Search

Sox9 plays an essential role in mammalian testis formation. It has been reported that gene expression in the testes is regulated by enhancers. Among them, mXYSRa/Enh13—which is located at far upstream of the transcription start site—plays a critical role, wherein its deletion causes complete male-to-female sex reversal in mice. It has been proposed that the binding sites (BSs) of SOX9 and SRY, the latter of which is the sex determining gene on the Y chromosome, are associated with mXYSRa/Enh13. They function as an enhancer, whereby the sequences are evolutionarily conserved and in vivo binding of SOX9 and SRY to mXYSRa/Enh13 has been demonstrated previously. However, their precise in vivo functions have not been examined to date. To this end, this study generated mice with substitutions on the SOX9 and SRY BSs to reveal their in vivo functions. Homozygous mutants of SOX9 and SRY BS were indistinguishable from XY males, whereas double mutants had small testes, suggesting that these functions are redundant and that there is another functional sequence on mXYSRa/Enh13, since mXYSRa/Enh13 deletion mice are XY females. In addition, the majority of hemizygous mice with substitutions in SOX9 BS and SRY BS were female and male, respectively, suggesting that SOX9 BS contributes more to SRY BS for mXYSRa/Enh13 to function. The additive effect of SOX9 and SRY via these BSs was verified using an in vitro assay. In conclusion, SOX9 BS and SRY BS function redundantly in vivo, and at least one more functional sequence should exist in mXYSRa/Enh13.

Pituitary gigantism is a rare endocrinopathy characterized by tall stature due to growth hormone (GH) hypersecretion. This condition is generally linked to a genetic predisposition to tumors that produce GH or GH-releasing hormone (GHRH). Here, we report a Japanese woman who exhibited prominent body growth from infancy to reach an adult height of 197.4 cm (+7.4 standard deviation). Her blood GH levels were markedly elevated. She carried no pathogenic variants in known growth-controlling genes but had a hitherto unreported 752 kb heterozygous deletion at 20q11.23. The microdeletion was located 8.9 kb upstream of GHRH and encompassed exons 2–9 of a ubiquitously expressed gene TTI1 together with 12 other genes, pseudogenes and non-coding RNAs. Transcript analyses of the patient’s leukocytes showed that the microdeletion produced chimeric mRNAs consisting of exon 1 of TTI1 and all coding exons of GHRH. In silico analysis detected promoter-associated genomic features around TTI1 exon 1. Genome-edited mice carrying the same microdeletion recapitulated accelerated body growth from a few weeks after birth. The mutant mice developed pituitary hyperplasia and exhibited ectopic Ghrh expression in all tissues examined. Thus, the extreme phenotype of pituitary gigantism in the patient likely reflects GHRH overexpression driven by an acquired promoter. The results of this study indicate that germline submicroscopic deletions have the potential to cause conspicuous developmental abnormalities due to gene overexpression. Furthermore, this study provides evidence that constitutive expression of a hormone-encoding gene can result in congenital disease.

The eye and brain are composed of elaborately organized tissues, development of which is supported by spatiotemporally precise expression of a number of transcription factors and developmental regulators. Here we report the molecular and genetic characterization of Integrator complex subunit 15 (INTS15). INTS15 was identified in search for the causative gene(s) for an autosomal-dominant eye disease with variable individual manifestation found in a large pedigree. While homozygous INTS15 knockout mice are embryonic lethal, mutant mice lacking a small C-terminal region of INTS15 show ocular malformations similar to the human patients. INTS15 is highly expressed in the eye and brain during embryogenesis and stably interacts with the Integrator complex to support snRNA 3′ end processing. Its knockdown resulted in missplicing of a large number of genes, probably as a secondary consequence, and substantially affected genes associated with eye and brain development. Moreover, studies using human iPS-derived neural progenitor cells showed that INTS15 is critical for axonal outgrowth in retinal ganglion cells. This study suggests a new link between general transcription machinery and a highly specific hereditary disease.

PurposeWolf-Hirschhorn syndrome (WHS), a contiguous gene syndrome caused by the hemizygous deletion of the distal short arm of chromosome 4 whereNSD2is, reportedly exhibits specific DNA methylation signatures in peripheral blood cells. However, responsible genomic loci for signatures are unreported. The objective of the study is to define the loci of WHS-related DNA methylation signatures and to explore the role ofNSD2for the signatures.MethodsWe conducted genome-wide methylation analysis of individuals with WHS orNSD2variants using array. We studied genome-edited knock in mice or induced pluripotent stem cells to explore the function ofNSD2variants which are observed in congenital anomaly cases.ResultsThree undiagnosed cases withNSD2variants showed WHS-related DNA methylation signatures. These variants were validated to beNSD2loss-of-function in induced pluripotent stem cells or genome-edited knock-in mice. p.Pro905Leu variant decreased Nsd2 protein levels, and changed Histone H3-Lysine 36 demethylation levels in similar way in the same genomic regions asNsd2knock out mice regulated.Nsd2knock out mice exhibited common DNA methylation changes.ConclusionThese results revealed that WHS-related DNA methylation signatures are dependent onNSD2dysfunction and are useful in diagnosingNSD2variants of unknown significance.

