Douglas B. Menke

In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premeiotic DNA replication

The transition from mitosis to meiosis is a defining juncture in the life cycle of sexually reproducing organisms. In yeast, the decision to enter meiosis is made before the single round of DNA replication that precedes the two meiotic divisions. We present genetic evidence of an analogous decision point in the germ line of a multicellular organism. The mouse Stra8 gene is expressed in germ cells of embryonic ovaries, where meiosis is initiated, but not in those of embryonic testes, where meiosis does not begin until after birth. Here we report that in female embryos lacking Stra8 gene function, the early, mitotic development of germ cells is normal, but these cells then fail to undergo premeiotic DNA replication, meiotic chromosome condensation, cohesion, synapsis and recombination. Combined with previous findings, these genetic data suggest that active differentiation of ovarian germ cells commences at a regulatory point upstream of premeiotic DNA replication.

Follistatin operates downstream of Wnt4 in mammalian ovary organogenesis.

Wnt4‐/‐ XX gonads display features normally associated with testis differentiation, suggesting that WNT4 actively represses elements of the male pathway during ovarian development. Here, we show that follistatin (Fst), which encodes a TGFβ superfamily binding protein, is a downstream component of Wnt4 signaling. Fst inhibits formation of the XY‐specific coelomic vessel in XX gonads. In addition, germ cells in the ovarian cortex are almost completely lost in both Wnt4 and Fst null gonads before birth. Thus, we propose that WNT4 acts through FST to regulate vascular boundaries and maintain germ cell survival in the ovary. Developmental Dynamics 230:210–215, 2004.

Sexual differentiation of germ cells in XX mouse gonads occurs in an anterior-to-posterior wave.

Differentiation of mouse embryonic germ cells as male or female is dependent on the somatic environment of the gonad rather than the sex chromosome constitution of the germ cell. However, little is known about the initiation of germ cell sexual differentiation. Here, we traced the initiation of germ cell sexual differentiation in XX gonads using the Stra8 gene, which we demonstrate is an early molecular marker of female germ cell development. Stra8 is upregulated in embryonic germ cells of XX gonads prior to meiotic entry and is not expressed in male embryonic germ cells. A developmental time course of Stra8 expression in germ cells of XX gonads has revealed an anterior-to-posterior wave of differentiation that lasts approximately 4 days, from embryonic days 12.5 to 16.5. Consistent with these results, we find that embryonic ovarian germ cells upregulate the meiotic gene Dmc1 and downregulate the Oct4 transcription factor in an anterior-to-posterior wave. In complementary experiments, we find that embryonic XX gonads upregulate certain gene markers of somatic female differentiation in an anterior-to-posterior pattern, while others display a center-to-pole pattern of regulation. Thus, sexual differentiation and meiotic entry of germ cells in embryonic XX gonads progress in an anterior-to-posterior pattern that may reflect local environmental cues that are present in the embryonic XX gonad.

Human-specific loss of regulatory DNA and the evolution of human-specific traits

Humans differ from other animals in many aspects of anatomy, physiology, and behaviour; however, the genotypic basis of most human-specific traits remains unknown. Recent whole-genome comparisons have made it possible to identify genes with elevated rates of amino acid change or divergent expression in humans, and non-coding sequences with accelerated base pair changes. Regulatory alterations may be particularly likely to produce phenotypic effects while preserving viability, and are known to underlie interesting evolutionary differences in other species. Here we identify molecular events particularly likely to produce significant regulatory changes in humans: complete deletion of sequences otherwise highly conserved between chimpanzees and other mammals. We confirm 510 such deletions in humans, which fall almost exclusively in non-coding regions and are enriched near genes involved in steroid hormone signalling and neural function. One deletion removes a sensory vibrissae and penile spine enhancer from the human androgen receptor (AR) gene, a molecular change correlated with anatomical loss of androgen-dependent sensory vibrissae and penile spines in the human lineage. Another deletion removes a forebrain subventricular zone enhancer near the tumour suppressor gene growth arrest and DNA-damage-inducible, gamma (GADD45G), a loss correlated with expansion of specific brain regions in humans. Deletions of tissue-specific enhancers may thus accompany both loss and gain traits in the human lineage, and provide specific examples of the kinds of regulatory alterations and inactivation events long proposed to have an important role in human evolutionary divergence.

