Macrophage-enriched Sectm1a promotes efficient efferocytosis to attenuate ischemia/reperfusion-induced cardiac injury.

Efficient clearance and degradation of apoptotic cardiomyocytes by macrophages (collectively termed efferocytosis) is critical for inflammation resolution and restoration of cardiac function after myocardial ischemia/reperfusion (I/R). Here, we define secreted and transmembrane protein 1a (Sectm1a), a cardiac macrophage-enriched gene, as a modulator of macrophage efferocytosis in I/R-injured hearts. Upon myocardial I/R, Sectm1a-KO mice exhibited impaired macrophage efferocytosis, leading to massive accumulation of apoptotic cardiomyocytes, cardiac inflammation, fibrosis, and consequently, exaggerated cardiac dysfunction. By contrast, therapeutic administration of recombinant SECTM1A protein significantly enhanced macrophage efferocytosis and improved cardiac function. Mechanistically, SECTM1A could elicit autocrine effects on the activation of glucocorticoid-induced TNF receptor (GITR) at the surface of macrophages, leading to the upregulation of liver X receptor α (LXRα) and its downstream efferocytosis-related genes and lysosomal enzyme genes. Our study suggests that Sectm1a-mediated activation of the Gitr/LXRα axis could be a promising approach to enhance macrophage efferocytosis for the treatment of myocardial I/R injury.

A common allele increases endometrial Wnt4 expression, with antagonistic implications for pregnancy, reproductive cancers, and endometriosis.

The common human SNP rs3820282 is associated with multiple phenotypes including gestational length and likelihood of endometriosis and cancer, presenting a paradigmatic pleiotropic variant. Deleterious pleiotropic mutations cause the co-occurrence of disorders either within individuals, or across population. When adverse and advantageous effects are combined, pleiotropy can maintain high population frequencies of deleterious alleles. To reveal the causal molecular mechanisms of this pleiotropic SNP, we introduced this substitution into the mouse genome by CRISPR/Cas 9. Previous work showed that rs3820282 introduces a high-affinity estrogen receptor alpha-binding site at the Wnt4 locus. Here, we show that this mutation upregulates Wnt4 transcription in endometrial stroma, following the preovulatory estrogen peak. Effects on uterine transcription include downregulation of epithelial proliferation and induction of progesterone-regulated pro-implantation genes. We propose that these changes increase uterine permissiveness to embryo invasion, whereas they decrease resistance to invasion by cancer and endometriotic foci in other estrogen-responsive tissues.

Self-complementary AAV vector therapy for treating corneal cloudiness of mucopolysaccharidosis type VII (MPS VII).

PURPOSE: To design a novel efficacious scAAV-Gusb viral vector for treating Mucopolysaccharidosis Type VII (MPS VII) caused by a mutation in the ß-Glu gene (Gusb allele). METHODS: ß-Glu expression of single-stranded AAV-Gusb (ssAAV-Gusb) and self-complementary AAV (scAAV-Gusb) vectors are tested with cultured murine Gusb fibroblasts. The scAAV-Gusb vector was chosen in further studies to prolong the life span and treat corneal pathology of Gusb mice via intrahepatic injection of neonates and intrastromal injection in adults, respectively. Corneal pathology was studied using HRT2 in vivo confocal microscope and histochemistry in mice corneas. RESULTS: Both ssAAV-Gusb and scAAV-Gusb vectors expressed murine ß-Glu in cultured Gusb fibroblasts. The scAAV-Gusb vector had higher transduction efficiency than the ssAAV-Gusb vector. To prolong the life span of Gusb mice, neonates (3 days old) were administered with scAAV-Gusb virus via intrahepatic injection. The treatment improves the survival rate of Gusb mice, prolonging the median survival rate from 22.5 weeks (untreated) to 50 weeks (treated). Thereafter, we determined the efficacy of the scAAV-Gusb virus in ameliorating corneal cloudiness observed in aged Gusb mice. Both corneal cloudiness and stroma thickness decreased, and there was the presence of ß-Glu enzyme activity in the Gusb corneas receiving scAAV-Gusb virus associated with morphology change of amoeboid stromal cells in untreated to characteristic dendritic keratocytes morphology after 4-12 weeks of scAAV-Gusb virus injection. CONCLUSION: Intrahepatic injection of scAAV-Gusb is efficacious in prolonging the life span of Gusb mice, and intrastromal injection can ameliorate corneal phenotypes. Both strategies can be adapted for treating other MPS.

