The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription.

DNA replication stress can cause chromosomal instability and tumor progression. One key pathway that counteracts replication stress and promotes faithful DNA replication consists of the Fanconi anemia (FA) proteins. However, how these proteins limit replication stress remains largely elusive. Here we show that conflicts between replication and transcription activate the FA pathway. Inhibition of transcription or enzymatic degradation of transcription-associated R-loops (DNA:RNA hybrids) suppresses replication fork arrest and DNA damage occurring in the absence of a functional FA pathway. Furthermore, we show that simple aldehydes, known to cause leukemia in FA-deficient mice, induce DNA:RNA hybrids in FA-depleted cells. Finally, we demonstrate that the molecular mechanism by which the FA pathway limits R-loop accumulation requires FANCM translocase activity. Failure to activate a response to physiologically occurring DNA:RNA hybrids may critically contribute to the heightened cancer predisposition and bone marrow failure of individuals with mutated FA proteins.

GLI3 resides at the intersection of hedgehog and androgen action to promote male sex differentiation.

Urogenital tract abnormalities are among the most common congenital defects in humans. Male urogenital development requires Hedgehog-GLI signaling and testicular hormones, but how these pathways interact is unclear. We found that Gli3XtJ mutant mice exhibit cryptorchidism and hypospadias due to local effects of GLI3 loss and systemic effects of testicular hormone deficiency. Fetal Leydig cells, the sole source of these hormones in developing testis, were reduced in numbers in Gli3XtJ testes, and their functional identity diminished over time. Androgen supplementation partially rescued testicular descent but not hypospadias in Gli3XtJ mutants, decoupling local effects of GLI3 loss from systemic effects of androgen insufficiency. Reintroduction of GLI3 activator (GLI3A) into Gli3XtJ testes restored expression of Hedgehog pathway and steroidogenic genes. Together, our results show a novel function for the activated form of GLI3 that translates Hedgehog signals to reinforce fetal Leydig cell identity and stimulate timely INSL3 and testosterone synthesis in the developing testis. In turn, exquisite timing and concentrations of testosterone are required to work alongside local GLI3 activity to control development of a functionally integrated male urogenital tract.

Transcription Factors YAP/TAZ and SRF Cooperate To Specify Renal Myofibroblasts in the Developing Mouse Kidney.

BACKGROUND: The embryonic renal stroma consists of multiple molecularly distinct cell subpopulations, the functional significance of which is largely unknown. Previous work has demonstrated that the transcription factors YAP and TAZ play roles in the development and morphogenesis of the nephrons, collecting ducts, and nephron progenitor cells. METHODS: In embryonic mouse kidneys, we identified a subpopulation of stromal cells with enriched activity in YAP and TAZ. To evaluate the function of these cell types, we genetically ablated both Yap and Taz from the stromal progenitor population and examined how gene activity and development of YAP/TAZ mutant kidneys are affected over a developmental time course. RESULTS: We found that YAP and TAZ are active in a subset of renal interstitium and that stromal-specific coablation of YAP/TAZ disrupts cortical fibroblast, pericyte, and myofibroblast development, with secondary effects on peritubular capillary differentiation. We also demonstrated that the transcription factor SRF cooperates with YAP/TAZ to drive expression of at least a subset of renal myofibroblast target genes and to specify myofibroblasts but not cortical fibroblasts or pericytes. CONCLUSIONS: These findings reveal a critical role for YAP/TAZ in specific embryonic stromal cells and suggest that interaction with cofactors, such as SRF, influence the expression of cell type-specific target genes, thus driving stromal heterogeneity. Further, this work reveals functional roles for renal stroma heterogeneity in creating unique microenvironments that influence the differentiation and maintenance of the renal parenchyma.

Single cell transcriptomic analysis of external genitalia reveals complex and sexually dimorphic cell populations in the early genital tubercle.