Sexually dimorphic gene expression in the developing mouse gonad.

Over the course of a few days, the bipotential embryonic mouse gonad differentiates into either a testis or an ovary. Though a few gene expression differences that underlie gonadal sex differentiation have been identified, additional components of the testicular and ovarian developmental pathways must be identified to understand this process. Here we report the use of a PCR-based cDNA subtraction to investigate expression differences that arise during gonadal sex differentiation. Subtraction of embryonic day 12.5 (E12.5) XY gonadal cDNA with E12.5 XX gonadal cDNA yielded 19 genes that are expressed at significantly higher levels in XY gonads. These genes display a variety of expression patterns within the embryonic testis and encode a broad range of proteins. A reciprocal subtraction (of E12.5 XX gonadal cDNA with E12.5 XY gonadal cDNA) yielded two genes, follistatin and Adamts19, that are expressed at higher levels in XX gonads. Follistatin is a well-known antagonist of TGFbeta family members while Adamts19 encodes a new member of the ADAMTS family of secreted metalloproteases.

Dual hindlimb control elements in the Tbx4 gene and region-specific control of bone size in vertebrate limbs.

The Tbx4 transcription factor is crucial for normal hindlimb and vascular development, yet little is known about how its highly conserved expression patterns are generated. We have used comparative genomics and functional scanning in transgenic mice to identify a dispersed group of enhancers controlling Tbx4 expression in different tissues. Two independent enhancers control hindlimb expression, one located upstream and one downstream of the Tbx4 coding exons. These two enhancers, hindlimb enhancer A and hindlimb enhancer B (HLEA and HLEB), differ in their primary sequence, in their precise patterns of activity within the hindlimb, and in their degree of sequence conservation across animals. HLEB is highly conserved from fish to mammals. Although Tbx4 expression and hindlimb development occur at different axial levels in fish and mammals, HLEB cloned from either fish or mouse is capable of driving expression at the appropriate position of hindlimb development in mouse embryos. HLEA is highly conserved only in mammals. Deletion of HLEA from the endogenous mouse locus reduces expression of Tbx4 in the hindlimb during embryogenesis, bypasses the embryonic lethality of Tbx4-null mutations, and produces viable, fertile mice with characteristic changes in the size of bones in the hindlimb but not the forelimb. We speculate that dual hindlimb enhancers provide a flexible genomic mechanism for altering the strength and location of Tbx4 expression during normal development, making it possible to separately modify the size of forelimb and hindlimb bones during vertebrate evolution.