Development of functional resident macrophages in human pluripotent stem cell-derived colonic organoids and human fetal colon.

Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.

The gene therapy for corneal pathology with novel nonsense cystinosis mouse lines created by CRISPR Gene Editing.

PURPOSE: Cystinosis is an autosomal recessive lysosomal storage disease (LSDs) caused by mutations in the gene encoding cystinosin (CTNS) that leads to cystine crystal accumulation in the lysosome that compromises cellular functions resulting in tissue damage and organ failure, especially in kidneys and eyes. However, the underlying molecular mechanism of its pathogenesis remains elusive. Two novel mice lines created via CRISPR are used to examine the pathogenesis of cystinosis in the kidney and cornea and the treatment efficacy of corneal pathology using self-complimentary Adeno-associated viral (scAAV-CTNS) vector. METHODS: The CRISPR technique generated two novel cystinotic mouse lines, Ctnsis1 (an insertional mutation) and Ctnsis2 (a nonsense mutation). Immune histochemistry, renal functions test and HRT2 in vivo confocal microscopy were used to evaluate the age-related renal pathogenesis and treatment efficacy of the scAAV-CTNS virus in corneal pathology. RESULTS: Both mutations lead to the production of truncated Ctns proteins. Ctnsis1 and Ctnsis 2 mice exhibit the characteristic of cystinotic corneal crystal phenotype at four-week-old. Treatment with the scAAV-CTNS viral vector decreased the corneal crystals in the treated mice cornea. Ctnsis 1 show renal abnormalities manifested by increased urine volume, reduced urine osmolality, and the loss of response to Desmopressin (dDAVP) at 22-month-old but Ctnsis2 don't manifest renal pathology up to 2 years of age. CONCLUSIONS: Both Ctnsis1 and Ctnsis2 mice exhibit phenotypes resembling human intermediate nephropathic and ocular cystinosis, respectively. scAAV-CTNS viral vectors reduce the corneal cystine crystals and have a great potential as a therapeutic strategy for treating patients suffering from cystinosis.

Human iPSC-derived hepatocyte system models cholestasis with tight junction protein 2 deficiency.

Background & Aims: The truncating mutations in tight junction protein 2 (TJP2) cause progressive cholestasis, liver failure, and hepatocyte carcinogenesis. Due to the lack of effective model systems, there are no targeted medications for the liver pathology with TJP2 deficiency. We leveraged the technologies of patient-specific induced pluripotent stem cells (iPSC) and CRISPR genome-editing, and we aim to establish a disease model which recapitulates phenotypes of patients with TJP2 deficiency. Methods: We differentiated iPSC to hepatocyte-like cells (iHep) on the Transwell membrane in a polarized monolayer. Immunofluorescent staining of polarity markers was detected by a confocal microscope. The epithelial barrier function and bile acid transport of bile canaliculi were quantified between the two chambers of Transwell. The morphology of bile canaliculi was measured in iHep cultured in the Matrigel sandwich system using a fluorescent probe and live-confocal imaging. Results: The iHep differentiated from iPSC with TJP2 mutations exhibited intracellular inclusions of disrupted apical membrane structures, distorted canalicular networks, altered distribution of apical and basolateral markers/transporters. The directional bile acid transport of bile canaliculi was compromised in the mutant hepatocytes, resembling the disease phenotypes observed in the liver of patients. Conclusions: Our iPSC-derived in vitro hepatocyte system revealed canalicular membrane disruption in TJP2 deficient hepatocytes and demonstrated the ability to model cholestatic disease with TJP2 deficiency to serve as a platform for further pathophysiologic study and drug discovery. Lay summary: We investigated a genetic liver disease, progressive familial intrahepatic cholestasis (PFIC), which causes severe liver disease in newborns and infants due to a lack of gene called TJP2. By using cutting-edge stem cell technology and genome editing methods, we established a novel disease modeling system in cell culture experiments. Our experiments demonstrated that the lack of TJP2 induced abnormal cell polarity and disrupted bile acid transport. These findings will lead to the subsequent investigation to further understand disease mechanisms and develop an effective treatment.

