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Temporal dynamics of gene expression inform cellular and molecular perturbations associated with disease development and evolution. Given the complexity of high-dimensional temporal genomic data, an analytic framework guided by a robust theory is needed to interpret time-sequential changes and to predict system dynamics. Here we model temporal dynamics of the transcriptome of peripheral blood mononuclear cells in a two-dimensional state-space representing states of health and leukemia using time-sequential bulk RNA-seq data from a murine model of acute myeloid leukemia (AML). The state-transition model identified critical points that accurately predict AML development and identifies stepwise transcriptomic perturbations that drive leukemia progression. The geometry of the transcriptome state-space provided a biological interpretation of gene dynamics, aligned gene signals that are not synchronized in time across mice, and allowed quantification of gene and pathway contributions to leukemia development. Our state-transition model synthesizes information from multiple cell types in the peripheral blood and identifies critical points in the transition from health to leukemia to guide interpretation of changes in the transcriptome as a whole to predict disease progression.Significance:These findings apply the theory of state transitions to model the initiation and development of acute myeloid leukemia, identifying transcriptomic perturbations that accurately predict time to disease development.See related commentary by Kuijjer, p. 3072

Table S1. Differentially expressed genes for c_1 vs c_1^* Table S2. Differentially expressed genes for c_2 vs c_1^* Table S3. Differentially expressed genes for c_3 vs c_1^* Table S4. Differentially expressed genes for c_2 vs c_1 Table S5. Differentially expressed genes for c_3 vs c_1 Table S6. Differentially expressed genes for c_3 vs c_2 Tables S7-S10. Differentially expressed genes for early, transition, persistent, and leukemia events. Tables S11-S14. GO terms enriched for early, transition, persistent, and leukemia events. Table S15. Top 1% of eigengenes.

Figure S1. Details of animal model and fusion gene CM, Figure S2. Ex vivo flow cytometry analysis of bone marrow, Figure S3. Details of principal component analysis, Figure S4. Comparison of state-space construction with different dimension reduction methods, Figure S5. Hierarchical clustering of time-series RNA-seq data and relation to state-space, Figure S6. Details of critical point estimation, construction of quasi-potential, and state-space dynamics, Figure S7. Correlation of state-space geometry with Kit and CM gene expression. Sensitivity of state-space geometry to inclusion of Kit and CM, Figure S8. Volcano plots of critical-point based differential gene expression analysis, Figure S9. Heatmaps of selected GO pathways in early, transition, persistent, and leukemic events, Figure S10. Computation of eigengene angle in state-space, Figure S11. Details of principal component analysis of validation cohorts and state-space dynamics, Figure S12. Mean-squared displacement analysis of particle trajectories in state-space and calibration of Fokker-Planck diffusion coefficient with training cohort, Figure S13. Sensitivity analysis of state-space construction to sample and normalization thresholds, Figure S14. Bootstrap cross validation of state-space construction.

In this supplement we provide additional data and methods to support our results. In particular, we show: details of our mouse model of AML; demonstrate the robustness of our state-space construction to normalization methods and gene selection criteria; compare dimension reduction methods; show bootstrap cross-validation of our predictions; compare hierarchical clustering with our state-space approach; and finally, provide full lists of differentially expressed genes and gene ontology (GO) term enrichment.

BACKGROUND: RNA-based predictive biomarkers are not assessed in the clinical diagnostic setting. Potential biomarkers include long noncoding RNAs (lncRNAs), a class of molecules emerging as critical regulators of diverse cellular functions, including therapy resistance. Advances in sequencing technologies have made whole transcriptome profiling (RNA-seq) possible in formalin fixed, paraffin embedded (FFPE) tissue. In this study, we assessed the feasibility of detecting lncRNAs in clinical FFPE specimens, using clinical grade RNA-seq technology offered at a CLIA-certified laboratory. Our second aim was to investigate expression of lncRNAs contributing to endocrine resistance in a cohort of metastatic breast cancer patients. METHODS: RNA-seq was performed on clinical FFPE specimens as a physician-ordered diagnostic test in a CLIA-certified laboratory. RNA-seq libraries were constructed using ribosomal RNA depletion method and > 100 M reads were sequenced for each library. We checked for contaminating sequences using fastqScreen against multiple genomes, including human and E. coli among others. To assess the quality of the sequencing libraries, we used the nf-core RNASeq pipeline. Trimmed reads were mapped to the GRCh38 reference and abundance of transcripts from the Gencode comprehensive annotation (v33) was estimated. Each library was assessed for quality control (QC). Mapped reads were merged into a matrix of counts per gene for each sample. Differential expression was assessed between metastatic breast cancers with and without endocrine resistance. RESULTS: We obtained twenty-five cases (23 patients) of ER-positive, HER2 negative metastatic breast carcinoma that had undergone whole transcriptome sequencing at a CLIA-certified molecular laboratory. Of these, 6 (24%) cases had failed QC: 5 patients’ specimens had low RNA content (all core biopsies); 1 patient had total RNA content comprised of less than 75% human reads (lumpectomy). The mean age for specimens that failed sequencing QC was significantly different from those that passed (637.2 days vs 193.7 days, t-test, p = 0.04). After QC, a total of 19 cases were analyzed for differentially expressed lncRNA profiles. Of these, 5 cases were clinically annotated as hormone sensitive; 14 cases had acquired secondary hormone resistance. Thirty-six lncRNAs were identified as differentially expressed (7 antisense; 25 intergenic; 3 intronic; 1 sense-overlapping). Of these, lncRNAs which were found to be over-expressed in endocrine resistant tumors compared to endocrine sensitive tumors are involved in various oncogenic pathways such as cell cycle regulation (Linc0051, MIR205HG, ST8SOA6-AS1), tumor progression (LINC00885, LINC00319), and chemoresistance (SIRLNT). LncRNAs which were downregulated in endocrine resistant tumors compared to endocrine sensitive tumors are involved in stem cell state (CCDC144NL.AS1) and endocrine resistance (MIR2052HG). CONCLUSION: It is feasible to detect lncRNA biomarkers in clinical FFPE tissue specimens, using a laboratory-developed RNA sequencing pipeline in a CLIA-certified genomic laboratory. FFPE-RNA-seq pipeline can potentially expand the molecular information that could be gleamed from clinical FFPE specimens to answer pertinent clinical questions, such as therapy resistance. Table 1.Summary data of metastatic ER+ breast cancer evaluated in this studyFailed RNA QC Success RNA QCNumber of samples (%)6 (24%)19 (76%)Mean specimen age (range)27 days (11-44)194 days (3-1331)Number of specimens which are biopsies (%)5 (83%)17 (89%)Number of specimens derived from bone source (%)2 (33%)1 (5%)Number of specimens derived from visceral organs (%)2 (33%)12 (63%)Number of specimens derived from soft tissue or lymphoid sources (%)2 (33%)6 (32%)Acquired endocrine resistance5 (83%)14 (74%)Endocrine sensitive1 (17%)5 (26%) Citation Format: Christina H. Wei, Denis O'Meally, Michele Kirschenbaum, Allen Mao, Chetan Kancharla, Kevin V. Morris, Daphne Stewart. Feasibility of detecting long noncoding RNAs in clinical FFPE specimens using clinical grade RNA-sequencing pipeline [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-10-05.

