Biology subjects

Koopman, P.

Publications and source records attributed to Koopman, P..

7 recordsLinked to original sources

bioRxiv2022

Functional genomics analysis identifies impairment of HNF1B function as a cause of Mayer-Rokitansky-Küster-Hauser syndrome.

Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome is a congenital condition characterized by aplasia or hypoplasia of the uterus and vagina in women with a typical 46,XX karyotype. This condition can occur as type I when isolated or as type II when associated with extragenital anomalies including kidney and skeletal abnormalities. The genetic basis of MRKH syndrome remains unexplained and several candidate genes have been proposed to play a role in its etiology, including HNF1B, LHX1, and WNT4. Here, we conducted a genomic analysis of 13 women affected by MRKH syndrome, resulting in the identification of candidate genes, including several novel candidates. We focused on HNF1B for further investigation due to its known association with, but unknown etiological role in, MRKH syndrome. We ablated Hnf1b specifically in the epithelium of the Mullerian ducts in mice, and found that this caused hypoplastic development of both the epithelial and stromal compartments of the uterus, as well as kidney anomalies, closely mirroring the MRKH type II phenotype. Using single-cell RNA sequencing of uterine tissue in the Hnf1b-ablated embryos, we analyzed the molecules and pathways downstream of Hnf1b, revealing a dysregulation of processes associated with cell proliferation, migration, and differentiation. Thus, we establish that loss of Hnf1b function leads to an MRKH phenotype, and generate the first mouse model of MRKH syndrome type II. Our results support the diagnostic value of HNF1B in clinical genetic testing for MRKH syndrome, and shed new light on the genetic causes of this poorly understood condition in womens reproductive health.

developmental biology↗

bioRxiv2021

Functional analysis of Mmd2 and related PAQR genes during sex determination in mice

Sex determination in eutherian mammals is controlled by the Y-linked gene Sry, which drives the formation of testes in male embryos. Despite extensive study, the genetic steps linking Sry action and male sex determination remain largely unknown. Here, we focused on Mmd2, a gene that encodes a member of the progestin and adipoQ receptor (PAQR) family. We show that Mmd2 is expressed during the sex-determining period in XY but not XX gonads, specifically in the Sertoli cell lineage which orchestrates early testis development. Analysis of knockout mice deficient in Sox9 and Sf1 revealed that Mmd2 operates downstream of these known sex-determining genes. However, when we used CRISPR to ablate Mmd2 in the mouse, fetal testis development appeared to progress normally. To determine if other genes might have compensated for the loss of Mmd2, we identified the closely related PAQR family members Paqr8 and Mmd as also being expressed during testis development. We used CRISPR to generate mouse strains deficient in Paqr8 and Mmd, but both knockout lines appeared phenotypically normal and fertile. Finally, we generated Mmd2;Mmd and Mmd2;Paqr8 double-null embryos and again observed normal testis development. These results may reflect functional redundancy among these factors. Our findings highlight the difficulties involved in identifying genes with a functional role in sex determination and gonadal development through expression screening and loss-of-function analyses of individual candidate genes, and may help to explain the paucity of genes in which variations have been found to cause human disorders/differences of sex development.

developmental biology↗

bioRxiv2021

Generation and mutational analysis of a transgenic mouse model of human SRY

SRY is the Y-chromosomal gene that determines male sex development in humans and most other mammals. After three decades of study, we still lack a detailed understanding of which domains of the SRY protein are required to engage pathway of gene activity leading to testis development. Some insight has been gained from the study of genetic variations underlying differences/disorders of sex determination (DSD), but the lack of a system of experimentally generating SRY mutations and studying their consequences in vivo has limited progress in the field. To address this issue, we generated a mouse model carrying a human SRY transgene able to drive male sex determination in XX mice. Using CRISPR-Cas9 gene editing, we generated novel genetic modifications in each of SRYs three domains (N-terminal, HMG box, and C-terminal) and performed detailed analysis of their molecular and cellular effects on embryonic testis development. Our results provide new functional insights unique to human SRY and the causes of DSD, and present a versatile and powerful system in which to demonstrate causality of SRY variations in DSD, to functionally study the SRY variation database, and to characterize new pathogenic SRY variations found in DSD.

developmental biology↗

bioRxiv2020

A dominant-negative SOX18 mutant disrupts multiple regulatory layers essential to transcription factor activity.

Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged opossum and the human genetic disorder Hypotrichosis-Lymphedema-Telangiectasia-Renal Syndrome. Combing three single-molecule imaging assays in living cells, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its chromatin-binding dynamics. The dominant-negative effect is further amplified by recruiting the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular etiology of dominant-negative transcription factor function.

cell biology↗

bioRxiv2019

Sox17 expression in endocardium precursor cells regulates heart development in mice

The endocardium is the endothelial component of the vertebrate heart and plays a key role in heart development. Cardiac progenitor cells (CPCs) that express the homeobox gene Nkx2-5 give rise to the endocardium. Where, when, and how the endocardium segregates during embryogenesis have remained largely unknown, however. We now show that Nkx2-5+ CPCs that express the Sry-type HMG box gene Sox17 specifically differentiate into the endocardium in mouse embryos. Approximately 20% to 30% of Nkx2-5+CPCs transiently express Sox17 from embryonic day (E) 7.5 to E8.5.Although Sox17 is not essential or sufficient for endocardium fate, it can bias the fate of CPCs toward the endocardium. On the other hand, Sox17 expression in the endocardium is required for heart development. Deletion of Sox17 specifically in the mesoderm markedly impaired endocardium development with regard to cell proliferation and behavior. The proliferation of cardiomyocytes, ventricular trabeculation, and myocardium thickening were also impaired in a non-cell-autonomous manner in the Sox17 mutant, resulting in anomalous morphology of the heart, likely as a consequence of down-regulation of NOTCH signaling. Changes in gene expression profile in both the endocardium and myocardium preceded the reduction in NOTCH-related gene expression in the mutant embryos, suggesting that Sox17 expression in the endocardium regulates an unknown signal required for nurturing of the myocardium. Our results thus provide insight into differentiation of the endocardium and its role in heart development. SignificanceThe endocardium is vital for vertebrate heart development; however, the molecular mechanisms regulating fate determination and differentiation remain largely unknown. Here, we show that a part of the earliest cardiac progenitor cells (CPCs) transiently and exclusively express Sry-type HMG box gene Sox17 in the mouse embryo. Sox17-expressing CPCs specifically differentiate to the endocardium. Sox17 biases the fate of CPCs toward the endocardium, and regulates proliferation and cellular behavior cell autonomously. Conversely, Sox17 in the endocardium regulates the myocardium non-cell autonomously. Notably, Sox17 is required for the ventricular trabeculation via the NOTCH signal that is not directly induced but maintained by Sox17. This study, thus, sheds light on endocardium development.

developmental biology↗

bioRxiv2018

Nr5a1 suppression during the fetal period optimizes ovarian development by fine-tuning of Notch signaling

The nuclear receptor NR5A1 (also known as Ad4BP, or SF1) is essential for the initial steps of mammalian gonadal development. The Nr5a1 gene is equally expressed in XX and XY gonadal primordia, but after sex determination, is up-regulated in XY and down-regulated in XX gonads. We recently reported a case of 46, XX disorder of sex development (DSD) in which ectopically expressed NR5A1 in XX gonads led to an ovo-testicular phenotype, suggesting that excess NR5A1 can direct the development of immature XX gonads towards testicular formation. However, a direct causal relationship has not been demonstrated in an animal model. Here, using a Wt1-BAC (bacterial artificial chromosome) transgene system, we generated two lines of mice overexpressing Nr5a1 in the fetal gonads at different levels. One of these lines (Tg-S), highly expressing Nr5a1, revealed that enforced Nr5a1 expression alone is insufficient to switch the fate of the 46,XX gonads toward testicular formation in mice. In the other line (Tg-A) expressing Nr5a1 at lower level, ovarian development was compromised, with multi-oocyte follicles, reduced number of matured follicles, and impaired expression of Wnt4, resulting in late onset infertility at 20 weeks after birth. The phenotype was similar to that of genetically modified mice with impaired Notch signaling. Indeed, the expression level of Notch2 and 3 was significantly reduced in Tg-A mice, and the ovarian phenotype in Tg-A mice was almost completely rescued by in utero treatment with a Notch2 agonist HMN2-29. We conclude that suppression of Nr5a1 during the fetal period optimizes ovarian development by fine tuning of Notch signaling levels.\n\nAUTHOR SUMMARYSexual development is a process of differentiation from undifferentiated bipotential gonads, and insight into sexual differentiation will bring important new knowledge to our understanding of organogenesis. The nuclear receptor NR5A1 which is essential for mammalian gonadal development, is equally expressed in both gonadal primordia, but after sex determination, is up-regulated in XY and down-regulated in XX gonads. We have recently demonstrated that this down-regulation is mediated by ovarian transcription factor, Forkhead box L2 (FOXL2). This finding raised two key questions, whether Nr5a1 can function as a male sex-determining factor, and whether the repression is essential for appropriate ovarian development. By generating two lines Tg mice in XX gonads with different enforced expression levels of Nr5a1, our present study revealed that alterations in Nr5a1 dosage, either reduced or excessive, result in pathological effects in ovarian development and female fertility, indicating that the precise control of Nr5a1 at the transcriptional level is essential for optimal ovarian development. We envisage that the improved understanding of how this pathway regulates ovarian development and female fertility would aid the development of artificial somatic ovarian cells, which in turn may provide a valuable treatment option in reproductive medicine.\n\nABBREVIATIONS

developmental biology↗

bioRxiv2017

Gene editing of the multi-copy H2A.B gene family by a single pair of TALENS

In view of the controversy related to the generation of off-target mutations by gene editing approaches, we tested the specificity of TALENs by disrupting a multi-copy gene family using only one pair of TALENS. We show here that TALENS do display a high level of specificity by simultaneously knocking out the function of the three genes that encode for H2A.B.3. This represents the first described knockout of this histone variant.

genomics↗