External genital organs are among the most recognizable sexually dimorphic characters. The penis and clitoris develop from the embryonic genital tubercle, an outgrowth at the anterior margin of the cloaca that undergoes an extensive period of development in male and female embryos prior to the onset of sexual differentiation. In mice, differentiation into the penis and clitoris begins around embryonic day (E)15.5. Current knowledge of cell types that comprise the genital tubercle is limited to a few studies that have fate mapped derivatives of endoderm, mesoderm, and ectoderm. Here we use single cell transcriptomics to characterize the cell populations in the genital tubercles of male and female mouse embryos at E14.5, approximately 24 â€‹h before the onset of sexual differentiation, and we present the first comprehensive atlas of single-cell gene expression during external genital development. Clustering analyses and annotation using marker genes shows 19 distinct cell populations in E14.5 genital tubercles. Mapping of cell clusters to anatomical locations using in situ gene expression patterns revealed granularity of cellular specializations and positional identities. Although E14.5 precedes sexually dimorphic morphogenesis of the genital tubercle, comparative analysis of males and females identified sexual dimorphisms at the single cell level, including male-specific cell clusters with transcriptional signatures of smooth muscle and bone progenitors, both of which are known to be sexually dimorphic in adult genitalia, as well as immune cells. These results provide a new resource for classification of external genital cell types based on gene expression profiles and reveal sex-specific cellular specializations in the early genital tubercle.

Anomalous incisor morphology indicates tissue-specific roles for Tfap2a and Tfap2b in tooth development.

Mice possess two types of teeth that differ in their cusp patterns; incisors have one cusp and molars have multiple cusps. The patterning of these two types of teeth relies on fine-tuning of the reciprocal molecular signaling between dental epithelial and mesenchymal tissues during embryonic development. The AP-2 transcription factors, particularly Tfap2a and Tfap2b, are essential components of such epithelial-mesenchymal signaling interactions that coordinate craniofacial development in mice and other vertebrates, but little is known about their roles in the regulation of tooth development and shape. Here we demonstrate that incisors and molars differ in their temporal and spatial expression of Tfap2a and Tfap2b. At the bud stage, Tfap2a is expressed in both the epithelium and mesenchyme of the incisors and molars, but Tfap2b expression is restricted to the molar mesenchyme, only later appearing in the incisor epithelium. Tissue-specific deletions show that loss of the epithelial domain of Tfap2a and Tfap2b affects the number and spatial arrangement of the incisors, notably resulting in duplicated lower incisors. In contrast, deletion of these two genes in the mesenchymal domain has little effect on tooth development. Collectively these results implicate epithelial expression of Tfap2a and Tfap2b in regulating the extent of the dental lamina associated with patterning the incisors and suggest that these genes contribute to morphological differences between anterior (incisor) and posterior (molar) teeth within the mammalian dentition.

Domestic cat embryos reveal unique transcriptomes of developing incisor, canine, and premolar teeth.

Division of the dentition into morphologically distinct classes of teeth (incisors, canines, premolars, and molars) and the acquisition of tribosphenic molars facilitated precise occlusion between the teeth early in mammal evolution. Despite the evolutionary and ecological importance of distinct classes of teeth with unique cusp, crest, and basin morphologies, relatively little is known about the genetic basis for the development of different tooth classes within the embryo. Here we investigated genetic differences between developing deciduous incisor, canine, and premolar teeth in the domestic cat (Felis catus), which we propose to be a new model for tooth development. We examined differences in both developmental timing and crown morphology between the three tooth classes. Using RNA sequencing of early bell stage tooth germs, we showed that each of the three deciduous tooth classes possess a unique transcriptional profile. Three notable groups of genes emerged from our differential expression analysis; genes involved in the extracellular matrix (ECM), Wnt pathway signaling, and members of multiple homeobox gene families (Lhx, Dlx, Alx, and Nkx). Our results suggest that ECM genes may play a previously under-appreciated role in shaping the surface of the tooth crown during development. Differential regulation of these genes likely underlies differences in tooth crown shape and size, although subtle temporal differences in development between the tooth germs could also be responsible. This study provides foundational data for future experiments to examine the function of these candidate genes in tooth development to directly test their potential effects on crown morphology.

The genetic program for cartilage development has deep homology within Bilateria.