BACKGROUND As a rare subtype of glioblastoma (GBM), the genetic features and outcomes in cystic GBM (cGBM) are largely unknown. This study aimed to evaluate the survival of cystic features, investigate genomic patterns and the immune microenvironment in patients with cGBM. METHODS Gd T1-weighted, T2-weighted, T2-Flair, DWI or ADC images were used to classify cGBM or noncystic GBM (non-cGBM). Clinical information from IvyGAP, EGA databases and Chinese cohort were used for survival analysis. Fresh cystic fluid and residual tumor tissue were collected to evaluate immunological components, pathological and genomic differences between cGBM and non-cGBM. Data from IvyGAP were included to determine difference of molecular feature among substructure. RESULTS 143 cases with cGBM and non-cGBM were screened to compare survival outcomes. Cystic features were to confer a survival benefit. It was an independent prognostic factor irrespective of IDH, MGMT and therapeutic regimen. cGBM patients have a hypomethylation state compared to non-cGBM. Active immune cells such as CD4+, CD8+ T cells were present in the cystic fluid and infiltrated into tumor. 10 cytokines/chemokines were only detected in cystic fluid, 8 cytokines/chemokines increased two-fold in cystic fluid than in peripheral blood serum (PBPs). Much differential methylated position (DMPs), differentially expressed genes (DEGs), Gene Set Enrichment Analysis (GSAE), Functional Epigenetic Modules (FEM) networks and molecular feature of substructural regions were different between cGBM and non-cGBM. CONCLUSION These results imply that cGBM could be a new subtype and has distinctive immune microenvironment which may have potential value correlating with prognosis.

MicroRNAs (miRNAs) have been shown to hold prognostic value in acute myeloid leukemia (AML); however, the temporal dynamics of miRNA expression in AML are poorly understood. Using serial samples from a mouse model of AML to generate time-series miRNA sequencing data, we are the first to show that the miRNA transcriptome undergoes state-transition during AML initiation and progression. We modeled AML state-transition as a particle undergoing Brownian motion in a quasi-potential and validated the AML state-space and state-transition model to accurately predict time to AML in an independent cohort of mice. The critical points of the model provided a framework to align samples from mice that developed AML at different rates. Our mathematical approach allowed discovery of dynamic processes involved during AML development and, if translated to humans, has the potential to predict an individual’s disease trajectory.

Human immunodeficiency virus type 1 (HIV-1) causes a persistent viral infection resulting in the demise of immune regulatory cells. Clearance of HIV-1 infection results in integration of proviral DNA into the genome of host cells, which provides a means for evasion and long-term persistence. A therapeutic compound that specifically targets and sustainably activates a latent HIV-1 provirus could be transformative and is the goal for the “shock-and-kill” approach to a functional cure for HIV-1. Substantial progress has been made toward the development of recombinant proteins that target specific genomic loci for gene activation, repression, or inactivation by directed mutations. However, most of these modalities are too large or too complex for efficient therapeutic application. We describe here the development and testing of a novel recombinant zinc finger protein transactivator, ZFP-362-VPR, which specifically and potently enhances proviral HIV-1 transcription both in established latency models and activity across different viral clades. Additionally, ZFP-362-VPR-activated HIV-1 reporter gene expression in a well-established primary human CD4+ T cell latency model and off-target pathways were determined by transcriptome analyses. This study provides clear proof of concept for the application of a novel, therapeutically relevant, protein transactivator to purge cellular reservoirs of HIV-1., Graphical Abstract, Described here is a “shock-and-kill” approach toward HIV-1 through the development of a novel recombinant zinc finger protein transactivator, ZFP-362-VPR, that targets the NF-κB doublet in the 5¢ LTR of HIV, in order to specifically and potently activate proviral HIV-1 transcription in latently infected cells.

Significance Reptiles have an extraordinary variety of mechanisms to determine sex. The best candidate sex-determining gene in our model reptile (the Australian central bearded dragon) is the key vertebrate sex gene nr5a1 (coding for the steroidogenic factor 1). There are no sex-specific sequence differences between nr5a1 alleles on the sex chromosomes, but the Z- and W-borne alleles are transcribed into remarkably different alternative transcripts. We propose that altered configuration of the repeat-laden W chromosome affects the conformation of the primary transcript to generate more diverse and potentially inhibitory W-borne isoforms that suppress testis determination. This is a mechanism for vertebrate sex determination, in which epigenetic control regulates the action of a gene present on both sex chromosomes., Pogona vitticeps has female heterogamety (ZZ/ZW), but the master sex-determining gene is unknown, as it is for all reptiles. We show that nr5a1 (Nuclear Receptor Subfamily 5 Group A Member 1), a gene that is essential in mammalian sex determination, has alleles on the Z and W chromosomes (Z-nr5a1 and W-nr5a1), which are both expressed and can recombine. Three transcript isoforms of Z-nr5a1 were detected in gonads of adult ZZ males, two of which encode a functional protein. However, ZW females produced 16 isoforms, most of which contained premature stop codons. The array of transcripts produced by the W-borne allele (W-nr5a1) is likely to produce truncated polypeptides that contain a structurally normal DNA-binding domain and could act as a competitive inhibitor to the full-length intact protein. We hypothesize that an altered configuration of the W chromosome affects the conformation of the primary transcript generating inhibitory W-borne isoforms that suppress testis determination. Under this hypothesis, the genetic sex determination (GSD) system of P. vitticeps is a W-borne dominant female-determining gene that may be controlled epigenetically.

