Phosphorylation by ATR triggers FANCD2 chromatin loading and activates the Fanconi anemia pathway.

The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs) in humans. Activation of the pathway relies on loading of the FANCD2/FANCI complex onto chromosomes, where it is fully activated by subsequent monoubiquitination. However, the mechanism for loading the complex onto chromosomes remains unclear. Here, we identify 10 SQ/TQ phosphorylation sites on FANCD2, which are phosphorylated by ATR in response to ICLs. Using a range of biochemical assays complemented with live-cell imaging including super-resolution single-molecule tracking, we show that these phosphorylation events are critical for loading of the complex onto chromosomes and for its subsequent monoubiquitination. We uncover how the phosphorylation events are tightly regulated in cells and that mimicking their constant phosphorylation leads to an uncontrolled active state of FANCD2, which is loaded onto chromosomes in an unrestrained fashion. Taken together, we describe a mechanism where ATR triggers FANCD2/FANCI loading onto chromosomes.

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.

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.

Lessons learned from the COVID-19 pandemic: Improving initial investigations with the implementation of a COVID-19 blood request panel.

BACKGROUND: During the COVID-19 pandemic, the Hillingdon Hospitals NHS Foundation Trust produced trust guidelines for the initial blood investigation of COVID-19 inpatients. However, insufficient education meant inconsistent adherence to this guidance. OBJECTIVE: To examine whether the implementation of a COVID-19 blood request panel improves adherence to local trust guidelines. METHOD: Between March and April 2020, initial blood investigations performed for positive COVID-19 cases were compared to guidelines. Results were presented locally and a COVID-19 panel was added to the electronic system that provided prompts for appropriate investigations. A re-audit between May and June 2020 was conducted to assess adherence post-intervention. RESULTS: 383 patients were identified in the initial audit cohort and a sample of 20 patients were re-audited. Adherence to Full Blood Count, Urea and Electrolytes, C-reactive Protein and Liver Function Tests increased to 100% from 99.7% (p = 0.8), 99.2% (p = 0.69), 98.7% (p = 0.61), and 96.6% (p = 0.4) respectively. Coagulation screen adherence increased to 90% from 72.8% (p = 0.09). Appropriate requesting of D dimers increased to 50% from 19.9% (p = 0.001). Inappropriate troponin requesting decreased to 26.3% from 38.9% (p = 0.23). CONCLUSION: A user-friendly COVID-19 panel of investigations resulted in improved adherence to guidelines. Clear communication and education are essential to help alleviate uncertainty during a pandemic.

Convergent developmental patterns underlie the repeated evolution of adhesive toe pads among lizards.

How developmental modifications produce key innovations, which subsequently allow for rapid diversification of a clade into new adaptive zones, has received much attention. However, few studies have used a robust comparative framework to investigate the influence of evolutionary and developmental constraints on the origin of key innovations, such as the adhesive toe pad of lizards. Adhesive toe pads evolved independently at least 16 times in lizards, allowing us to examine whether the patterns observed are general evolutionary phenomena or unique, lineage-specific events. We performed a high-resolution comparison of plantar scale development in 14 lizard species in Anolis and geckos, encompassing five independent origins of toe pads (one in Anolis, four in geckos). Despite substantial evolutionary divergence between Anolis and geckos, we find that these clades have undergone similar developmental modifications to generate their adhesive toe pads. Relative to the ancestral plantar scale development, in which scale ridges form synchronously along the digit, both padded geckos and Anolis exhibit scansor formation in a distal-to-proximal direction. Both clades have undergone developmental repatterning and, following their origin, modifications in toe pad morphology occurred through relatively minor developmental modifications, suggesting that developmental constraints governed the diversification of the adhesive toe pad in lizards.

Spatial regulation by multiple Gremlin1 enhancers provides digit development with cis-regulatory robustness and evolutionary plasticity.

Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development. We establish that transcript levels are controlled in an additive manner while spatial regulation requires synergistic interactions among multiple enhancers. Disrupting these interactions shows that altered spatial regulation rather than reduced Grem1 transcript levels prefigures digit fusions and loss. Two of the enhancers are evolutionary ancient and highly conserved from basal fishes to mammals. Analysing these enhancers from different species reveal the substantial spatial plasticity in Grem1 regulation in tetrapods and basal fishes, which provides insights into the fin-to-limb transition and evolutionary diversification of pentadactyl limbs.

