"Nothing in biology makes sense except in the light of evolution"

Theodosius Dobzhansky, 1973


"A hen is only an egg's way of making another egg"

Samuel Butler, 1878


Our long-term objective is to understand the genetic differences between males and females, both within and beyond the reproductive tract, and ultimately the biological and medical ramifications of these differences. We aim to understand these differences and ramifications in broad context, through comparative biological, evolutionary, developmental, and clinically focused analyses. The questions we are currently pursuing fall into two broad categories:  


1. Sex chromosomes and sex differences

     What dynamics shaped the structure and gene content of mammalian and avian sex chromosomes?

     What are the implications for male and female biology?


2. Germ cell origins and development

     How is meiosis initiated and regulated?

     Does germ cell chromatin state contribute to the totipotency of the zygote?






Sex chromosomes and sex differences


In many animals, males and females differ by one chromosome. In mammals, females are XX and males are XY. In birds, which are the closest living relatives of mammals, females are ZW and males are ZZ. The last common ancestor of mammals and birds lived about 320 million years ago. We and other investigators around the world established that the mammalian X and Y chromosomes had evolved, over the last 200-300 million years, from ordinary autosomes that persist as autosomes in modern birds; in parallel, the avian Z and W chromosomes evolved from different autosomes that remain autosomes in mammals. The present-day Y and W chromosomes retain only a small fraction of the ancestral genes that they once shared with the X and Z, respectively.


As we have discovered, mammalian and avian sex chromosomes exhibit extraordinarily high levels of intrachromosomal sequence identity -- so high that they cannot be accurately and fully sequenced using conventional approaches. To understand the structural intricacies of sex chromosomes, and explore their biology, our multi-center team (the Page Lab at Whitehead Institute and the genome sequencing centers at Washington University and Baylor) has pioneered and refined a super-resolution sequencing method that is known technically as SHIMS, or Single-Haplotype Iterative Mapping and Sequencing. We began developing this method about 18 years ago, and our ongoing development of super-resolution sequencing has led to new and unforeseen biological insights into sex chromosomes:


   The myth of the disappearing Y. Our comparison of super-resolution assemblies of the human and rhesus macaque Y chromosomes demonstrated that they carried nearly identical sets of surviving ancestral genes (i.e. genes derived from the ancestral autosomes that gave rise to the mammalian X and Y), despite 25 million years of genetic separation (Hughes et al. 2012). We concluded that Y-chromosome gene loss had essentially ceased at least 25 million years ago, overturning long-standing views that the Y chromosomes is headed for extinction. An interview with Stephen Colbert helped spread the news of our findings.


   A new appreciation of the Y chromosome's organismal roles. We broadened our comparison of super-resolution Y assemblies to include eight mammals: human, chimpanzee, rhesus, marmoset, mouse, rat, bull, and opossum (Bellott et al. 2014). We found that mammalian Y chromosomes had preferentially retained a small subset of ancestral genes that are broadly expressed across the body, are dosage-sensitive, and encode proteins involved in chromatin modification, transcription, splicing, translation, and protein stability. Many of these surviving ancestral genes have broadly expressed X-linked counterparts that encode related but non-identical proteins. We surmised that, in most if not all tissues and organs, XX and XY cells differ biochemically as a direct consequence of genetic differences between X and Y chromosomes that arose during their differentiation from ordinary autosomes. This may contribute to the well-documented phenotypic differences that exist between human males and females, in health and disease, throughout the body.


To watch David Page's TEDx talk "Why Sex Really Matters" click here.


   Selfish chromosomes at war: X-Y arms races. For decades, the X chromosome was deemed the most evolutionarily conservative of mammalian chromosomes. During the past two years, super-resolution projects in our lab on human X and mouse Y have unexpectedly converged, leading us to recast the mammalian X as an evolutionary dynamo, and perhaps even as a selfish adversary of the Y. Specifically, we reported that the human and mouse X chromosomes are rife with independently acquired and amplified gene families, expressed in testicular germ cells (Mueller et al. 2013). These findings took on new meaning when we discovered, through super-resolution sequencing of the mouse Y chromosome, that the mouse Y had independently acquired, and massively amplified, genes homologous to several of the mouse X-amplified, testis-expressed gene families (Soh et al. 2014). We interpreted these co-amplifications on mouse X and Y as evidence of an arms race, and of sex-linked meiotic drive -- a selfishly motivated battle between X and Y for transmission to the next generation.





Triangular dot plot of DNA sequence identity within the 90-megabase mouse Y chromosome. Each dot represents 100% intrachromosomal identity within a 200-bp window. Direct repeats appear as horizontal lines, inverted repeats as vertical lines. Below plot, schematic representation of chromosome is shown.



Germ cell origins and development


Germ cells, which give rise to differentiated gametes (sperm in males and eggs in females), are the most fundamental expression of sex differences. The essential function of germ cells is to carry the genome from parent to offspring, thereby providing a continuous link between generations. In order to perform this function, germ cells must complete a complex developmental program, which includes maintaining the diploid genome throughout embryogenesis, halving the genome during meiosis, and preparing the haploid genome for fertilization. Through this process, germ cells undergo extensive cellular differentiation and specialization, but they are still capable of generating a totipotent embryo at fertilization. Our current work focuses on two areas of germ cell biology:


   Meiotic initiation and regulation. We use the mouse as an experimental model to genetically dissect the process of meiotic initiation, which is a critical juncture in mammalian development, both female and male. Over the past decade, our work identified Dazl as a competence factor for meiotic initiation (Lin et al. 2008), retinoic acid (RA) as an extrinsic meiosis inducer (Koubova et al. 2006), and Stra8 as an RA-induced gatekeeper for premeiotic replication and meiotic prophase (Baltus et al. 2006). These key findings have led to a spate of recent discoveries, including 1) RA activates not one but two molecular pathways required for meiosis in ovarian germ cells (Koubova et al. 2014); 2) In females, oocyte-like differentiation is genetically dissociable from meiosis (Dokshin et al. 2013); and 3) In males, RA-Stra8 signaling governs germ cell differentiation at two points -- both at meiotic initiation and when cells exit the stem cell pool (Endo et al. 2015). We have also recently characterized a larger gene regulatory network regulating meiotic prophase through a systematic genome-wide study (Soh et al. 2015).


   The role of poised chromatin in germ cells. Poised chromatin, which bears histone modifications associated with both transcriptional activation (H3K4me3) and repression (H3K27me3), was originally described in cultured embryonic stem cells (ESCs) where this epigenetic feature is prominent at the promoters of developmental regulatory genes. We recently put the role of poised chromatin in an in vivo context. We showed that key regulators of somatic development, including genes involved in specification of all three germ layers and extraembryonic tissue, are maintained in a poised state throughout germ cell differentiation in mouse (Lesch et al. 2013). None of these germline-poised developmental regulatory genes are involved in germ cell development or gametogenesis, but the majority are expressed in somatic lineages during embryogenesis, suggesting that poising may prepare somatic developmental genes for regulated activation upon fertilization. We hypothesize that maintenance of the poised chromatin state through germ cell development to fertilization is essential for promoting the critical transition from differentiated gamete to totipotent zygote, and that disruption of this process leads to infertility and congenital disorders.




Mouse embryonic testis section, showing organization of testicular tubules. Germ cells that will ultimately become sperm are shown in red; organizer cells, or Sertoli cells, are shown in blue; interstitial cells, or Leydig cells, which are adjacent to tubules, are shown in green.


To watch David Page's HHMI lectures on sex determination click here.