As a rare subtype of glioblastomas (GBM), genetic features in cystic GBM (cGBM) are still largely unknown. We have previously identified a series of active T cell subsets and positive cytokines/chemocytokines in the cystic fluid of cGBMs, including IFN-gamma, IL-2, IL-6, IL-8, TNF-alpha, and MIP-1alpha/beta. This study aimed to evaluate the prognosis of cGBM through DNA methylation patterns and immune microenvironment transcriptional signatures. IvyGAP, EGA and fresh samples from a prospective Chinese cohort were collected to investigate genomic signatures, immune microenvironment and survival outcomes in patients with cGBMs. Gd T1-weighted, T2-weighted, T2-Flair, DWI or ADC images were used to classify cGBM. 143 cases with MGMT/IDH record were used to compare survival time and genomic analysis. Among the three cohorts, Kaplan-Meier analysis showed cystic features have benefit to overall survival (OS). Moreover, cyst feature is the forth factor (HR=0.65, [95% CI 0.42, 1.01]) of survival after IDH, MGMT and Stupp therapeutic regimen. Interestingly, in IDH-wild/MGMT-unmethyled subtype, cGBM patients have longer OS vs noncystic GBM (noncGBM) patients (HR = 0.57, [95% CI 0.33, 0.99]). Compared to noncGBM, cGBM patients have hypomethylation state both in whole gene region and cpG islands. Ivygap database showed between cystic and noncystic group, in sub-structures such as CT (cellular tumor region), CTpan (pseudopalisading cells around necrosis) and CTpnz (perinecrotic zone) there are differential gene expressions and different enrichment pathways. GSEA analysis showed within cGBM group, many gene sets associated with immune function activation such as TRL1/2/3/7 and IFN pathways. These results suggest that cysts of GBM may be associated with hypomethylation status an activated immune microenvironment which is associated with longer survival and may define a unique subgroup of GBM with intrinsically different biology and prognosis.

Neuroinflammation is initiated by activation of the brain’s innate immune system in response to an inflammatory challenge. Insufficient control of neuroinflammation leads to enhanced or prolonged pathology in various neurological conditions including multiple sclerosis and Alzheimer’s disease. Nicotinamide adenine dinucleotide (NAD(+)) plays critical roles in cellular energy metabolism and calcium homeostasis. Our previous study demonstrated that deletion of CD38, which consumes NAD(+), suppressed cuprizone-induced demyelination, neuroinflammation and glial activation. However, it is still unknown whether CD38 directly affects neuroinflammation through regulating brain NAD(+) level. In this study, we investigated the effect of CD38 deletion and inhibition and supplementation of NAD(+) on lipopolysaccharide (LPS)-induced neuroinflammation in mice. First, intracerebroventricular injection of LPS significantly increased CD38 expression especially in the hippocampus. Deletion of CD38 decreased LPS-induced inflammatory responses and glial activation. Pre-administration of apigenin, a flavonoid with CD38 inhibitory activity, or nicotinamide riboside (NR), an NAD(+) precursor, increased NAD(+) level, and significantly suppressed induction of cytokines and chemokines, glial activation and subsequent neurodegeneration after LPS administration. In cell culture, LPS-induced inflammatory responses were suppressed by treatment of primary astrocytes or microglia with apigenin, NAD(+), NR or 78c, the latter a specific CD38 inhibitor. Finally, all these compounds suppressed NF-κB signaling pathway in microglia. These results suggest that CD38-mediated neuroinflammation is linked to NAD(+) consumption and that boosting NAD(+) by CD38 inhibition and NR supplementation directly suppress neuroinflammation in the brain.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective siRNA therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle delivery system. Multiple small-interfering RNAs (siRNAs) targeting highly conserved regions of the SARS-CoV-2 virus were screened and three candidate siRNAs emerged that effectively inhibit virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel lipid nanoparticle formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.

Background Homologous sex chromosomes can differentiate over time because recombination is suppressed in the region of the sex determining locus, leading to the accumulation of repeats, progressive loss of genes that lack differential influence on the sexes and sequence divergence on the hemizygous homolog. Divergence in the non-recombining regions leads to the accumulation of Y or W specific sequence useful for developing sex-linked markers. Here we use in silico whole-genome subtraction to identify putative sex-linked sequences in the scincid lizard Bassiana duperreyi which has heteromorphic XY sex chromosomes. Results We generated 96.7 × 109 150 bp paired-end genomic sequence reads from a XY male and 81.4 × 109 paired-end reads from an XX female for in silico whole genome subtraction to yield Y enriched contigs. We identified 7 reliable markers which were validated as Y chromosome specific by polymerase chain reaction (PCR) against a panel of 20 males and 20 females. Conclusions The sex of B. duperreyi can be reversed by low temperatures (XX genotype reversed to a male phenotype). We have developed sex-specific markers to identify the underlying genotypic sex and its concordance or discordance with phenotypic sex in wild populations of B. duperreyi. Our pipeline can be applied to isolate Y or W chromosome-specific sequences of any organism and is not restricted to sequence residing within single-copy genes. This study greatly improves our knowledge of the Y chromosome in B. duperreyi and will enhance future studies of reptile sex determination and sex chromosome evolution.