The evolution of novel cell types led to the emergence of new tissues and organs during the diversification of animals. The origin of the chondrocyte, the cell type that synthesizes cartilage matrix, was central to the evolution of the vertebrate endoskeleton. Cartilage-like tissues also exist outside the vertebrates, although their relationship to vertebrate cartilage is enigmatic. Here we show that protostome and deuterostome cartilage share structural and chemical properties, and that the mechanisms of cartilage development are extensively conserved--from induction of chondroprogenitor cells by Hedgehog and ß-catenin signalling, to chondrocyte differentiation and matrix synthesis by SoxE and SoxD regulation of clade A fibrillar collagen (ColA) genes--suggesting that the chondrogenic gene regulatory network evolved in the common ancestor of Bilateria. These results reveal deep homology of the genetic program for cartilage development in Bilateria and suggest that activation of this ancient core chondrogenic network underlies the parallel evolution of cartilage tissues in Ecdysozoa, Lophotrochozoa and Deuterostomia.

Foxa1 and Foxa2 orchestrate development of the urethral tube and division of the embryonic cloaca through an autoregulatory loop with Shh.

Congenital anomalies of external genitalia affect approximately 1 in 125 live male births. Development of the genital tubercle, the precursor of the penis and clitoris, is regulated by the urethral plate epithelium, an endodermal signaling center. Signaling activity of the urethral plate is mediated by Sonic hedgehog (SHH), which coordinates outgrowth and patterning of the genital tubercle by controlling cell cycle kinetics and expression of downstream genes. The mechanisms that govern Shh transcription in urethral plate cells are largely unknown. Here we show that deletion of Foxa1 and Foxa2 results in persistent cloaca, an incomplete separation of urinary, genital, and anorectal tracts, and severe hypospadias, a failure of urethral tubulogenesis. Loss of Foxa2 and only one copy of Foxa1 results in urethral fistula, an additional opening of the penile urethra. Foxa1/a2 participate in an autoregulatory feedback loop with Shh, in which FOXA1 and FOXA2 positively regulate transcription of Shh in the urethra, and SHH feeds back to negatively regulate Foxa1 and Foxa2 expression. These findings reveal novel roles for Foxa genes in development of the urethral tube and in division of the embryonic cloaca.

UBR5 interacts with the replication fork and protects DNA replication from DNA polymerase η toxicity.

Accurate DNA replication is critical for the maintenance of genome integrity and cellular survival. Cancer-associated alterations often involve key players of DNA replication and of the DNA damage-signalling cascade. Post-translational modifications play a fundamental role in coordinating replication and repair and central among them is ubiquitylation. We show that the E3 ligase UBR5 interacts with components of the replication fork, including the translesion synthesis (TLS) polymerase polη. Depletion of UBR5 leads to replication problems, such as slower S-phase progression, resulting in the accumulation of single stranded DNA. The effect of UBR5 knockdown is related to a mis-regulation in the pathway that controls the ubiquitylation of histone H2A (UbiH2A) and blocking this modification is sufficient to rescue the cells from replication problems. We show that the presence of polη is the main cause of replication defects and cell death when UBR5 is silenced. Finally, we unveil a novel interaction between polη and H2A suggesting that UbiH2A could be involved in polη recruitment to the chromatin and the regulation of TLS.

Identification of UHRF2 as a novel DNA interstrand crosslink sensor protein.

The Fanconi Anemia (FA) pathway is important for repairing interstrand crosslinks (ICLs) between the Watson-Crick strands of the DNA double helix. An initial and essential stage in the repair process is the detection of the ICL. Here, we report the identification of UHRF2, a paralogue of UHRF1, as an ICL sensor protein. UHRF2 is recruited to ICLs in the genome within seconds of their appearance. We show that UHRF2 cooperates with UHRF1, to ensure recruitment of FANCD2 to ICLs. A direct protein-protein interaction is formed between UHRF1 and UHRF2, and between either UHRF1 and UHRF2, and FANCD2. Importantly, we demonstrate that the essential monoubiquitination of FANCD2 is stimulated by UHRF1/UHRF2. The stimulation is mediating by a retention of FANCD2 on chromatin, allowing for its monoubiquitination by the FA core complex. Taken together, we uncover a mechanism of ICL sensing by UHRF2, leading to FANCD2 recruitment and retention at ICLs, in turn facilitating activation of FANCD2 by monoubiquitination.