Mammalian sex chromosomes are highly conserved, and sex is determined by

SOX9, a transcription factor, is expressed in the undifferentiated XX and XY gonads. SRY induces significant upregulation of SOX9 expression in XY gonads. Loss-of-function SOX9 variants cause testicular dysgenesis in 46,XY patients, while duplication of the total gene or the upstream regulatory region results in testicular development in 46,XX patients. However, gain-of-function (GoF) SOX9 variants have not been reported previously. We report the case of a 16-year-old female patient with a 46,XX karyotype who had masculinized external genitalia and unilateral ovotestis. Next-generation sequencing-based genetic screening for disorders of sex development led to the identification of a novel SOX9 variant (p.Glu50Lys), transmitted from the phenotypically normal father. Expression analysis showed that E50K-SOX9 enhanced transactivation of the luciferase reporter containing the testis enhancer sequence core element compared with that containing the wildtype-SOX9. This GoF activity was not observed in the luciferase reporter containing Amh, the gene for anti-Mullerian hormone. We genetically engineered female mice (Sox9E50K/E50K ), and they showed no abnormalities in the external genitalia or ovaries. In conclusion, a novel SOX9 variant with a promoter-specific GoF activity was identified in vitro; however, the disease phenotype was not recapitulated by the mouse model. At present, the association between the GoF SOX9 variant and the ovotestis phenotype remains unclear. Future studies are needed to verify the possible association.

In the CNS, oligodendrocyte precursor cells differentiate into oligodendrocytes to wrap their plasma membranes around neuronal axons, generating mature neural networks with myelin sheaths according to spatial and temporal patterns. While myelination is known to be one of the most dynamic cell morphological changes, the overall intrinsic and extrinsic molecular cues controlling myelination remain to be fully clarified. Here, we describe the biphasic roles of Rnd2, an atypical branch of the Rho family GTPase, in oligodendrocyte myelination during development and after maturation in mice. Compared with littermate controls, oligodendrocyte-specific Rnd2 knockout mice exhibit decreased myelin thickness at the onset of myelination but increased myelin thickness in the later period. Larger proportions of Rho kinase and its substrate Mbs, the signaling unit that negatively regulates oligodendrocyte myelination, are phosphorylated at the onset of myelination, while their smaller proportions are phosphorylated in the later period. In addition, we confirm the biphasic role of Rnd2 through experiments with oligodendrocyte-specific Rnd2 transgenic mice. We conclude that Rnd2 positively regulates myelination in the early myelinating period and negatively regulates myelination in the later period. This unique modulator thus plays different roles depending on the myelination period.

POU Class 1 Homeobox1 (POU1F1/Pou1f1) is a well-established pituitary-specific transcription factor, and causes, when mutated, combined pituitary hormone deficiency in humans and mice. POU1F1/Pou1f1 has 2 isoforms: the alpha and beta isoforms. Recently, pathogenic variants in the unique coding region of the beta isoform (beta domain) and the intron near the exon–intron boundary for the beta domain were reported, although their functional consequences remain obscure. In this study, we generated mice carrying the Pou1f1 c.143-83A>G substitution that recapitulates the human intronic variant near the exon–intron boundary for the beta domain. Homozygous mice showed postnatal growth failure, with an average body weight that was 35% of wild-type littermates at 12 weeks, which was accompanied by anterior pituitary hypoplasia and deficiency of circulating insulin-like growth factor 1 and thyroxine. The results of RNA-seq analysis of the pituitary gland were consistent with reduction of somatotrophs, and this was confirmed immunohistochemically. Reverse transcription polymerase chain reaction of pituitary Pou1f1 mRNA showed abnormal splicing in homozygous mice, with a decrease in the alpha isoform, an increase in the beta isoform, and the emergence of the exon-skipped transcript. We further characterized artificial variants in or near the beta domain, which were candidate positions of the branch site in pre-mRNA, using cultured cell–basis analysis and found that only c.143-83A>G produced transcripts similar to the mice model. Our report is the first to show that the c.143-83A>G variant leads to splicing disruption and causes morphological and functional abnormalities in the pituitary gland. Furthermore, our mice will contribute understanding the role of POU1F1/Pou1f1 transcripts in pituitary development.