Temporal dynamics of gene expression inform cellular and molecular perturbations associated with disease development and evolution. Given the complexity of high-dimensional temporal genomic data, an analytic framework guided by a robust theory is needed to interpret time-sequential changes and to predict system dynamics. Here we model temporal dynamics of the transcriptome of peripheral blood mononuclear cells in a two-dimensional state-space representing states of health and leukemia using time-sequential bulk RNA-seq data from a murine model of acute myeloid leukemia (AML). The state-transition model identified critical points that accurately predict AML development and identifies stepwise transcriptomic perturbations that drive leukemia progression. The geometry of the transcriptome state-space provided a biological interpretation of gene dynamics, aligned gene signals that are not synchronized in time across mice, and allowed quantification of gene and pathway contributions to leukemia development. Our state-transition model synthesizes information from multiple cell types in the peripheral blood and identifies critical points in the transition from health to leukemia to guide interpretation of changes in the transcriptome as a whole to predict disease progression. Significance: These findings apply the theory of state transitions to model the initiation and development of acute myeloid leukemia, identifying transcriptomic perturbations that accurately predict time to disease development. See related commentary by Kuijjer, p. 3072

Hibernation is a physiological state employed by many animals that are exposed to limited food and adverse winter conditions. Controlling tissue-specific and organism wide changes in metabolism and cellular function requires precise regulation of gene expression, including by microRNAs (miRNAs). Here we profile miRNA expression in the central bearded dragon (Pogona vitticeps) using small RNA sequencing of brain, heart, and skeletal muscle from individuals in late hibernation and four days post-arousal. A total of 1295 miRNAs were identified in the central bearded dragon genome; 664 of which were novel to central bearded dragon. We identified differentially expressed miRNAs (DEmiRs) in all tissues and correlated mRNA expression with known and predicted target mRNAs. Functional analysis of DEmiR targets revealed an enrichment of differentially expressed mRNA targets involved in metabolic processes. However, we failed to reveal biologically relevant tissue-specific processes subjected to miRNA-mediated regulation in heart and skeletal muscle. In brain, neuroprotective pathways were identified as potential targets regulated by miRNAs. Our data suggests that miRNAs are necessary for modulating the shift in cellular metabolism during hibernation and regulating neuroprotection in the brain. This study is the first of its kind in a hibernating reptile and provides key insight into this ephemeral phenotype.

Early pregnancy is a critical time for successful reproduction; up to half of human pregnancies fail before the development of the definitive chorioallantoic placenta. Unlike the situation in eutherian mammals, marsupial pregnancy is characterised by a long pre-implantation period prior to the development of the short-lived placenta, making them ideal models for study of the uterine environment promoting embryonic survival pre-implantation. Here we present a transcriptomic study of pre-implantation marsupial pregnancy, and identify differentially expressed genes in the Sminthopsis crassicaudata uterus involved in metabolism and biosynthesis, transport, immunity, tissue remodelling, and uterine receptivity. Interestingly, almost one quarter of the top 50 genes that are differentially upregulated in early pregnancy are putatively involved in histotrophy, highlighting the importance of nutrient transport to the conceptus prior to the development of the placenta. This work furthers our understanding of the mechanisms underlying survival of pre-implantation embryos in the earliest live bearing ancestors of mammals.

Long non-coding RNAs (LncRNAs) have been found to play important roles in maintaining and promoting cellular proliferation in cancerous cells. One such lncRNA, LINC00963 has been found to promote cellular proliferation, migration and tumorigenesis in prostate, ovarian, breast and non-small cell lung (NSCLC) cancer. In this study, we aim to identify important functional regions of this lncRNA with a view to identify unique regions for targeted drug therapy. In order to achieve this, we sought to investigate this lncRNA in the A549 NSCLC cell line. We found that LINC00963 was highly expressed and when targeted with either siRNAs or antisense oligonucleotides, cells rapidly undergo apoptosis. Using cell death as a proxy for loss-of-function, we characterized LINC00963 at the genomic level using a CRIPSRScan approach. CRISPRScan is a novel way of determining lncRNA functional domains through the introduction of local mutations. These mutations could potentially disrupt lncRNA function, which for LINC00963 would lead to apoptosis of the cell. We created a unique lentivirus library comprised of 1784 single guide RNAs spanning the length of LINC00963 in order to precisely identify regions of functional importance. Upon transduction in a stably expressing Cas9-A549 cell line, we collected cellular fractions over time, and using high-throughput sequencing (NextSeq, Illumina), evaluated the depletion of these sgRNAs as a proxy for their importance in LINC00963 function. The identification of these key genomic regions that may render LINC00963 non-functional may offer new lncRNA target sites for anti-cancer therapies in the future. Additionally, the utilization of an in-depth CRISPR-Cas9 approach to determine important lncRNA domains provides a novel and precise way in which to evaluate lncRNA function. Citation Format: Roslyn M. Ray, Lu Lang, Sheela P. Pokharel, Denis O'Meally, Tristan A. Scott, Chun-Wei Chen, Kevin V. Morris. The systematic evaluation of the oncogenic lncRNA LINC00963 using a CRISPRScan technique [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2393.

DNA methylation plays a key role in maintaining transcriptional silence on the inactive X chromosome of eutherian mammals. Beyond eutherians, there are limited genome wide data on DNA methylation from other vertebrates. Previous studies of X borne genes in various marsupial models revealed no differential DNA methylation of promoters between the sexes, leading to the conclusion that CpG methylation plays no role in marsupial X-inactivation. Using reduced representation bisulfite sequencing, we generated male and female CpG methylation profiles in four representative vertebrates (mouse, gray short-tailed opossum, platypus, and chicken). A variety of DNA methylation patterns were observed. Platypus and chicken displayed no large-scale differential DNA methylation between the sexes on the autosomes or the sex chromosomes. As expected, a metagene analysis revealed hypermethylation at transcription start sites (TSS) of genes subject to X-inactivation in female mice. This contrasted with the opossum, in which metagene analysis did not detect differential DNA methylation between the sexes at TSSs of genes subject to X-inactivation. However, regions flanking TSSs of these genes were hypomethylated. Our data are the first to demonstrate that, for genes subject to X-inactivation in both eutherian and marsupial mammals, there is a consistent difference between DNA methylation levels at TSSs and immediate flanking regions, which we propose has a silencing effect in both groups.