Cartilaginous Fishes Provide Insights into the Origin, Diversification, and Sexually Dimorphic Expression of Vertebrate Estrogen Receptor Genes.

Vertebrate estrogen receptors (ERs) perform numerous cell signaling and transcriptional regulatory functions. ERɑ (Esr1) and ERß (Esr2) likely evolved from an ancestral receptor that duplicated and diverged at the protein and cis-regulatory levels, but the evolutionary history of ERs, including the timing of proposed duplications, remains unresolved. Here we report on identification of two distinct ERs in cartilaginous fishes and demonstrate their orthology to ERα and ERß. Phylogenetic analyses place the ERα/ERß duplication near the base of crown gnathostomes (jawed vertebrates). We find that ERα and ERß from little skate (Leucoraja erinacea) and mammals share key subtype-specific residues, indicating conserved protein evolution. In contrast, jawless fishes have multiple non-orthologous Esr genes that arose by parallel duplications. Esr1 and Esr2 are expressed in subtype-specific and sexually dimorphic patterns in skate embryos, suggesting that ERs might have functioned in sexually dimorphic development before the divergence of cartilaginous and bony fishes.

Phosphorylation regulates human polη stability and damage bypass throughout the cell cycle.

DNA translesion synthesis (TLS) is a crucial damage tolerance pathway that oversees the completion of DNA replication in the presence of DNA damage. TLS polymerases are capable of bypassing a distorted template but they are generally considered inaccurate and they need to be tightly regulated. We have previously shown that polη is phosphorylated on Serine 601 after DNA damage and we have demonstrated that this modification is important for efficient damage bypass. Here we report that polη is also phosphorylated by CDK2, in the absence of damage, in a cell cycle-dependent manner and we identify serine 687 as an important residue targeted by the kinase. We discover that phosphorylation on serine 687 regulates the stability of the polymerase during the cell cycle, allowing it to accumulate in late S and G2 when productive TLS is critical for cell survival. Furthermore, we show that alongside the phosphorylation of S601, the phosphorylation of S687 and S510, S512 and/or S514 are important for damage bypass and cell survival after UV irradiation. Taken together our results provide new insights into how cells can, at different times, modulate DNA TLS for improved cell survival.

Spatiotemporal dynamics of androgen signaling underlie sexual differentiation and congenital malformations of the urethra and vagina.

Disorders of sex development (DSDs) are congenital anomalies that affect sexual differentiation of genitourinary organs and secondary sex characters. A common cause of female genital virilization is congenital adrenal hyperplasia (CAH), in which excess androgen production during development of 46XX females can result in vaginal atresia, masculinization of the urethra, a single urogenital sinus, and clitoral hypertrophy or ambiguous external genitalia. Development of the vagina depends on sexual differentiation of the urogenital sinus ridge, an epithelial thickening that forms where the sex ducts attach to the anterior urethra. In females, the sinus ridge descends posteriorly to allow the vaginal opening to form in the vulva, whereas in males and in females with CAH, androgens inhibit descent of the sinus ridge. The mechanisms that regulate development of the female urethra and vagina are largely unknown. Here we show that the timing and duration of, and the cell population targeted by, androgen signaling determine the position of vaginal attachment to the urethra. Manipulations of androgen signaling in utero reveal a temporal window of development when sinus ridge fate is determined. Cell type-specific genetic deletions of androgen receptor (Ar) identify a subpopulation of mesenchymal cells that regulate sinus ridge morphogenesis. These results reveal a common mechanism that coordinates development of the vagina and feminization of the urethra, which may account for development of a single urogenital sinus in females exposed to excessive androgen during a critical period of prenatal development.

Developmental, genetic, and genomic insights into the evolutionary loss of limbs in snakes.

