of drug-seeking. Expression of the Bdnf gene is highly regulated via interactions with non-coding RNA, which themselves are altered following drug exposure. Here we investigate whether a novel long non-coding RNA antisense to Bdnf prevents extinction of drug-seeking. METHODS: Strand-specific RNA sequencing identified a novel long non-coding RNA antisense to exon IV of the Bdnf gene in the ventromedial prefrontal cortex of 8 adult male rats. We then assessed asBdnf-IV expression using strand-specific reverse transcription and quantitative polymerase chain reaction following acquisition, extinction or abstinence from intravenous nicotine self-administration (N = 116). A functional role of the asBdnf-IV in extinction of nicotine-seeking was established by infusing gapmer oligonucleotides into the infralimbic cortex prior to extinction and testing for the effect of these infusions on reinstatement and reacquisition of nicotine-seeking (N = 36). RESULTSRNA sequencing identified the presence of a novel long non-coding RNA antisense to exon IV of the Bdnf gene (asBdnf-IV). Expression of asBdnf-IV was elevated following intravenous nicotine self-administration but not experimenter-administered nicotine. Elevated asBdnf-IV persisted across abstinence and to a greater extent following extinction training, suggesting an interaction between abstinence and extinction learning. In support of this, knockdown of the asBdnf-IV across extinction, but not abstinence, significantly attenuated nicotine-primed reinstatement of nicotine-seeking. CONCLUSIONSasBdnf-IV accumulates in the infralimbic cortex across self-administration training, interferes with the inhibitory learning that underpins extinction of drug-seeking, and predisposes animals to drug relapse.

neuroscience↗

bioRxiv2019

Non-linear sequence similarity between the Xist and Rsx long noncoding RNAs suggests shared functions of tandem repeat domains

The marsupial inactive X chromosome expresses a long noncoding RNA (lncRNA) called Rsx that has been proposed to be the functional analogue of eutherian Xist. Despite the possibility that Xist and Rsx encode related functions, the two lncRNAs harbor no linear sequence similarity. However, both lncRNAs harbor domains of tandemly repeated sequence. In Xist, these repeat domains are known to be critical for function. Using k-mer based comparison, we show that the repeat domains of Xist and Rsx unexpectedly partition into two major clusters that each harbor substantial levels of non-linear sequence similarity. Xist Repeats B, C and D were most similar to each other and to Rsx Repeat 1, whereas Xist Repeats A and E were most similar to each other and to Rsx Repeats 2, 3, and 4. Similarities at the level of k-mers corresponded to domain-specific enrichment of protein-binding motifs. Within individual domains, protein-binding motifs were often enriched to extreme levels. Our data support the hypothesis that Xist and Rsx encode similar functions through different spatial arrangements of functionally analogous protein-binding domains. We propose that the two clusters of repeat domains in Xist and Rsx function in part to cooperatively recruit PRC1 and PRC2 to chromatin. The physical manner in which these domains engage with protein cofactors may be just as critical to the function of the domains as the protein cofactors themselves. The general approaches we outline in this report should prove useful in the study of any set of RNAs.

genetics↗