Sex chromosomes in some reptiles share synteny with distantly related amniotes in regions orthologous to squamate chromosome 2. The latter finding suggests that chromosome 2 was formerly part of a larger ancestral (amniote) super-sex chromosome and raises questions about how sex chromosomes are formed and modified in reptiles. Australian dragon lizards (Agamidae) are emerging as an excellent model for studying these processes. In particular, they exhibit both genotypic (GSD) and temperature-dependent (TSD) sex determination, show evidence of transitions between the two modes and have evolved non-homologous ZW sex microchromosomes even within the same evolutionary lineage. They therefore represent an excellent group to probe further the idea of a shared ancestral super-sex chromosome and to investigate mechanisms for transition between different sex chromosome forms. Here, we compare sex chromosome homology among eight dragon lizard species from five genera to identify key cytological differences and the mechanisms that may be driving sex chromosome evolution in this group. We performed fluorescence in situ hybridisation to physically map bacterial artificial chromosome (BAC) clones from the bearded dragon, Pogona vitticeps&rsquo, ZW sex chromosomes and a nucleolar organising region (NOR) probe in males and females of eight Agamid species exhibiting either GSD or TSD. We show that the sex chromosome derived BAC clone hybridises near the telomere of chromosome 2q in all eight species examined. This clone also hybridises to the sex microchromosomes of three species (P vitticeps, P. barbata and Diporiphora nobbi) and a pair of microchromosomes in three others (Ctenophorus pictus, Amphibolurus norrisi and Amphibolurus muricatus). No other chromosomes are marked by the probe in two species from the closely related genus Physignathus. A probe bearing nucleolar organising region (NOR) sequences maps close to the telomere of chromosome 2q in all eight species, and to the ZW pair in P. vitticeps and P. barbata, the W microchromosome in D. nobbi, and several microchromosomes in P. cocincinus. Our findings provide evidence of sequence homology between chromosome 2 and the sex chromosomes of multiple agamids. These data support the hypothesis that there was an ancestral sex chromosome in amniotes that gave rise to squamate chromosome 2 and raises the prospect that some particular property of this chromosome has favoured its role as a sex chromosome in amniotes. It is likely that the amplification of repetitive sequences associated with this region has driven the high level of heterochromatinisation of the sex-specific chromosomes in three species of agamid. Our data suggest a possible mechanism for chromosome rearrangement, including inversion and duplication near the telomeric regions of the ancestral chromosome 2 and subsequent translocation to the ZW sex microchromosomes in three agamid species. It is plausible that these chromosome rearrangements involving sex chromosomes also drove speciation in this group.

Devastating fires in Australia over 2019–20 decimated native fauna and flora, including koalas. The resulting population bottleneck, combined with significant loss of habitat, increases the vulnerability of remaining koala populations to threats which include disease. Chlamydia is one disease which causes significant morbidity and mortality in koalas. The predominant pathogenic species, Chlamydia pecorum, causes severe ocular, urogenital and reproductive tract disease. In marsupials, including the koala, gene expansions of an antimicrobial peptide family known as cathelicidins have enabled protection of immunologically naïve pouch young during early development. We propose that koala cathelicidins are active against Chlamydia and other bacteria and fungi. Here we describe ten koala cathelicidins, five of which contained full length coding sequences that were widely expressed in tissues throughout the body. Focusing on these five, we investigate their antimicrobial activity against two koala C. pecorum isolates from distinct serovars; MarsBar and IPTaLE, as well as other bacteria and fungi. One cathelicidin, PhciCath5, inactivated C. pecorum IPTaLE and MarsBar elementary bodies and significantly reduced the number of inclusions compared to the control (pChlamydia, natural PhciCath5 concentrations may be inadequate in vivo to prevent or control C. pecorum infections in koalas. PhciCath5 also displayed antimicrobial activity against fungi and Gram negative and positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Electrostatic interactions likely drive PhciCath5 adherence to the pathogen cell membrane, followed by membrane permeabilisation leading to cell death. Activity against E. coli was reduced in the presence of 10% serum and 20% whole blood. Future modification of the PhciCath5 peptide to enhance activity, including in the presence of serum/blood, may provide a novel solution to Chlamydia infection in koalas and other species.

Defensins comprise a family of cysteine-rich antimicrobial peptides with important roles in innate and adaptive immune defense in vertebrates. We characterized alpha and beta defensin genes in three Australian marsupials: the Tasmanian devil (Sarcophilus harrisii), koala (Phascolarctos cinereus), and tammar wallaby (Macropus eugenii) and identified 48, 34, and 39 defensins, respectively. One hundred and twelve have the classical antimicrobial peptides characteristics required for pathogen membrane targeting, including cationic charge (between 1+ and 15+) and a high proportion ofhydrophobic residues (>30%). Phylogenetic analysis shows that gene duplication has driven unique and species-specific expansions of devil, koala, and tammar wallaby beta defensins and devil alpha defensins. Defensin genes are arranged in three genomic clusters in marsupials, whereas further duplications and translocations have occurred in eutherians resulting in four and five gene clusters in mice and humans, respectively. Marsupial defensins are generally under purifying selection, particularly residues essential for defensin structural stability. Certain hydrophobic or positively charged sites, predominantly found in the defensin loop, are positively selected, which may have functional significance in defensin-target interaction and membrane insertion. This research was supported by the Australian Research Council. We thank Rebecca Johnson, Marc Wilkins, and Peter Timms and other colleagues in the Koala Genome Consortium for providing access to koala sequence data prior to public release.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The koala, the only extant species of the marsupial family Phascolarctidae, is classified as ‘vulnerable’ due to habitat loss and widespread disease. We sequenced the koala genome, producing a complete and contiguous marsupial reference genome, including centromeres. We reveal that the koala’s ability to detoxify eucalypt foliage may be due to expansions within a cytochrome P450 gene family, and its ability to smell, taste and moderate ingestion of plant secondary metabolites may be due to expansions in the vomeronasal and taste receptors. We characterized novel lactation proteins that protect young in the pouch and annotated immune genes important for response to chlamydial disease. Historical demography showed a substantial population crash coincident with the decline of Australian megafauna, while contemporary populations had biogeographic boundaries and increased inbreeding in populations affected by historic translocations. We identified genetically diverse populations that require habitat corridors and instituting of translocation programs to aid the koala’s survival in the wild., コアラの全ゲノム配列の解読に成功 --コアラはなぜ猛毒のユーカリを食べるのか？--. 京都大学プレスリリース. 2018-07-04.