Defining a mesenchymal progenitor niche at single-cell resolution

INTRODUCTION In most vertebrate organs, epithelial tubes or sacs are surrounded by support and stromal tissues—including smooth muscle, cartilage, pericytes, fibroblasts, and mesothelium—that form during development from a loose collection of undifferentiated progenitor cells called mesenchyme. Although the behavior and regulation of epithelial progenitors and their niches have begun to be elucidated, much less is known about the identity and behavior of the progenitors of support and stromal tissues. This is critical not only because of the key cell types that they form but also because support and stromal cells can signal to epithelial stem cells and tumors and contribute to other serious diseases such as fibrosis and asthma. Mesenchyme cells are generally thought to represent highly proliferative, migratory, and multipotent cells that condense around epithelia to generate support and stromal cell types and do not form organized progenitor pools. Elucidating their behavior has been limited by the inability to track the fate of individual mesenchymal cells in development. Diverse mechanisms generate mesenchymal cell derivatives. (Top) Single mesenchyme cells are labeled early in lung development. The labeled cell (green) proliferates, and daughter cells disperse to seed progenitor “niches” that generate support and stromal cell types. (Bottom) Photomicrographs of individual clones and schematics of mesenchymal niches, highlighting the distinct modes of recruitment. Airway epithelium, white (schematic), blue (photomicrographs); mesenchyme, light gray (schematic); mesothelium, dark gray (schematic outline); smooth muscle, red (schematic); endothelium, blue (schematic). Scale bars, 10 μm. Diverse mechanisms generate mesenchymal cell derivatives. (Top) Single mesenchyme cells are labeled early in lung development. The labeled cell (green) proliferates, and daughter cells disperse to seed progenitor “niches” that generate support and stromal cell types. (Bottom) Photomicrographs of individual clones and schematics of mesenchymal niches, highlighting the distinct modes of recruitment. Airway epithelium, white (schematic), blue (photomicrographs); mesenchyme, light gray (schematic); mesothelium, dark gray (schematic outline); smooth muscle, red (schematic); endothelium, blue (schematic). Scale bars, 10 μm. RATIONALE We adapted clonal cell labeling strategies with multicolor reporters in mice to probe the behavior and potential of individual and sibling mesenchyme cells in lung development. This was used to define the proliferation, migration, and differentiation behavior of individual mesenchyme cells and to map the locations and behavior of mesenchymal progenitors at single-cell resolution. RESULTS We show that although mesenchymal cells are highly proliferative, as classical studies suggested, there is a surprising diversity of mesenchymal progenitor populations with different locations, patterns of migration, recruitment mechanisms, and lineage boundaries. We focus on airway smooth muscle progenitors, which map exclusively to the mesenchyme just ahead of budding and bifurcating airway branches. Progenitors are recruited from these tip pools to the branch stalk, where they differentiate into circumferentially oriented airway smooth muscle cells. There is a lineage boundary that prevents mesenchymal cells surrounding airway stalks from becoming airway smooth muscle from branch sides, but this stalk mesenchyme can be induced in the presence of a newly budded airway branch to generate a new smooth muscle progenitor pool dedicated to the new branch. Micrografting experiments show that the airway tip alone is not sufficient to induce a new smooth muscle progenitor pool in stalk mesenchyme. The missing mesenchymal signal can be provided by a focal Wnt signal, and delocalized Wnt pathway activity expands or alters the progenitor pool, causing ectopic smooth muscle formation on nearby endothelial cells. CONCLUSIONS Lung mesenchyme is neither a large homogeneous progenitor pool, as it has classically been viewed, nor a collection of discrete, isolated, and unchanging progenitor niches. Rather, the progenitors of each differentiated cell type occupy different locations and display varied modes of recruitment. The localized and carefully controlled domains of airway smooth muscle progenitors rival epithelial progenitor niches in regulatory sophistication. Most vertebrate organs are composed of epithelium surrounded by support and stromal tissues formed from mesenchyme cells, which are not generally thought to form organized progenitor pools. Here, we use clonal cell labeling with multicolor reporters to characterize individual mesenchymal progenitors in the developing mouse lung. We observe a diversity of mesenchymal progenitor populations with different locations, movements, and lineage boundaries. Airway smooth muscle (ASM) progenitors map exclusively to mesenchyme ahead of budding airways. Progenitors recruited from these tip pools differentiate into ASM around airway stalks; flanking stalk mesenchyme can be induced to form an ASM niche by a lateral bud or by an airway tip plus focal Wnt signal. Thus, mesenchymal progenitors can be organized into localized and carefully controlled domains that rival epithelial progenitor niches in regulatory sophistication. Clonal cell labeling with multicolor reporters reveals individual stem cells in the developing mouse lung. [Also see Perspective by Lee and Kim] How lung mesenchymal cells behave Despite the variety of organ systems, there is a common theme: Stromal tissues support and maintain most vertebrate organs. These stromal tissues form from mesenchymal stem cells. Kumar et al. used clonal cell labeling in mice to identify and characterize stromal progenitors in the developing mouse lung at single-cell resolution (see the Perspective by Lee and Kim). Progenitor populations occupied different locations and displayed a variety of movements and lineage boundaries. Airway smooth muscle progenitors are located just ahead of budding branches in the bronchial tree and are organized into carefully controlled domains. Science, this issue 10.1126/science.1258810; see also p. 810

Engineering subtle targeted mutations into the mouse genome.

