Biology subjects

Page, D. C.

Publications and source records attributed to Page, D. C..

4 recordsLinked to original sources

bioRxiv2018

Isolating mitotic and meiotic germ cells from male mice by developmental synchronization, staging, and sorting

Isolating discrete populations of germ cells from the mouse testis is challenging, because the adult testis contains germ cells at every step of spermatogenesis, in addition to somatic cells. We present a novel method for isolating precise, high-purity populations of male germ cells. We first synchronize germ cell development in vivo by manipulating retinoic acid metabolism, and perform histological staging to verify synchronization. We use fluorescence-activated cell sorting to separate the synchronized differentiating germ cells from contaminating somatic and germline stem cells. We achieve [~]90% purity at each step of development from the germline stem cell pool through late meiotic prophase. Utilizing this \"3S\" method (synchronize, stage, and sort), we can separate germ cell types that were previously challenging or impossible to distinguish, with sufficient yield for epigenetic and biochemical studies. The 3S method should enable detailed characterization of molecular changes that occur during the mitotic and meiotic phases of spermatogenesis.

developmental biology↗

bioRxiv2017

Cost-effective, high-throughput, single-haplotype iterative mapping and sequencing for complex genomic structures

Reference sequence of structurally complex regions can only be obtained through highly accurate clone-based approaches. We and others have successfully employed Single-Haplotype Iterative Mapping and Sequencing (SHIMS 1.0) to assemble structurally complex regions across the sex chromosomes of several vertebrate species and in targeted improvements to the reference sequences of human autosomes. However, SHIMS 1.0 was expensive and time consuming, requiring the resources that only a genome center could command. Here we introduce SHIMS 2.0, an improved SHIMS protocol to allow even a small laboratory to generate high-quality reference sequence from complex genomic regions. Using a streamlined and parallelized library preparation protocol, and taking advantage of high-throughput, inexpensive, short-read sequencing technologies, a small group can sequence and assemble hundreds of clones in a week. Relative to SHIMS 1.0, SHIMS 2.0 reduces the cost and time required by two orders of magnitude, while preserving high sequencing accuracy.

genomics↗

bioRxiv2017

Preexisting heterogeneities in gene dosage sensitivity shaped sex chromosome evolution in mammals and birds

Mammalian X and Y chromosomes evolved from an ordinary autosomal pair; genetic decay decimated the Y, which in turn necessitated X chromosome inactivation (XCI). Genes of the ancestral autosomes are often assumed to have undertaken these transitions on uniform terms, but we hypothesized that they varied in their dosage constraints. We inferred such constraints from conservation of microRNA (miRNA)-mediated repression, validated by analysis of experimental data. X-linked genes with a surviving Y homolog have the most conserved miRNA target sites, followed by genes with no Y homolog and subject to XCI, and then genes with no Y homolog but escaping XCI; this heterogeneity existed on the ancestral autosomes. Similar results for avian Z-linked genes, with or without a W homolog, lead to a model of XY/ZW evolution incorporating preexisting dosage sensitivities of individual genes in determining their evolutionary fates, and ultimately shaping the mammalian and avian sex chromosomes.

genetics↗

bioRxiv2017

Preexisting heterogeneities in gene dosage sensitivity shaped sex chromosome evolution in mammals and birds

Mammalian X and Y chromosomes evolved from an ordinary autosomal pair. Genetic decay of the Y led to X chromosome inactivation (XCI) in females, but some Y-linked genes were retained during the course of sex chromosome evolution, and many X-linked genes did not become subject to XCI. We reconstructed gene-by-gene dosage sensitivities on the ancestral autosomes through phylogenetic analysis of microRNA (miRNA) target sites and compared these preexisting characteristics to the current status of Y-linked and X-linked genes in mammals. Preexisting heterogeneities in dosage sensitivity, manifesting as differences in the extent of miRNA-mediated repression, predicted either the retention of a Y homolog or the acquisition of XCI following Y gene decay. Analogous heterogeneities among avian Z-linked genes predicted either the retention of a W homolog or gene-specific dosage compensation following W gene decay. Genome-wide analyses of human copy number variation indicate that these heterogeneities consisted of sensitivity to both increases and decreases in dosage. We propose a model of XY/ZW evolution incorporating such preexisting dosage sensitivities in determining the evolutionary fates of individual genes. Our findings thus provide a more complete view of the role of dosage sensitivity in shaping the mammalian and avian sex chromosomes, and reveal an important role for post-transcriptional regulatory sequences (miRNA target sites) in sex chromosome evolution.

genetics↗