Bandicoots are omnivorous marsupials of the order Peramelemorphia. Conservation concerns and their unique biological characteristics suggest peramelomorphs are worthy research subjects, but knowledge of their genetics and immunology has lagged behind that of other high-profile marsupials. Here, we characterise the transcriptome of the long-nose bandicoot (Perameles nasuta), the first high-throughput data set from any peramelomorph. We investigate the immune gene repertoire of the bandicoot, with a focus on key immune gene families, and compare to previously characterised marsupial and mammalian species. We find that the immune gene complement in bandicoot is often conserved with respect to other marsupials; however, the diversity of expressed transcripts in several key families, such as major histocompatibility complex, T cell receptor μ and natural killer cell receptors, appears greater in the bandicoot than other Australian marsupials, including devil and koala. This transcriptome is an important first step for future studies of bandicoots and the bilby, allowing for population level analysis and construction of bandicoot-specific immunological reagents and assays. Such studies will be critical to understanding the immunology and physiology of Peramelemorphia and to inform the conservation of these unique marsupials.

Background The cane toad (Rhinella marina formerly Bufo marinus) is a species native to Central and South America that has spread across many regions of the globe. Cane toads are known for their rapid adaptation and deleterious impacts on native fauna in invaded regions. However, despite an iconic status, there are major gaps in our understanding of cane toad genetics. The availability of a genome would help to close these gaps and accelerate cane toad research. Findings We report a draft genome assembly for R. marina, the first of its kind for the Bufonidae family. We used a combination of long-read Pacific Biosciences RS II and short-read Illumina HiSeq X sequencing to generate 359.5 Gb of raw sequence data. The final hybrid assembly of 31,392 scaffolds was 2.55 Gb in length with a scaffold N50 of 168 kb. BUSCO analysis revealed that the assembly included full length or partial fragments of 90.6% of tetrapod universal single-copy orthologs (n = 3950), illustrating that the gene-containing regions have been well assembled. Annotation predicted 25,846 protein coding genes with similarity to known proteins in Swiss-Prot. Repeat sequences were estimated to account for 63.9% of the assembly. Conclusions The R. marina draft genome assembly will be an invaluable resource that can be used to further probe the biology of this invasive species. Future analysis of the genome will provide insights into cane toad evolution and enrich our understanding of their interplay with the ecosystem at large.

Temporal dynamics of gene expression are informative of changes associated with disease development and evolution. Given the complexity of high-dimensional temporal datasets, an analytical framework guided by a robust theory is needed to interpret time-sequential changes and to predict system dynamics. Herein, we use acute myeloid leukemia as a proof-of-principle to model gene expression dynamics in a transcriptome state-space constructed based on time-sequential RNA-sequencing data. We describe the construction of a state-transition model to identify state-transition critical points which accurately predicts leukemia development. We show an analytical approach based on state-transition critical points identified step-wise transcriptomic perturbations driving leukemia progression. Furthermore, the gene(s) trajectory and geometry of the transcriptome state-space provides biologically-relevant gene expression signals that are not synchronized in time, and allows quantification of gene(s) contribution to leukemia development. Therefore, our state-transition model can synthesize information, identify critical points to guide interpretation of transcriptome trajectories and predict disease development.Graphical AbstractIn briefThe theory of state-transition is applied to acute myeloid leukemia (AML) to model transcriptome dynamics and trajectories in a state-space, and is used to identify critical points corresponding to critical transcriptomic perturbations that predict leukemia development.HighlightsLeukemia transcriptome dynamics are modeled as movement in transcriptome state-spaceState-transition model and critical points accurately predicts leukemia developmentCritical point-based approach identifies step-wise transcriptome events in leukemiaState-based geometric analysis provides quantification of leukemogenic contribution

The sex chromosomes in Sauropsida (reptiles and birds) have evolved independently many times. They show astonishing diversity in morphology ranging from cryptic to highly differentiated sex chromosomes with male (XX/XY) and female heterogamety (ZZ/ZW). Comparing such diverse sex chromosome systems thus provides unparalleled opportunities to capture evolution of morphologically differentiated sex chromosomes in action. Here, we describe chromosomal mapping of 18 microsatellite repeat motifs in eight species of Sauropsida. More than two microsatellite repeat motifs were amplified on the sex-specific chromosome, W or Y, in five species (Bassiana duperreyi, Aprasia parapulchella, Notechis scutatus, Chelodina longicollis, and Gallus gallus) of which the sex-specific chromosomes were heteromorphic and heterochromatic. Motifs (AAGG)n and (ATCC)n were amplified on the W chromosome of Pogona vitticeps and the Y chromosome of Emydura macquarii, respectively. By contrast, no motifs were amplified on the W chromosome of Christinus marmoratus, which is not much differentiated from the Z chromosome. Taken together with previously published studies, our results suggest that the amplification of microsatellite repeats is tightly associated with the differentiation and heterochromatinization of sex-specific chromosomes in sauropsids as well as in other taxa. Although some motifs were common between the sex-specific chromosomes of multiple species, no correlation was observed between this commonality and the species phylogeny. Furthermore, comparative analysis of sex chromosome homology and chromosomal distribution of microsatellite repeats between two closely related chelid turtles, C. longicollis and E. macquarii, identified different ancestry and differentiation history. These suggest multiple evolutions of sex chromosomes in the Sauropsida.

Major histocompatibility complex (MHC) genes are a central component of the vertebrate immune system and usually exist in a single genomic region. However, considerable differences in MHC organization and size exist between different vertebrate lineages. Reptiles occupy a key evolutionary position for understanding how variation in MHC structure evolved in vertebrates, but information on the structure of the MHC region in reptiles is limited. In this study, we investigate the organization and cytogenetic location of MHC genes in the tuatara (Sphenodon punctatus), the sole extant representative of the early-diverging reptilian order Rhynchocephalia. Sequencing and mapping of 12 clones containing class I and II MHC genes from a bacterial artificial chromosome library indicated that the core MHC region is located on chromosome 13q. However, duplication and translocation of MHC genes outside of the core region was evident, because additional class I MHC genes were located on chromosome 4p. We found a total of seven class I sequences and 11 class II β sequences, with evidence for duplication and pseudogenization of genes within the tuatara lineage. The tuatara MHC is characterized by high repeat content and low gene density compared with other species and we found no antigen processing or MHC framework genes on the MHC gene-containing clones. Our findings indicate substantial differences in MHC organization in tuatara compared with mammalian and avian MHCs and highlight the dynamic nature of the MHC. Further sequencing and annotation of tuatara and other reptile MHCs will determine if the tuatara MHC is representative of nonavian reptiles in general.

