scholarly journals In-vitro cellular reprogramming to model gonad development and its disorders

2021 ◽  
Author(s):  
Nitzan Gonen ◽  
Caroline Eozenou ◽  
Richard Mitter ◽  
Andreia Bernardo ◽  
Almira Chervova ◽  
...  
Keyword(s):  
Sex Determination ◽  
Gonad Development ◽  
Disorders Of Sex Development ◽  
Cellular Reprogramming ◽  
Tubule Formation ◽  
Sex Development ◽  
Human Disorders ◽  
Induced Pluripotent

During embryonic development, mutually antagonistic signaling cascades determine the fate of the bipotential gonad towards a testicular or ovarian identity. Errors in this process result in human Disorders of Sex Development (DSDs), where there is discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in-vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells towards gonadal progenitors. Transcriptomic analysis reveals that the in-vitro-derived murine gonadal cells are equivalent to E11.5 in-vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete AMH, migrate and form tubular structures. The cells derived from a 46,XY DSD female hiPSCs, carrying a NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR/Cas9-mediated correction of the variant rescued the phenotype. This is a robust tool to understand mechanisms of sex-determination and model DSDs.

177. A MECHANISM UNDERLYING DISORDERS OF SEX DEVELOPMENT CAUSED BY DAX1 DUPLICATION

2009 ◽  
Vol 21 (9) ◽  
pp. 95
Author(s):  
L. Ludbrook ◽  
R. Sekido ◽  
R. Lovell-Badge ◽  
V. Harley
Keyword(s):  
Sex Determination ◽  
Disorders Of Sex Development ◽  
Nuclear Hormone Receptor ◽  
Testicular Development ◽  
Transcriptional Level ◽  
Sox9 Expression ◽  
Transgenic Mouse Line ◽  
Sex Development

The DAX1 protein is an orphan nuclear hormone receptor expressed in developing and adult hypothalamic, pituitary, adrenal and gonadal tissues. In humans, duplication of the DAX1 gene at locus Xp21 causes Disorders of Sex Development (DSD), whereby XY individuals develop as females, due to the failure of testicular development. DAX1 acts as a co-factor for nuclear receptor-mediated transcription of steroidogenic genes. In mice, overexpression of a Dax1 transgene causes delayed testis cord formation, a milder phenotype than that seen in human (1). Exactly how DAX1 duplication interferes with typical testicular development is unclear but a ‘window' of DAX1 activity was proposed (2). In order to identify the mechanism of DAX1 action when overexpressed in the developing XY gonad, we have used both in vivo and in vitro approaches. We hypothesised that, when present in excess, DAX1 must repress the action of early testis-forming genes. We investigated the effect of Dax1 over expression, using the Dax1 transgenic mouse line, Dax1812 (1), on expression of Sox9, a critical testis-forming gene. Immunostaining of Dax1812 gonads revealed reduced Sox9 expression, suggesting excess Dax1 antagonises Sox9 upregulation during the early stages of sex determination. To determine whether antagonism of Sox9 was occurring at the transcriptional level we assessed the effect of excess Dax1 on the activity of the Testis-Specific Enhancer of Sox9 (TES), which drives Sox9 transcription in the developing XY gonad (3). In combination, the in vivo and in vitro evidence strongly suggests that Dax1, when present in excess, can repress Sox9 expression through TES and that this repression occurs through inhibition of Steroidogenic Factor-1 activity. With this work we have identified a potential mechanism for disruption of the male-specific sex determination pathway caused by DAX1 duplication and leading to DSD in XY individuals.

scholarly journals Transcriptomic-Based Quantification of the Epithelial-Hybrid-Mesenchymal Spectrum Across Biological Contexts

Biomolecules ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 29
Author(s):  
Susmita Mandal ◽  
Tanishq Tejaswi ◽  
Rohini Janivara ◽  
Syamanthak Srikrishnan ◽  
Pradipti Thakur ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cancer Metastasis ◽  
Expression Profiles ◽  
Gene List ◽  
Cellular Reprogramming ◽  
Induced Pluripotent ◽  
Patient Prognosis ◽  
High Degree

Epithelial-mesenchymal plasticity (EMP) underlies embryonic development, wound healing, and cancer metastasis and fibrosis. Cancer cells exhibiting EMP often have more aggressive behavior, characterized by drug resistance, and tumor-initiating and immuno-evasive traits. Thus, the EMP status of cancer cells can be a critical indicator of patient prognosis. Here, we compare three distinct transcriptomic-based metrics—each derived using a different gene list and algorithm—that quantify the EMP spectrum. Our results for over 80 cancer-related RNA-seq datasets reveal a high degree of concordance among these metrics in quantifying the extent of EMP. Moreover, each metric, despite being trained on cancer expression profiles, recapitulates the expected changes in EMP scores for non-cancer contexts such as lung fibrosis and cellular reprogramming into induced pluripotent stem cells. Thus, we offer a scoring platform to quantify the extent of EMP in vitro and in vivo for diverse biological applications including cancer.