Leydig cells in fetal testes play crucial roles in masculinizing fetuses through androgen production. Gene knockout studies have revealed that growth factors are implicated in fetal Leydig cell (FLC) differentiation, but little is known about the mechanisms regulating this process. We investigated this issue by characterizing FLC progenitor cells using single-cell RNA sequencing. The sequence datasets suggested that thymosin β10 (Tmsb10) was transiently upregulated in the progenitors. While studying the function of Tmsb10, we revealed that platelet-derived growth factor (PDGF) regulated ciliogenesis through the RAS/ERK and PI3K/AKT pathways, and thereby promoted desert hedgehog (DHH)-dependent FLC differentiation. Tmsb10 expressed in the progenitor cells induced their differentiation into FLCs by suppressing the RAS/ERK pathway. Through characterizing the transiently expressed Tmsb10 in the FLC progenitors, this study unveiled the molecular process of FLC differentiation and showed that it is cooperatively induced by DHH and PDGF.

Generation of mutant imprinting control region (ICR) mice using genome editing is an important approach for elucidating ICR functions. IG-DMR is an ICR in the Dlk1-Dio3 imprinted domain that contains functional regions—in both parental alleles—that are essential for embryonic development. One drawback of this approach is that embryonic lethality can occur from aberrant expression of the imprinted genes if IG-DMR gets mutated in either the paternal or maternal allele. To overcome this problem, we generated mosaic mice that contained cells with modified IG-DMR alleles and wild-type cells using the 2CC method that allowed for microinjection of the CRISPR/Cas9 constructs into a blastomere of 2-cell embryos. This method improved the birth rate of the founder pups relative to that obtained using the standard protocol. We also successfully produced mosaic mice in which the tandem repeat array sequence in the IG-DMR had been replaced by homology directed repair. Additionally, paternal transmission of the replaced allele caused aberrant expression of the imprinted genes due to hypomethylation of the IG-DMR, indicating that the replaced allele recapitulated our deletion model. Our results indicate that this method is useful for the generation of mutant mice in which a genomic locus essential for normal development has been genetically edited.

Nuclear factor of activated T-cells 5 (NFAT5) directly binds to the promoter of the RING finger protein 183 (RNF183) gene and induces its transcription under hypertonic conditions in mouse inner-medullary collecting duct (mIMCD-3) cells. However, there is no specific anti-RNF183 antibody for immunostaining; therefore, it is unclear whether NFAT5 regulates RNF183 expression in vivo and where RNF183 is localized in the kidney. This study investigated NFAT5-regulated in vivo RNF183 expression and localization using CRISPR/Cas9-mediated RNF183-green fluorescent protein (RNF183-GFP) knock-in mice. GFP with linker sequences was introduced upstream of an RNF183 open reading frame in exon 3 by homologous recombination through a donor plasmid. Immunofluorescence staining using GFP antibody revealed that GFP signals gradually increase from the outer medulla down to the inner medulla and colocalize with aquaporin-2. Furosemide treatment dramatically decreased RNF183 expression in the renal medulla, consistent with the decrease in NFAT5 protein and target gene mRNA expression. Furosemide treatment of mIMCD-3 cells did not affect mRNA expression and RNF183 promoter activities. These results indicated that RNF183 is predominantly expressed in the renal medullary collecting ducts, and that decreased renal medullary tonicity by furosemide treatment decreases RNF183 expression by NFAT5 downregulation.

Background The stimulatory G-protein α -subunit encoded by GNAS exons 1–13 ( GNAS -Gs α ) mediates signal transduction of multiple G protein–coupled receptors, including arginine vasopressin receptor 2 (AVPR2). Various germline-derived loss-of-function GNAS -Gs α variants of maternal and paternal origin have been found in pseudohypoparathyroidism type Ia and pseudopseudohypoparathyroidism, respectively. Specific somatic gain-of-function GNAS -Gs α variants have been detected in McCune–Albright syndrome and may result in phosphate wasting. However, no germline-derived gain-of-function variant has been identified, implying that such a variant causes embryonic lethality. Methods We performed whole-exome sequencing in two families with dominantly inherited nephrogenic syndrome of inappropriate antidiuresis (NSIAD) as a salient phenotype after excluding a gain-of-function variant of AVPR2 and functional studies for identified variants. Results Whole-exome sequencing revealed two GNAS -Gs α candidate variants for NSIAD: GNAS -Gs α p.(F68_G70del) in one family and GNAS -Gs α p.(M255V) in one family. Both variants were absent from public and in-house databases. Of genes with rare variants, GNAS -Gs α alone was involved in AVPR2 signaling and shared by the families. Protein structural analyses revealed a gain-of-function–compatible conformational property for p.M255V-Gs α , although such assessment was not possible for p.F68_G70del-Gs α . Both variants had gain-of-function effects that were significantly milder than those of McCune–Albright syndrome–specific somatic Gs α variants. Model mice for p.F68_G70del-Gs α showed normal survivability and NSIAD-compatible phenotype, whereas those for p.M255V-Gs α exhibited severe failure to thrive. Conclusions This study shows that germline-derived gain-of-function rare variants of GNAS -Gs α exist and cause NSIAD as a novel Gs α -mediated genetic disease. It is likely that AVPR2 signaling is most sensitive to GNAS -Gs α ’s gain-of-function effects.