The first report of reptile sex reversal in the wild and rapid transition between genetic and environmental sex determination in the Australian bearded dragon (Pogona vitticeps) There have been repeated evolutionary transitions in reptiles between genetic and temperature-dependent sex determination, the regulatory process that initiates differentiation of the gonads in the early embryo to form either testes or ovaries. Various mechanisms have been proposed to explain the transition, including a role for sex reversal. Clare Holleley et al. present the first report of reptile sex reversal in the wild, associated with rapid transition between genetic and environmental sex determination. In a study of the Australian bearded dragon (Pogona vitticeps), they observe sex reversal at the warmer end of the animals geographic range. When sex-reversed females mate with normal males, the chromosomal sex determination system is lost and temperature-dependent sex determination is established. It is not known whether climate-induced changes in sex determination are advantageous or detrimental to the process of evolutionary adaptation. Sex determination in animals is amazingly plastic. Vertebrates display contrasting strategies ranging from complete genetic control of sex (genotypic sex determination) to environmentally determined sex (for example, temperature-dependent sex determination)1. Phylogenetic analyses suggest frequent evolutionary transitions between genotypic and temperature-dependent sex determination in environmentally sensitive lineages, including reptiles2. These transitions are thought to involve a genotypic system becoming sensitive to temperature, with sex determined by gene–environment interactions3. Most mechanistic models of transitions invoke a role for sex reversal3,4,5. Sex reversal has not yet been demonstrated in nature for any amniote, although it occurs in fish6 and rarely in amphibians7,8. Here we make the first report of reptile sex reversal in the wild, in the Australian bearded dragon (Pogona vitticeps), and use sex-reversed animals to experimentally induce a rapid transition from genotypic to temperature-dependent sex determination. Controlled mating of normal males to sex-reversed females produces viable and fertile offspring whose phenotypic sex is determined solely by temperature (temperature-dependent sex determination). The W sex chromosome is eliminated from this lineage in the first generation. The instantaneous creation of a lineage of ZZ temperature-sensitive animals reveals a novel, climate-induced pathway for the rapid transition between genetic and temperature-dependent sex determination, and adds to concern about adaptation to rapid global climate change.

Toxoplasma gondii and Neospora caninum (both Apicomplexa) are closely related cyst-forming coccidian parasites that differ significantly in their host ranges and ability to cause disease. Unlike eutherian mammals, Australian marsupials (metatherian mammals) have long been thought to be highly susceptible to toxoplasmosis and neosporosis because of their historical isolation from the parasites. In this study, the carnivorous fat-tailed dunnart ( Sminthopsis crassicaudata ) was used as a disease model to investigate the immune response and susceptibility to infection of an Australian marsupial to T. gondii and N. caninum . The disease outcome was more severe in N. caninum -infected dunnarts than in T. gondii -infected dunnarts, as shown by the severity of clinical and histopathological features of disease and higher tissue parasite burdens in the tissues evaluated. Transcriptome sequencing (RNA-seq) of spleens from infected dunnarts and mitogen-stimulated dunnart splenocytes was used to define the cytokine repertoires. Changes in mRNA expression during the time course of infection were measured using quantitative reverse transcription-PCR (qRT-PCR) for key Th1 (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]), Th2 (interleukin 4 [IL-4] and IL-6), and Th17 (IL-17A) cytokines. The results show qualitative differences in cytokine responses by the fat-tailed dunnart to infection with N. caninum and T. gondii . Dunnarts infected with T. gondii were capable of mounting a more effective Th1 immune response than those infected with N. caninum , indicating the role of the immune response in the outcome scenarios of parasite infection in this marsupial mammal.

Koala (Phascolarctos cinereus) populations are on the decline across the majority of Australia's mainland. Two major diseases threatening the long-term survival of affected koala populations are caused by obligate intracellular pathogens: Chlamydia and koala retrovirus (KoRV). To improve our understanding of the koala immune system, we characterised their major histocompatibility complex (MHC) class I genes, which are centrally involved in presenting foreign peptides derived from intracellular pathogens to cytotoxic T cells. A total of 11 class I genes were identified in the koala genome. Three genes, Phci-UA, UB and UC, showed relatively high genetic variability and were expressed in all 12 examined tissues, whereas the other eight genes had tissue-specific expression and limited polymorphism. Evidence of diversifying selection was detected in Phci-UA and UC, while gene conversion may have played a role in creating new alleles at Phci-UB. We propose that Phci-UA, UB and UC are likely classical MHC genes of koalas, and further research is needed to understand their role in koala chlamydial and KoRV infections.

Highlights • Pentastomids infected 59% of snakes surveyed in the Australian tropics. • Pentastomids were of the genera Raillietiella and Waddycephalus. • Raillietiella orientalis has invaded Australia via an unknown pathway. • Five species of Waddycephalus were present in the Australian tropics. • Morphological features are unreliable for distinguishing Waddycephalus species., Pentastomids are endoparasites of the respiratory system of vertebrates, maturing primarily in carnivorous reptiles. Adult and larval pentastomids can cause severe pathology resulting in the death of their intermediate and definitive hosts. The study of pentastomids is a neglected field, impaired by risk of zoonoses, difficulties in species identification, and life cycle complexities. We surveyed wild snakes in the tropics of Australia to clarify which host species possess these parasites, and then sought to identify these pentastomids using a combination of morphological and molecular techniques. We detected pentastomid infections in 59% of the 81 snakes surveyed. The ubiquity of pentastomid infections in snakes of the Australian tropics sampled in this study is alarmingly high considering the often-adverse consequences of infection and the recognized zoonotic potential of these parasites. The pentastomids were of the genera Raillietiella and Waddycephalus and infected a range of host taxa, encompassing seven snake species from three snake families. All seven snake species represent new host records for pentastomids of the genera Raillietiella and/or Waddycephalus. The arboreal colubrid Dendrelaphis punctulatus and the terrestrial elapid Demansia vestigiata had particularly high infection prevalences (79% and 100% infected, respectively). Raillietiella orientalis infected 38% of the snakes surveyed, especially frog-eating species, implying a frog intermediate host for this parasite. Raillietiella orientalis was previously known only from Asian snakes and has invaded Australia via an unknown pathway. Our molecular data indicated that five species of Waddycephalus infect 28% of snakes in the surveyed area. Our morphological data indicate that features of pentastomid anatomy previously utilised to identify species of the genus Waddycephalus are unreliable for distinguishing species, highlighting the need for additional taxonomic work on this genus.