Homologous recombination in embryonic stem (ES) cells offers an exquisitely precise mechanism to introduce targeted modifications to the mouse genome. This ability to produce specific alterations to the mouse genome has become an essential tool for the analysis of gene function and the development of mouse models of human disease. Of the many thousands of mouse alleles that have been generated by gene targeting, the majority are designed to completely ablate gene function, to create conditional alleles that are inactivated in the presence of Cre recombinase, or to produce reporter alleles that label‐specific tissues or cell populations (Eppig et al., 2012, Nucleic Acids Res 40:D881–D886). However, there is a variety of powerful motivations for the introduction of subtle targeted mutations (STMs) such as point mutations, small deletions, or small insertions into the mouse genome. The introduction of STMs allows the ablation of specific transcript isoforms, permits the functional investigation of particular domains or amino acids within a protein, provides the ability to study the role of specific sites with in cis‐regulatory elements, and can result in better mouse models of human genetic disorders. In this review, I examine the current strategies that are commonly used to introduce STMs into the mouse genome and highlight new gene targeting technologies, including TALENs and CRISPR/Cas, which are likely to influence the future of gene targeting in mice. genesis 51:605–618.

Comparison of the enhancement of tumor responses to fractionated irradiation by SR 4233 (Tirapazamine) and by nicotinamide with carbogen

PURPOSE This study was undertaken to compare in a fractionated regimen, with clinically relevant radiation doses, two radiation response modifiers that function by different mechanisms: SR 4233, a bioreductive agent toxic to hypoxic cells, and nicotinamide with carbogen, a combination that has been shown to improve tumor oxygenation. METHODS AND MATERIALS Cell survival assays were used to examine the response of three different tumors: KHT, RIF-1 and SCCVII/St in C3H/Km mice. Regrowth delay studies were also performed with the RIF-1 tumor. A fractionated irradiation schedule, consisting of twice daily 2.5 Gy treatments was investigated with and without drug pretreatment. SR 4233 was given IP at 0.12 mmol/kg one half hour before each irradiation. Nicotinamide (250, 500, 1000 mg/kg) was given IP 1 h before each irradiation with carbogen exposure 5 min prior to and during the irradiation. RESULTS Both treatment strategies enhanced the response of all three tumors to the fractionated radiation regimen. However, for two of the tumors (KHT and SCCVII), SR 4233 produced a significantly greater enhancement than did the combination of nicotinamide + carbogen. For the RIF-1 tumor (which has the lowest hypoxic fraction of the three), the response was comparable for the two modalities. For nicotinamide + carbogen, there was no significant change in the radiation enhancement at nicotinamide doses between 250 and 1000 mg/kg. CONCLUSION Adding the bioreductive cytotoxin SR 4233 or nicotinamide + carbogen to fractionated irradiation enhances the response of the three transplanted tumors used in this study to fractionated irradiation. The radiation enhancement was significantly greater, however, for SR 4233 for two of the tumors with comparable results in the third. The data are consistent with the prediction that killing tumor hypoxic cells can produce a similar or greater enhancement of the efficacy of fractionated radiation in enhancing tumor response than either oxygenating or radiosensitizing these cells.

Pitx1 broadly associates with limb enhancers and is enriched on hindlimb cis-regulatory elements

Extensive functional analyses have demonstrated that the pituitary homeodomain transcription factor Pitx1 plays a critical role in specifying hindlimb morphology in vertebrates. However, much less is known regarding the target genes and cis-regulatory elements through which Pitx1 acts. Earlier studies suggested that the hindlimb transcription factors Tbx4, HoxC10, and HoxC11 might be transcriptional targets of Pitx1, but definitive evidence for direct regulatory interactions has been lacking. Using ChIP-Seq on embryonic mouse hindlimbs, we have pinpointed the genome-wide location of Pitx1 binding sites during mouse hindlimb development and identified potential gene targets for Pitx1. We determined that Pitx1 binding is significantly enriched near genes involved in limb morphogenesis, including Tbx4, HoxC10, and HoxC11. Notably, Pitx1 is bound to the previously identified HLEA and HLEB hindlimb enhancers of the Tbx4 gene and to a newly identified Tbx2 hindlimb enhancer. Moreover, Pitx1 binding is significantly enriched on hindlimb relative to forelimb-specific cis-regulatory features that are differentially marked by H3K27ac. However, our analysis revealed that Pitx1 also strongly associates with many functionally verified limb enhancers that exhibit similar levels of activity in the embryonic mesenchyme of forelimbs and hindlimbs. We speculate that Pitx1 influences hindlimb morphology both through the activation of hindlimb-specific enhancers as well as through the hindlimb-specific modulation of enhancers that are active in both sets of limbs.