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Failure of embryo implantation accounts for a significant percentage of female infertility. Exquisitely coordinated molecular programs govern the interaction between the competent blastocyst and the receptive uterus. Decidualization, the rapid proliferation and differentiation of endometrial stromal cells into decidual cells, is required for implantation. Decidualization defects can cause poor placentation, intrauterine growth restriction, and early parturition leading to preterm birth. Decidualization has not yet been systematically studied at the genetic level due to the lack of a suitable high-throughput screening tool. Herein we describe the generation of an immortalized human endometrial stromal cell line that uses yellow fluorescent protein under the control of the prolactin promoter as a quantifiable visual readout of the decidualization response (hESC-PRLY cells). Using this cell line, we performed a genome-wide siRNA library screen, as well as a screen of 910 small molecules, to identify more than 4,000 previously unrecognized genetic and chemical modulators of decidualization. Ontology analysis revealed several groups of decidualization modulators, including many previously unappreciated transcription factors, sensory receptors, growth factors, and kinases. Expression studies of hits revealed that the majority of decidualization modulators are acutely sensitive to ovarian hormone exposure. Gradient treatment of exogenous factors was used to identify EC50 values of small-molecule hits, as well as verify several growth factor hits identified by the siRNA screen. The high-throughput decidualization reporter cell line and the findings described herein will aid in the development of patient-specific treatments for decidualization-based recurrent pregnancy loss, subfertility, and infertility.

Retinoic acid (RA) signaling has long been speculated to regulate embryo implantation, because many enzymes and proteins responsible for maintaining RA homeostasis and transducing RA signals are tightly regulated in the endometrium during this critical period. However, due to a lack of genetic data, it was unclear whether RA signaling is truly required for implantation and which specific RA signaling cascades are at play. Herein we utilize a genetic murine model that expresses a dominant-negative form of RA receptor (RAR) specifically in female reproductive organs to show that functional RA signaling is fundamental to female fertility, particularly implantation and decidualization. Reduction in RA signaling activity severely affects the ability of the uterus to achieve receptive status and decidualize, partially through dampening follistatin expression and downstream activin B/bone morphogenetic protein 2 signaling. To confirm translational relevance of these findings to humans, human endometrial stromal cells (hESCs) were treated with a pan-RAR antagonist to show that in vitro decidualization is impaired. RNA interference perturbation of individual RAR transcripts in hESCs revealed that RARα in particular was essential for proper decidualization. These data provide direct functional evidence that uterine RAR-mediated RA signaling was crucial for mammalian embryo implantation, and its disruption led to failure of uterine receptivity and decidualization, resulting in severely compromised fertility.

Wnt/β-catenin signaling plays an essential role in eye development. Faulty regulation of this pathway results in ocular malformations due to defects in cell fate determination and differentiation. Herein we show that disruption of Maz, the gene encoding Myc-associated zinc finger transcription factor, produces developmental eye defects in mice and humans. Expression of key genes involved in the Wnt cascade, Sfrp2, Wnt2b and Fzd4 were significantly increased in mice with targeted inactivation of Maz, resulting in abnormal peripheral eye formation with reduced proliferation of the progenitor cells in the region. Paradoxically, the Wnt reporter TCF-Lef1 displayed a significant downregulation in Maz-deficient eyes. Molecular analysis indicates that Maz is necessary for the activation of the Wnt/β-catenin pathway and participates in the network controlling ciliary margin patterning. Copy number variations and single nucleotide variants of MAZ were identified in humans that result in abnormal ocular development. The data support MAZ as a key contributor to the eye comorbidities associated with chromosome 16p11.2 copy number variants and as a transcriptional regulator of ocular development.