The sex-determining region of the Y chromosome, Sry/SRY, is an initiation factor for testis development in both humans and mice. Although the functional compatibility between murine SRY and human SRY was previously examined in transgenic mice, their equivalency remains inconclusive. Because molecular interaction and timeline of mammalian sex determination were mostly described in murine experiments, we generated a mouse model in which Sry was substituted with human SRY to verify the compatibility. The mouse model had the human SRY open reading frame at the locus of murine Sry exon 1—Sry(SRY) mice—and was generated using the CRISPR/Cas9 system. The reproductive system of the mice was analyzed. The expression of human SRY in the fetal gonadal ridge of Sry(SRY) mice was detected. The external and internal genitalia of adult Sry(SRY) mice were similar to those of wild-type females, without any significant difference in anogenital distance. Sry(SRY) mice obtained gonads, which were morphologically considered as ovaries. Histological analysis revealed that the cortical regions of gonads from adult Sry(SRY) mice contained few follicles. We successfully replaced genes on the Y chromosome with targeted genome editing using the CRISPR/Cas9 system. Because the Sry(SRY) XY mice did not develop testis, we concluded that human SRY was insufficient to drive testis development in mouse embryos. The difference in response elements and lack of glutamine-rich domains may have invalidated human SRY function in mice. Signal transduction between Sry/SRY expression and Sox9/SOX9 activation is possibly organized in a species-specific manner.

SOX9, a transcription factor, is expressed in the undifferentiated XX and XY gonads. SRY induces significant upregulation of SOX9 expression in XY gonads. Loss-of-function SOX9 variants cause testicular dysgenesis in 46,XY patients, while duplication of the total gene or the upstream regulatory region results in testicular development in 46,XX patients. However, gain-of-function (GoF) SOX9 variants have not been reported previously. We report the case of a 16-year-old female patient with a 46,XX karyotype who had masculinized external genitalia and unilateral ovotestis. Next-generation sequencing-based genetic screening for disorders of sex development led to the identification of a novel SOX9 variant (p.Glu50Lys), transmitted from the phenotypically normal father. Expression analysis showed that E50K-SOX9 enhanced transactivation of the luciferase reporter containing the testis enhancer sequence core element compared with that containing the wildtype-SOX9. This GoF activity was not observed in the luciferase reporter containing Amh, the gene for anti-Müllerian hormone. We genetically engineered female mice (Sox9

Oligodendrocyte precursor cells differentiate into oligodendrocytes to wrap their plasma membranes around axons. While myelination is known to be one of the most dynamic cell morphological changes, the overall molecular pictures remain to be elucidated. Herein we describe the biphasic roles of Rnd2, an atypical branch of the Rho GTPase, in oligodendrocyte myelination during development and after maturation. Oligodendrocyte-specific Rnd2 knockout mice exhibit decreased myelin thickness in the onset of myelination whereas the knockout increased thickness in the subsequent process. Consistently, the biphasic role of Rnd2 is obtained using oligodendrocyte-specific Rnd2 transgenic mice. Indeed, Rho kinase and the substrate Mbs, the signaling unit regulating negatively myelination, exist in the decreased phosphorylated forms in the early developmental stage and in the increased phosphorylated forms in the subsequent stage. Therefore, Rnd2 uniquely regulates myelination, demonstrating the presence of a modulator that plays different roles depending on the myelination process.