Protocol of the Cognitive Health in Ageing Register: Investigational, Observational and Trial Studies in Dementia Research (CHARIOT): Prospective Readiness cOhort (PRO) SubStudy.

INTRODUCTION: The Cognitive Health in Ageing Register: Investigational, Observational and Trial Studies in Dementia Research (CHARIOT): Prospective Readiness cOhort (PRO) SubStudy (CPSS), sponsored by Janssen Pharmaceutical Research & Development LLC, is an Alzheimer's disease (AD) biomarker enriched observational study that began 3 July 2015 CPSS aims to identify and validate determinants of AD, alongside cognitive, functional and biological changes in older adults with or without detectable evidence of AD pathology at baseline. METHODS AND ANALYSIS: CPSS is a dual-site longitudinal cohort (3.5 years) assessed quarterly. Cognitively normal participants (60-85 years) were recruited across Greater London and Edinburgh. Participants are classified as high, medium (amnestic or non-amnestic) or low risk for developing mild cognitive impairment-Alzheimer's disease based on their Repeatable Battery for the Assessment of Neuropsychological Status performance at screening. Additional AD-related assessments include: a novel cognitive composite, the Global Preclinical Alzheimer's Cognitive Composite, brain MRI and positron emission tomography and cerebrospinal fluid analysis. Lifestyle, other cognitive and functional data, as well as biosamples (blood, urine, and saliva) are collected. Primarily, study analyses will evaluate longitudinal change in cognitive and functional outcomes. Annual interim analyses for descriptive data occur throughout the course of the study, although inferential statistics are conducted as required. ETHICS AND DISSEMINATION: CPSS received ethical approvals from the London-Central Research Ethics Committee (15/LO/0711) and the Administration of Radioactive Substances Advisory Committee (RPC 630/3764/33110) The study is at the forefront of global AD prevention efforts, with frequent and robust sampling of the well-characterised cohort, allowing for detection of incipient pathophysiological, cognitive and functional changes that could inform therapeutic strategies to prevent and/or delay cognitive impairment and dementia. Dissemination of results will target the scientific community, research participants, volunteer community, public, industry, regulatory authorities and policymakers. On study completion, and following a predetermined embargo period, CPSS data are planned to be made accessible for analysis to facilitate further research into the determinants of AD pathology, onset of symptomatology and progression. TRIAL REGISTRATION NUMBER: The CHARIOT:PRO SubStudy is registered with clinicaltrials.gov (NCT02114372). Notices of protocol modifications will be made available through this trial registry.

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.

Purification of DNA repair protein complexes from mammalian cells.

Cells possess multiple DNA repair pathways to tackle a variety of DNA lesions. Often, DNA repair proteins function as large protein complexes. Here, we describe a protocol to purify DNA repair protein complexes from nuclei of mammalian cells. The method permits purification of protein complexes containing stable as well as transiently associated proteins, which subsequently can be identified by mass-spectrometry analysis. This protocol can be applied to uncover the functions and mechanism of DNA repair pathways. For complete information on the use and execution of this protocol, please refer to Socha et al. (2020).

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.

Meeting report on the NIDDK/AUA Workshop on Congenital Anomalies of External Genitalia: challenges and opportunities for translational research.

Congenital anomalies of the external genitalia (CAEG) are a prevalent and serious public health concern with lifelong impacts on the urinary function, sexual health, fertility, tumor development, and psychosocial wellbeing of affected individuals. Complications of treatment are frequent, and data reflecting long-term outcomes in adulthood are limited. To identify a path forward to improve treatments and realize the possibility of preventing CAEG, the National Institute of Diabetes and Digestive and Kidney Diseases and the American Urological Association convened researchers from a range of disciplines to coordinate research efforts to fully understand the different etiologies of these common conditions, subsequent variation in clinical phenotypes, and best practices for long term surgical success. Meeting participants concluded that a central data hub for clinical evaluations, including collection of DNA samples from patients and their parents, and short interviews to determine familial penetrance (small pedigrees), would accelerate research in this field. Such a centralized datahub will advance efforts to develop detailed multi-dimensional phenotyping and will enable access to genome sequence analyses and associated metadata to define the genetic bases for these conditions. Inclusion of tissue samples and integration of clinical studies with basic research using human cells and animal models will advance efforts to identify the developmental mechanisms that are disrupted during development and will add cellular and molecular granularity to phenotyping CAEG. While the discussion focuses heavily on hypospadias, this can be seen as a potential template for other conditions in the realm of CAEG, including cryptorchidism or the exstrophy-epispadias complex. Taken together with long-term clinical follow-up, these data could inform surgical choices and improve likelihood for long-term success.