Reptiles, as the sister group to birds and mammals, are particularly valuable for comparative genomic studies among amniotes. The Australian central bearded dragon (Pogona vitticeps) is being developed as a reptilian model for such comparisons, with whole-genome sequencing near completion. The karyotype consists of 6 pairs of macrochromosomes and 10 pairs microchromosomes (2n = 32), including a female heterogametic ZW sex microchromosome pair. Here, we present a molecular cytogenetic map for P. vitticeps comprising 87 anchor bacterial artificial chromosome clones that together span each macro- and microchromosome. It is the first comprehensive cytogenetic map for any non-avian reptile. We identified an active nucleolus organizer region (NOR) on the sub-telomeric region of 2q by mapping 18S rDNA and Ag-NOR staining. We identified interstitial telomeric sequences in two microchromosome pairs and the W chromosome, indicating that microchromosome fusion has been a mechanism of karyotypic evolution in Australian agamids within the last 21 to 19 million years. Orthology searches against the chicken genome revealed an intrachromosomal rearrangement of P. vitticeps 1q, identified regions orthologous to chicken Z on P. vitticeps 2q, snake Z on P. vitticeps 6q and the autosomal microchromosome pair in P. vitticeps orthologous to turtle Pelodiscus sinensis ZW and lizard Anolis carolinensis XY. This cytogenetic map will be a valuable reference tool for future gene mapping studies and will provide the framework for the work currently underway to physically anchor genome sequences to chromosomes for this model Australian squamate.

Production of milk is a key characteristic of mammals, but the features of lactation vary greatly between monotreme, marsupial and eutherian mammals. Marsupials have a short gestation followed by a long lactation period, and milk constituents vary greatly across lactation. Marsupials are born immunologically naïve and rely on their mother’s milk for immunological protection. Koalas (Phascolarctos cinereus) are an iconic Australian species that are increasingly threatened by disease. Here we use a mammary transcriptome, two milk proteomes and the koala genome to comprehensively characterise the protein components of koala milk across lactation, with a focus on immune constituents. The most abundant proteins were well-characterised milk proteins, including β-lactoglobulin and lactotransferrin. In the mammary transcriptome, 851 immune transcripts were expressed, including immunoglobulins and complement components. We identified many abundant antimicrobial peptides, as well as novel proteins with potential antimicrobial roles. We discovered that marsupial VELP is an ortholog of eutherian Glycam1, and likely has an antimicrobial function in milk. We also identified highly-abundant koala endogenous-retrovirus sequences, identifying a potential transmission route from mother to young. Characterising the immune components of milk is key to understanding protection of marsupial young, and the novel immune compounds identified may have applications in clinical research.

Tasmanian devil (Sarcophilus harrisii) pouch young, like other marsupials, are born underdeveloped and immunologically naïve, and are unable to mount an adaptive immune response. The mother’s milk provides nutrients for growth and development as well as providing passive immunity. To better understand immune response in this endangered species, we set out to characterise the genes involved in passive immunity by sequencing and annotating the transcriptome of a devil milk sample collected during mid-lactation. At mid-lactation we expect the young to have heightened immune responses, as they have emerged from the pouch, encountering new pathogens. A total of 233,660 transcripts were identified, including approximately 17,827 unique protein-coding genes and 846 immune genes. The most highly expressed transcripts were dominated by milk protein genes such as those encoding early lactation protein, late lactation proteins,α-lactalbumin,α-casein andβ-casein. There were numerous highly expressed immune genes including lysozyme, whey acidic protein, ferritin and major histocompatibility complex I and II. Genes encoding immunoglobulins, antimicrobial peptides, chemokines and immune cell receptors were also identified. The array of immune genes identified in this study reflects the importance of the milk in providing immune protection to Tasmanian devil young and provides the first insight into Tasmanian devil milk.

Several recent studies have produced comparative maps of genes on amniote sex chromosomes, revealing homology of gene content and arrangement across lineages as divergent as mammals and lizards. For example, the chicken Z chromosome, which shares homology with the sex chromosomes of all birds, monotremes, and a gecko, is a striking example of stability of genome organization and retention, or independent acquisition, of function in sex determination. In other lineages, such as snakes and therian mammals, well conserved but independently evolved sex chromosome systems have arisen. Among lizards, novel sex chromosomes appear frequently, even in congeneric species. Here, we review recent gene mapping data, examine the evolutionary relationships of amniote sex chromosomes and argue that gene content can predispose some chromosomes to a specialized role in sex determination.

We report here the isolation and sequencing of 10 Y-specific tammar wallaby (Macropus eugenii) BAC clones, revealing five hitherto undescribed tammar wallaby Y genes (in addition to the five genes already described) and several pseudogenes. Some genes on the wallaby Y display testis-specific expression, but most have low widespread expression. All have partners on the tammar X, along with homologs on the human X. Nonsynonymous and synonymous substitution ratios for nine of the tammar XY gene pairs indicate that they are each under purifying selection. All 10 were also identified as being on the Y in Tasmanian devil (Sarcophilus harrisii; a distantly related Australian marsupial); however, seven have been lost from the human Y. Maximum likelihood phylogenetic analyses of the wallaby YX genes, with respective homologs from other vertebrate representatives, revealed that three marsupial Y genes (HCFC1X/Y, MECP2X/Y, and HUWE1X/Y) were members of the ancestral therian pseudoautosomal region (PAR) at the time of the marsupial/eutherian split; three XY pairs (SOX3/SRY, RBMX/Y, and ATRX/Y) were isolated from each other before the marsupial/eutherian split, and the remaining three (RPL10X/Y, PHF6X/Y, and UBA1/UBE1Y) have a more complex evolutionary history. Thus, the small marsupial Y chromosome is surprisingly rich in ancient genes that are retained in at least Australian marsupials and evolved from testis–brain expressed genes on the X.