Sox9 plays critical roles in testis formation. By mapping four familial cases of disorders of sexual development, a 32.5 kb sequence located far upstream of SOX9 was previously identified as being a commonly deleted region and named the XY sex reversal region (XYSR). To narrow down a responsible sequence in XYSR, we generated mutant mice with a series of deletions in XYSR by application of the CRISPR/Cas9 system, using a mixture of sgRNAs targeting several kilobase (kb) intervals in the region. When the whole XYSR corresponding sequence in mice was deleted in XY karyotype individuals, the mutation resulted in female offspring, suggesting that an expression mechanism of SOX9/Sox9 through XYSR is conserved in human and mouse. Male-to-female sex reversal was found in mice with a 4.8 kb deletion. We identified a sequence conserved among humans, mice, and opossum, the deletion of which (783 bp) in mice resulted in male-to-female sex reversal. The sequence includes a recently reported critical gonad enhancer for Sox9. Although it cannot be concluded that the human sequence is responsible for XYSR, it is likely. This method is applicable for fine mapping of responsible sequences for disease-causing deletions especially with regard to rare diseases.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for genome editing. In this study, using a microinjection-based CRISPR/Cas9 system, we efficiently generated mouse lines carrying mutations at the Irx3 and Irx5 loci, which are located in close proximity on a chromosome and are functionally redundant. During the generation of Irx3/Irx5 double mutant mice, a deletion of ~0.5 Mb between the Irx3 and Irx5 loci was unintentionally identified in 6 out of 27 living pups by PCR based genotyping analysis. This deletion was confirmed by DNA fluorescence in situ hybridization analysis of fibroblasts. These results indicate that the mutant mice with a deletion of at least 0.5 Mb in their genome can be generated by the CRISPR/Cas9 system through microinjection into fertilized eggs. Our findings expand the utility of the CRISPR/Cas9 system in production of disease model animals with large deletions.

The CRISPR/Cas9 system is a powerful genome editing tool for the production of genetically modified animals. To produce mutant mice, chimeric single-guide RNA (sgRNA) is cloned in a plasmid vector and a mixture of sgRNA and Cas9 are microinjected into the fertilized eggs. An issue associated with gene manipulation using the CRISPR/Cas9 system is that there can be off-target effects. To simplify the production of mutant mice with low risks of off-target effects caused by the CRISPR/Cas9 system, we demonstrated that genetically modified mice can be efficiently obtained using chemically synthesized CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and modified Cas9s, such as the nickase version and FokI-fused catalytically inactive Cas9, by microinjection into fertilized eggs. Using this method, it is no longer necessary to clone sgRNA into a plasmid vector, and this enables high-throughput production of mutant mice.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for creation of mutant mice with mutations mirroring those in human patients. Various methods have been developed for this purpose, including deletions, inversions, and translocations. So far, mutant mice with deletions of up to 1.2 megabases (Mb) have been generated by microinjection of the CRISPR/Cas9 system into fertilized eggs; however, a method for generation of mutant mice with a deletion of more than several Mb size is necessary because such deletions have often been identified as possible causes of human diseases. With an aim to enable the generation of disease models carrying large deletions with a breakpoint in custom-designed sequences, we developed a method for induction of an Mb-sized deletion by microinjection of a pair of sgRNAs, Cas9, and a donor plasmid into fertilized eggs. Using this method, we efficiently and rapidly generated mutant mice carrying deletions up to 5 Mb.

Vascular growth is necessary for normal lung development. Although endothelial progenitor cells (EPCs) play an important role in vascularization, little is known about EPC function in congenital diaphragmatic hernia (CDH), a severe neonatal condition that is associated with pulmonary hypoplasia. We hypothesized that the function of endothelial colony-forming cells (ECFCs), a type of EPC, is impaired in CDH. Cord blood (CB) was collected from full-term CDH patients and healthy controls. We assessed CB progenitor cell populations as well as plasma vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1α (SDF1α) levels. CB ECFC clonogenicity; growth kinetics; migration; production of VEGF, SDF1α, and nitric oxide (NO); vasculogenic capacity; and mRNA expression of VEGF-A, fms-related tyrosine kinase 1 (FLT1), kinase insert domain receptor (KDR), nitric oxide synthase (NOS) 1–3, SDF1, and chemokine (C-X-C motif) receptor 4 (CXCR4) were also assessed. Compared with controls, CB ECFCs were decreased in CDH. CDH ECFCs had reduced potential for self-renewal, clonogenicity, proliferation, and migration. Their capacity for NO production was enhanced but their response to VEGF was blunted in CDH ECFCs. In vivo potential for de novo vasculogenesis was reduced in CDH ECFCs. There was no difference in CB plasma VEGF and SDF1α concentrations, VEGF and SDF1α production by ECFCs, and ECFC mRNA expression of VEGF-A, FLT1, KDR, NOS1-3, SDF1, and CXCR4 between CDH and control subjects. In conclusion, CB ECFC function is disrupted in CDH, but these changes may be caused by mechanisms other than alteration of VEGF-NO and SDF1-CXCR4 signaling.