Genitourinary (GU) birth defects are among the most common yet least studied congenital malformations. Congenital anomalies of the kidney and urinary tract (CAKUTs) have high morbidity and mortality rates and account for ∼30% of structural birth defects. Copy number variation (CNV) mapping revealed that 16p11.2 is a hotspot for GU development. The only gene covered collectively by all of themapped GU-patient CNVs was MYC-associated zinc finger transcription factor (MAZ), and MAZ CNV frequency is enriched in nonsyndromic GU-abnormal patients. Knockdown of MAZ in HEK293 cells results in differential expression of several WNT morphogens required for normal GU development, including Wnt11 and Wnt4. MAZ knockdown also prevents efficient transition into S phase, affects transcription of cell-cycle regulators, and abrogates growth of human embryonic kidney cells. Murine Maz is ubiquitously expressed, and a CRISPR-Cas9 mouse model of Maz deletion results in perinatal lethality with survival rates dependent on Maz copy number. Homozygous loss of Maz results in high penetrance of CAKUTs, and Maz is haploinsufficient for normal bladder development. MAZ, once thought to be a simple housekeeping gene, encodes a dosage-sensitive transcription factor that regulates urogenital development and contributes to both nonsyndromic congenital malformations of the GU tract as well as the 16p11.2 phenotype.

The spectrum of congenital anomalies affecting either the upper tract (kidneys and ureters) or lower tract (reproductive organs) of the genitourinary (GU) system are fundamentally linked by the developmental origin of multiple GU tissues, including the kidneys, gonads, and reproductive ductal systems: the intermediate mesoderm. Although ∼31% of DiGeorge/del22q11.2 syndrome patients exhibit GU defects, little focus has been placed on the molecular etiology of GU defects in this syndrome. Among del22q11.2 patients exhibiting GU anomalies, we have mapped the smallest relevant region to only five genes, including CRKL. CRKL encodes a srchomology adaptor protein implicated in mediating tyrosine kinase signaling, and is expressed in the developing GU-tract in mice and humans. Here we show that Crkl mutant embryos exhibit gene dosage-dependent growth restriction, and homozygous mutants exhibit upper GU defects at a microdissection-detectable rate of 23%. RNA-sequencing revealed that 52 genes are differentially regulated in response to uncoupling Crkl from its signaling pathways in the developing kidney, including a fivefold up-regulation of Foxd1, a known regulator of nephron progenitor differentiation. Additionally, Crkl heterozygous adult males exhibit cryptorchidism, lower testis weight, lower sperm count, and subfertility. Together, these data indicate that CRKL is intimately involved in normal development of both the upper and lower GU tracts, and disruption of CRKL contributes to the high incidence of GU defects associated with deletion at 22q11.2.

Sexually dimorphic outgrowth and differentiation of the embryonic genital tubercles (GTs) give rise to the penis in males and the clitoris in females. Defects in androgen production or in response to androgen signaling can lead to various congenital penile anomalies in both mice and humans. Due to lack of a high-throughput screening system, identification of crucial regulators of GT sexual differentiation has been slow. To overcome this research barrier, we isolated embryonic GT mesenchymal (GTme) cells to model genitalia growth and differentiation in vitro. Using either a mechanical or fluorescence-activated cell sorting–assisted purification method, GTme cells were isolated and assayed for their proliferation using a microscopy and image analysis system, on a single cell level over time. Male and female GTme cells inherently exhibit different cellular dynamics, consistent with their in-vivo behaviors. This system allows for the rapid quantitative analyses of numerous drug treatments, and enables the discovery of potential genetic modulators of GT morphogenesis on a large scale. Using this system, we completed a 438-compound library screen and identified 82 kinase inhibitor hits. In mice, in-utero exposure to one such candidate kinase inhibitor, Cediranib, resulted in embryos with severe genitalia defects, especially in males. Gene silencing by RNAi was optimized in this system, laying the foundation for future larger-scale genetic screenings. These findings demonstrate the power of this novel high-throughput system to rapidly and successfully identify modulators of genitalia growth and differentiation, expanding the toolbox for the study of functional genomics and environmental factors.