scholarly journals Pluripotent Cell Models for Gonadal Research

2019 ◽  
Vol 20 (21) ◽  
pp. 5495 ◽  
Author(s):  
Daniel Rodríguez Gutiérrez ◽  
Anna Biason-Lauber
Keyword(s):  
Pluripotent Stem Cells ◽  
Cellular Reprogramming ◽  
Patient Specific ◽  
Cell Models ◽  
In Vitro Differentiation ◽  
Sex Development ◽  
Complex Process ◽  
Differences Of Sex Development ◽  
Induced Pluripotent

Sex development is a complex process involving many genes and hormones. Defects in this process lead to Differences of Sex Development (DSD), a group of heterogeneous conditions not as rare as previously thought. Part of the obstacles in proper management of these patients is due to an incomplete understanding of the genetics programs and molecular pathways involved in sex development and DSD. Several challenges delay progress and the lack of a proper model system for the single patient severely hinders advances in understanding these diseases. The revolutionary techniques of cellular reprogramming and guided in vitro differentiation allow us now to exploit the versatility of induced pluripotent stem cells to create alternatives models for DSD, ideally on a patient-specific personalized basis.

scholarly journals Transcriptomic-based quantification of the epithelial-hybrid-mesenchymal spectrum across biological contexts

2021 ◽  
Author(s):  
Susmita Mandal ◽  
Tanishq Tejaswi ◽  
Rohini Janivara ◽  
Syamanthak Srikrishnan ◽  
Pradipti Thakur ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cancer Metastasis ◽  
Expression Profiles ◽  
Gene List ◽  
Cellular Reprogramming ◽  
Induced Pluripotent ◽  
Patient Prognosis ◽  
High Degree

AbstractEpithelial-mesenchymal plasticity (EMP) underlies embryonic development, wound healing, and cancer metastasis and fibrosis. Cancer cells exhibiting EMP often have more aggressive behavior, characterized by drug resistance, and tumor-initiating and immuno-evasive traits. Thus, the EMP status of cancer cells can be a critical indicator of patient prognosis. Here, we compare three distinct transcriptomic-based metrics – each derived using a different gene list and algorithm – that quantify the EMP spectrum. Our results for 96 cancer-related RNA-seq datasets reveal a high degree of concordance among these metrics in quantifying the extent of EMP. Moreover, each metric, despite being trained on cancer expression profiles, recapitulates the expected changes in EMP scores for non-cancer contexts such as lung fibrosis and cellular reprogramming into induced pluripotent stem cells. Thus, we offer a scoring platform to quantify the extent of EMP in vitro and in vivo for diverse biological applications including cancer.

Targeting the Non-Coding Genome for the Diagnosis of Disorders of Sex Development

2021 ◽  
pp. 1-19
Author(s):  
Gabby Atlas ◽  
Rajini Sreenivasan ◽  
Andrew Sinclair
Keyword(s):  
Genetic Diagnosis ◽  
Disorders Of Sex Development ◽  
Regulatory Elements ◽  
Gonadal Development ◽  
Comparative Genomic ◽  
Enhancer Activity ◽  
Sex Development ◽  
Genomic Regions

Disorders of sex development (DSD) are a complex group of conditions with highly variable clinical phenotypes, most often caused by failure of gonadal development. DSD are estimated to occur in around 1.7% of all live births. Whilst the understanding of genes involved in gonad development has increased exponentially, approximately 50% of patients with a DSD remain without a genetic diagnosis, possibly implicating non-coding genomic regions instead. Here, we review how variants in the non-coding genome of DSD patients can be identified using techniques such as array comparative genomic hybridization (CGH) to detect copy number variants (CNVs), and more recently, whole genome sequencing (WGS). Once a CNV in a patient’s non-coding genome is identified, putative regulatory elements such as enhancers need to be determined within these vast genomic regions. We will review the available online tools and databases that can be used to refine regions with potential enhancer activity based on chromosomal accessibility, histone modifications, transcription factor binding site analysis, chromatin conformation, and disease association. We will also review the current in vitro and in vivo techniques available to demonstrate the functionality of the identified enhancers. The review concludes with a clinical update on the enhancers linked to DSD.