The evolution of snakes involved dramatic modifications to the ancestral lizard body plan. Limb loss and elongation of the trunk are hallmarks of snakes, although convergent evolution of limb-reduced and trunk-elongated forms occurred multiple times in snake-like lizards. Advanced snakes are completely limbless, but intermediate and basal snakes have retained rudiments of hindlimbs and pelvic girdles. Moreover, the snake fossil record indicates that complete legs were re-acquired at least once, suggesting that the potential for limb development was retained in some limb-reduced taxa. Recent work has shown that python embryos initiate development of a transitory distal leg skeleton, including a footplate, and that the limb-specific enhancer of the Sonic hedgehog gene, known as the zone of polarizing activity regulatory sequence (ZRS), underwent gradual degeneration during snake evolution. In this article, we review historical and recent investigations into squamate limblessness, and we discuss how new genomic and functional genetic experiments have improved our understanding of the evolution of limblessness in snakes. Finally, we explore the idea that pleiotropy of cis-regulatory elements may illuminate the convergent genetic changes that occurred in snake-like lizards, and we discuss a number of challenges that remain to be addressed in future studies.

Tissue-specific roles of Fgfr2 in development of the external genitalia.

Congenital anomalies frequently occur in organs that undergo tubulogenesis. Hypospadias is a urethral tube defect defined by mislocalized, oversized, or multiple openings of the penile urethra. Deletion of Fgfr2 or its ligand Fgf10 results in severe hypospadias in mice, in which the entire urethral plate is open along the ventral side of the penis. In the genital tubercle, the embryonic precursor of the penis and clitoris, Fgfr2 is expressed in two epithelial populations: the endodermally derived urethral epithelium and the ectodermally derived surface epithelium. Here, we investigate the tissue-specific roles of Fgfr2 in external genital development by generating conditional deletions of Fgfr2 in each of these cell types. Conditional deletion of Fgfr2 results in two distinct phenotypes: endodermal Fgfr2 deletion causes mild hypospadias and inhibits maturation of a complex urethral epithelium, whereas loss of ectodermal Fgfr2 results in severe hypospadias and absence of the ventral prepuce. Although these cell type-specific mutants exhibit distinctive genital anomalies, cellular analysis reveals that Fgfr2 regulates epithelial maturation and cell cycle progression in the urethral endoderm and in the surface ectoderm. The unexpected finding that ectodermal deletion of Fgfr2 results in the most severe hypospadias highlights a major role for Fgfr2 in the developing genital surface epithelium, where epithelial maturation is required for maintenance of a closed urethral tube. These results demonstrate that urethral tubulogenesis, prepuce morphogenesis, and sexually dimorphic patterning of the lower urethra are controlled by discrete regions of Fgfr2 activity.

An illustrated anatomical ontology of the developing mouse lower urogenital tract.

Malformation of the urogenital tract represents a considerable paediatric burden, with many defects affecting the lower urinary tract (LUT), genital tubercle and associated structures. Understanding the molecular basis of such defects frequently draws on murine models. However, human anatomical terms do not always superimpose on the mouse, and the lack of accurate and standardised nomenclature is hampering the utility of such animal models. We previously developed an anatomical ontology for the murine urogenital system. Here, we present a comprehensive update of this ontology pertaining to mouse LUT, genital tubercle and associated reproductive structures (E10.5 to adult). Ontology changes were based on recently published insights into the cellular and gross anatomy of these structures, and on new analyses of epithelial cell types present in the pelvic urethra and regions of the bladder. Ontology changes include new structures, tissue layers and cell types within the LUT, external genitalia and lower reproductive structures. Representative illustrations, detailed text descriptions and molecular markers that selectively label muscle, nerves/ganglia and epithelia of the lower urogenital system are also presented. The revised ontology will be an important tool for researchers studying urogenital development/malformation in mouse models and will improve our capacity to appropriately interpret these with respect to the human situation.

Timing of androgen receptor disruption and estrogen exposure underlies a spectrum of congenital penile anomalies.