Ineffective embryo implantation accounts for a significant percentage of female infertility, and often renders IVF procedures unsuccessful. Decidualization, the dramatic uterine morphological response to ovarian hormone exposure, is a prerequisite for embryo implantation. Despite its significance in reproduction, the genetic framework of decidualization was not systematically studied until our recent development of a suitable high-throughput screening tool, immortalized human endometrial stromal cells (hESCs) that carry the yellow fluorescent protein gene under the control of the progesterone-sensitive prolactin promoter (PRL-Y cells). We recently used PRL-Y cells to perform a genome-wide siRNA functional screen and results revealed that 36 members of the homeodomain-containing family of transcription factors (HDTFs) are modulators of human decidualization. To determine which HDTFs are transcriptionally sensitive to ovarian hormone exposure, RT-PCR was performed on wildtype hESCs for the 36 HDTF hits over a 72-hour time course of E2/P4/cAMP exposure. Twenty HDTF hits (55%) were both detectable by PCR and showed variable expression in response to ovarian hormone treatment. Interestingly, all of these homeodomain factors, with a few distinct exceptions, exhibited decreased transcriptional expression in response to ovarian hormone treatment. This suggests that precious energy is used to transcribe these factors during the pre-decidualized phase, and that they may be required to maintain homeostasis during times of low hormone exposure. Because siRNA is not fully efficient, in order to confirm which HDTFs are required for normal decidualization, we generated a doxycyline-inducible Cas9-expressing hESC clone in order to subsequently generate individual knockout hESC lines for each HDTF hit. Cas9 expression was turned on 5 days prior to crRNA and trRNA transfection targeting the first exon of each HDTF. Five days after transfection, the cells were treated for 72 hours with ovarian hormone induction medium before RNA was isolated for gene expression analysis. As a pool of cells prior to any cloning (which likely includes knockout and wildtype cells in different ratios) the results indicate that several HDTFs are required for proper decidualization. The reporter transcripts of PRL and EREG are significantly abrogated or entirely undetectable in certain knockout lines. Interestingly these include some original siRNA HDTF hits whose expression is undetectably low by PCR. Sequencing validation will be necessary to confirm that knocking out such low levels of these transcripts genuinely has the robust effect on the human decidualization reaction that we are witnessing in these results. Together these findings comprise significant initial steps in characterizing the intricate upstream roles of HDTFs in human decidualization and female fertility.

Wnt/β-catenin signaling plays an essential role in eye development. Faulty regulation of this pathway results in ocular malformations due to defects in cell fate determination and differentiation. Herein we show that disruption of Maz, the gene encoding Myc-associated zinc finger transcription factor, produces developmental eye defects in mice and humans. Expression of key genes involved in the Wnt cascade, Sfrp2, Wnt2b and Fzd4 were significantly increased in mice with targeted inactivation of Maz, resulting in abnormal peripheral eye formation with reduced proliferation of the progenitor cells in the region. Paradoxically, the Wnt reporter TCF-Lef1 displayed a significant downregulation in Maz-deficient eyes. Molecular analysis indicates that Maz is necessary for the activation of the Wnt/β-catenin pathway and participates in the network controlling ciliary margin patterning. Copy number variations and single nucleotide variants of MAZ were identified in humans that result in abnormal ocular development. The data support MAZ as a key contributor to the eye comorbidities associated with chromosome 16p11.2 copy number variants and as a transcriptional regulator of ocular development.

Dinosaur tracks have a long history of discovery and study in the UK, but track sites for sauropodomorph dinosaurs—the group that included the giant, graviportal herbivorous sauropods—are comparatively rare. Here, we provide a description of a sauropod dinosaur track site at Spyway Quarry in Dorset, southern England. The tracks at Spyway were discovered in the late 1990s and occur in the Stair Hole Member of the Durlston Formation in the Purbeck Limestone Group, of earliest Cretaceous age. More than 130 individual tracks of large sauropod dinosaurs are present at the site, but they are generally poorly preserved and do not form clear trackways, although it is likely that they represent multiple individuals. They provide further evidence for sauropods living in or passing through coastal lagoonal environments. Although poorly preserved, Spyway represents the largest in situ dinosaur track site currently accessible within the Purbeck Group, with considerable potential for further discoveries through ongoing quarrying in the surrounding area.