Ovotesticular disorders of sex development in FGF9 mouse models of human synostosis syndromes

2020 ◽  
Vol 29 (13) ◽  
pp. 2148-2161
Author(s):  
Anthony D Bird ◽  
Brittany M Croft ◽  
Masayo Harada ◽  
Lingyun Tang ◽  
Liang Zhao ◽  
...  
Keyword(s):  
Disorders Of Sex Development ◽  
Gonadal Development ◽  
Testis Development ◽  
Sex Development ◽  
Mouse Mutants ◽  
Skeletal Defects ◽  
Xy Sex Reversal

Abstract In mice, male sex determination depends on FGF9 signalling via FGFR2c in the bipotential gonads to maintain the expression of the key testis gene SOX9. In humans, however, while FGFR2 mutations have been linked to 46,XY disorders of sex development (DSD), the role of FGF9 is unresolved. The only reported pathogenic mutations in human FGF9, FGF9S99N and FGF9R62G, are dominant and result in craniosynostosis (fusion of cranial sutures) or multiple synostoses (fusion of limb joints). Whether these synostosis-causing FGF9 mutations impact upon gonadal development and DSD etiology has not been explored. We therefore examined embryonic gonads in the well-characterized Fgf9 missense mouse mutants, Fgf9S99N and Fgf9N143T, which phenocopy the skeletal defects of FGF9S99N and FGF9R62G variants, respectively. XY Fgf9S99N/S99N and XY Fgf9N143T/N143T fetal mouse gonads showed severely disorganized testis cords and partial XY sex reversal at 12.5 days post coitum (dpc), suggesting loss of FGF9 function. By 15.5 dpc, testis development in both mutants had partly recovered. Mitotic analysis in vivo and in vitro suggested that the testicular phenotypes in these mutants arise in part through reduced proliferation of the gonadal supporting cells. These data raise the possibility that human FGF9 mutations causative for dominant skeletal conditions can also lead to loss of FGF9 function in the developing testis, at least in mice. Our data suggest that, in humans, testis development is largely tolerant of deleterious FGF9 mutations which lead to skeletal defects, thus offering an explanation as to why XY DSDs are rare in patients with pathogenic FGF9 variants.

scholarly journals Long-Range Regulation of Key Sex Determination Genes

2021 ◽  
pp. 1-21
Author(s):  
Roberta Migale ◽  
Michelle Neumann ◽  
Robin Lovell-Badge
Keyword(s):  
Sex Determination ◽  
Long Range ◽  
Current Knowledge ◽  
Disorders Of Sex Development ◽  
Sexually Dimorphic ◽  
Protein Coding ◽  
Sex Development ◽  
Human Disorders ◽  
The Many

The development of sexually dimorphic gonads is a unique process that starts with the specification of the bipotential genital ridges and culminates with the development of fully differentiated ovaries and testes in females and males, respectively. Research on sex determination has been mostly focused on the identification of sex determination genes, the majority of which encode for proteins and specifically transcription factors such as SOX9 in the testes and FOXL2 in the ovaries. Our understanding of which factors may be critical for sex determination have benefited from the study of human disorders of sex development (DSD) and animal models, such as the mouse and the goat, as these often replicate the same phenotypes observed in humans when mutations or chromosomic rearrangements arise in protein-coding genes. Despite the advances made so far in explaining the role of key factors such as SRY, SOX9, and FOXL2 and the genes they control, what may regulate these factors upstream is not entirely understood, often resulting in the inability to correctly diagnose DSD patients. The role of non-coding DNA, which represents 98% of the human genome, in sex determination has only recently begun to be fully appreciated. In this review, we summarize the current knowledge on the long-range regulation of 2 important sex determination genes, <i>SOX9</i> and <i>FOXL2</i>, and discuss the challenges that lie ahead and the many avenues of research yet to be explored in the sex determination field.

scholarly journals Modelling the Human Placental Interface In Vitro—A Review

Micromachines ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 884
Author(s):  
Marta Cherubini ◽  
Scott Erickson ◽  
Kristina Haase
Keyword(s):  
Drug Testing ◽  
Cell Types ◽  
In Vitro Models ◽  
Placental Development ◽  
Scientific Challenge ◽  
Induced Pluripotent ◽  
And Function ◽  
And Control

Acting as the primary link between mother and fetus, the placenta is involved in regulating nutrient, oxygen, and waste exchange; thus, healthy placental development is crucial for a successful pregnancy. In line with the increasing demands of the fetus, the placenta evolves throughout pregnancy, making it a particularly difficult organ to study. Research into placental development and dysfunction poses a unique scientific challenge due to ethical constraints and the differences in morphology and function that exist between species. Recently, there have been increased efforts towards generating in vitro models of the human placenta. Advancements in the differentiation of human induced pluripotent stem cells (hiPSCs), microfluidics, and bioprinting have each contributed to the development of new models, which can be designed to closely match physiological in vivo conditions. By including relevant placental cell types and control over the microenvironment, these new in vitro models promise to reveal clues to the pathogenesis of placental dysfunction and facilitate drug testing across the maternal–fetal interface. In this minireview, we aim to highlight current in vitro placental models and their applications in the study of disease and discuss future avenues for these in vitro models.

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