Congenital penile anomalies (CPAs) are among the most common human birth defects. Reports of CPAs, which include hypospadias, chordee, micropenis, and ambiguous genitalia, have risen sharply in recent decades, but the causes of these malformations are rarely identified. Both genetic anomalies and environmental factors, such as antiandrogenic and estrogenic endocrine disrupting chemicals (EDCs), are suspected to cause CPAs; however, little is known about the temporal window(s) of sensitivity to EDCs, or the tissue-specific roles and downstream targets of the androgen receptor (AR) in external genitalia. Here, we show that the full spectrum of CPAs can be produced by disrupting AR at different developmental stages and in specific cell types in the mouse genital tubercle. Inactivation of AR during a narrow window of prenatal development results in hypospadias and chordee, whereas earlier disruptions cause ambiguous genitalia and later disruptions cause micropenis. The neonatal phase of penile development is controlled by the balance of AR to estrogen receptor α (ERα) activity; either inhibition of androgen or augmentation of estrogen signaling can induce micropenis. AR and ERα have opposite effects on cell division, apoptosis, and regulation of Hedgehog, fibroblast growth factor, bone morphogenetic protein, and Wnt signaling in the genital tubercle. We identify Indian hedgehog (Ihh) as a novel downstream target of AR in external genitalia and show that conditional deletion of Ihh inhibits penile masculinization. These studies reveal previously unidentified cellular and molecular mechanisms by which antiandrogenic and estrogenic signals induce penile malformations and demonstrate that the timing of endocrine disruption can determine the type of CPA.

Cellular response to DNA interstrand crosslinks: the Fanconi anemia pathway.

Interstrand crosslinks (ICLs) are a highly toxic form of DNA damage. ICLs can interfere with vital biological processes requiring separation of the two DNA strands, such as replication and transcription. If ICLs are left unrepaired, it can lead to mutations, chromosome breakage and mitotic catastrophe. The Fanconi anemia (FA) pathway can repair this type of DNA lesion, ensuring genomic stability. In this review, we will provide an overview of the cellular response to ICLs. First, we will discuss the origin of ICLs, comparing various endogenous and exogenous sources. Second, we will describe FA proteins as well as FA-related proteins involved in ICL repair, and the post-translational modifications that regulate these proteins. Finally, we will review the process of how ICLs are repaired by both replication-dependent and replication-independent mechanisms.

Molecular Characterization of the Genital Organizer: Gene Expression Profile of the Mouse Urethral Plate Epithelium.

PURPOSE: Lower urinary tract malformations are among the most common congenital anomalies in humans. Molecular genetic studies of mouse external genital development have begun to identify mechanisms that pattern the genital tubercle and orchestrate urethral tubulogenesis. The urethral plate epithelium is an endodermal signaling region that has an essential role in external genital development. However, little is known about the molecular identity of this cell population or the genes that regulate its activity. MATERIALS AND METHODS: We used microarray analysis to characterize differences in gene expression between urethral plate epithelium and surrounding tissue in mouse genital tubercles. In situ hybridizations were performed to map gene expression patterns and ToppCluster (https://toppcluster.cchmc.org/) was used to analyze gene associations. RESULTS: A total of 84 genes were enriched at least 20-fold in urethral plate epithelium relative to surrounding tissue. The majority of these genes were expressed throughout the urethral plate in males and females at embryonic day 12.5 when the urethral plate is known to signal. Functional analysis using ToppCluster revealed genetic pathways with known functions in other organ systems but unknown roles in external genital development. Additionally, a 3-dimensional molecular atlas of genes enriched in urethral plate epithelium was generated and deposited at the GUDMAP (GenitoUrinary Development Molecular Anatomy Project) website (http://gudmap.org/). CONCLUSIONS: We identified dozens of genes previously unknown to be expressed in urethral plate epithelium at a crucial developmental period. It provides a novel panel of genes for analysis in animal models and in humans with external genital anomalies.

Chromatin recruitment of DNA repair proteins: lessons from the fanconi anemia and double-strand break repair pathways.

In response to DNA damage, eukaryotic cells must rapidly load DNA repair proteins onto damaged chromatin. Chromatin recruitment often entails ubiquitination of a damage-specific DNA repair protein, interaction with a ubiquitin binding factor, assembly of a multisubunit DNA repair complex, and eventually a deubiquitination event once the DNA repair reaction has been completed. This review focuses on the recent discoveries in the Fanconi Anemia (FA) and DNA double-strand break (DSB) repair pathways, which underscore the importance of regulated chromatin loading in the DNA damage response. Interestingly, these two pathways share several features, suggesting a more general mechanism for DNA-repair regulation.