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.

WRNIP1 Is Recruited to DNA Interstrand Crosslinks and Promotes Repair.

The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs). Many FA proteins are recruited to ICLs in a timely fashion so that coordinated repair can occur. However, the mechanism of this process is poorly understood. Here, we report the purification of a FANCD2-containing protein complex with multiple subunits, including WRNIP1. Using live-cell imaging, we show that WRNIP1 is recruited to ICLs quickly after their appearance, promoting repair. The observed recruitment facilitates subsequent recruitment of the FANCD2/FANCI complex. Depletion of WRNIP1 sensitizes cells to ICL-forming drugs. We find that ubiquitination of WRNIP1 and the activity of its UBZ domain are required to facilitate recruitment of FANCD2/FANCI and promote repair. Altogether, we describe a mechanism by which WRNIP1 is recruited rapidly to ICLs, resulting in chromatin loading of the FANCD2/FANCI complex in an unusual process entailing ubiquitination of WRNIP1 and the activity of its integral UBZ domain.

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.

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.

Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage.

Interstrand crosslinks (ICLs) of the DNA helix are a deleterious form of DNA damage. ICLs can be repaired by the Fanconi anemia pathway. At the center of the pathway is the FANCD2/FANCI complex, recruitment of which to DNA is a critical step for repair. After recruitment, monoubiquitination of both FANCD2 and FANCI leads to their retention on chromatin, ensuring subsequent repair. However, regulation of recruitment is poorly understood. Here, we report a cluster of phosphosites on FANCD2 whose phosphorylation by CK2 inhibits both FANCD2 recruitment to ICLs and its monoubiquitination in vitro and in vivo. We have found that phosphorylated FANCD2 possesses reduced DNA binding activity, explaining the previous observations. Thus, we describe a regulatory mechanism operating as a molecular switch, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway.

Evolution of limb development in cephalopod mollusks.

Cephalopod mollusks evolved numerous anatomical novelties, including arms and tentacles, but little is known about the developmental mechanisms underlying cephalopod limb evolution. Here we show that all three axes of cuttlefish limbs are patterned by the same signaling networks that act in vertebrates and arthropods, although they evolved limbs independently. In cuttlefish limb buds, Hedgehog is expressed anteriorly. Posterior transplantation of Hedgehog-expressing cells induced mirror-image limb duplications. Bmp and Wnt signals, which establish dorsoventral polarity in vertebrate and arthropod limbs, are similarly polarized in cuttlefish. Inhibition of Bmp2/4 dorsally caused ectopic expression of Notum, which marks the ventral sucker field, and ectopic sucker development. Cuttlefish also show proximodistal regionalization of Hth, Exd, Dll, Dac, Sp8/9, and Wnt expression, which delineates arm and tentacle sucker fields. These results suggest that cephalopod limbs evolved by parallel activation of a genetic program for appendage development that was present in the bilaterian common ancestor.

Molecular ontogeny of the stomach in the catshark Scyliorhinus canicula.

The origin of extracellular digestion in metazoans was accompanied by structural and physiological alterations of the gut. These adaptations culminated in the differentiation of a novel digestive structure in jawed vertebrates, the stomach. Specific endoderm/mesenchyme signalling is required for stomach differentiation, involving the growth and transcription factors: 1) Shh and Bmp4, required for stomach outgrowth; 2) Barx1, Sfrps and Sox2, required for gastric epithelium development and 3) Cdx1 and Cdx2, involved in intestinal versus gastric identity. Thus, modulation of endoderm/mesenchyme signalling emerges as a plausible mechanism linked to the origin of the stomach. In order to gain insight into the ancient mechanisms capable of generating this structure in jawed vertebrates, we characterised the development of the gut in the catshark Scyliorhinus canicula. As chondrichthyans, these animals retained plesiomorphic features of jawed vertebrates, including a well-differentiated stomach. We identified a clear molecular regionalization of their embryonic gut, characterised by the expression of barx1 and sox2 in the prospective stomach region and expression of cdx1 and cdx2 in the prospective intestine. Furthermore, we show that gastric gland development occurs close to hatching, accompanied by the onset of gastric proton pump activity. Our findings favour a scenario in which the developmental mechanisms involved in the origin of the stomach were present in the common ancestor of chondrichthyans and osteichthyans.

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.