Alx1 Deficient Mice Recapitulate Craniofacial Phenotype and Reveal Developmental Basis of ALX1-Related Frontonasal Dysplasia.

Loss of ALX1 function causes the frontonasal dysplasia syndrome FND3, characterized by severe facial clefting and microphthalmia. Whereas the laboratory mouse has been the preeminent animal model for studying developmental mechanisms of human craniofacial birth defects, the roles of ALX1 in mouse frontonasal development have not been well characterized because the only previously reported Alx1 mutant mouse line exhibited acrania due to a genetic background-dependent failure of cranial neural tube closure. Using CRISPR/Cas9-mediated genome editing, we have generated an Alx1-deletion mouse model that recapitulates the FND craniofacial malformations, including median orofacial clefting and disruption of development of the eyes and alae nasi. In situ hybridization analysis showed that Alx1 is strongly expressed in frontonasal neural crest cells that give rise to periocular and frontonasal mesenchyme. Alx1 del/del embryos exhibited increased apoptosis of periocular mesenchyme and decreased expression of ocular developmental regulators Pitx2 and Lmxb1 in the periocular mesenchyme, followed by defective optic stalk morphogenesis. Moreover, Alx1 del/del embryos exhibited disruption of frontonasal mesenchyme identity, with loss of expression of Pax7 and concomitant ectopic expression of the jaw mesenchyme regulators Lhx6 and Lhx8 in the developing lateral nasal processes. The function of ALX1 in patterning the frontonasal mesenchyme is partly complemented by ALX4, a paralogous ALX family transcription factor whose loss-of-function causes a milder and distinctive FND. Together, these data uncover previously unknown roles of ALX1 in periocular mesenchyme development and frontonasal mesenchyme patterning, providing novel insights into the pathogenic mechanisms of ALX1-related FND.

Endogenous retroviruses drive species-specific germline transcriptomes in mammals.

Gene regulation in the germline ensures the production of high-quality gametes, long-term maintenance of the species and speciation. Male germline transcriptomes undergo dynamic changes after the mitosis-to-meiosis transition and have been subject to evolutionary divergence among mammals. However, the mechanisms underlying germline regulatory divergence remain undetermined. Here, we show that endogenous retroviruses (ERVs) influence species-specific germline transcriptomes. After the mitosis-to-meiosis transition in male mice, specific ERVs function as active enhancers to drive germline genes, including a mouse-specific gene set, and bear binding motifs for critical regulators of spermatogenesis, such as A-MYB. This raises the possibility that a genome-wide transposition of ERVs rewired germline gene expression in a species-specific manner. Of note, independently evolved ERVs are associated with the expression of human-specific germline genes, demonstrating the prevalence of ERV-driven mechanisms in mammals. Together, we propose that ERVs fine-tune species-specific transcriptomes in the mammalian germline.

RNA Demethylase ALKBH5 Selectively Promotes Tumorigenesis and Cancer Stem Cell Self-Renewal in Acute Myeloid Leukemia.

N6-methyladenosine (m6A), the most abundant internal modification in mRNA, has been implicated in tumorigenesis. As an m6A demethylase, ALKBH5 has been shown to promote the development of breast cancer and brain tumors. However, in acute myeloid leukemia (AML), ALKBH5 was reported to be frequently deleted, implying a tumor-suppressor role. Here, we show that ALKBH5 deletion is rare in human AML; instead, ALKBH5 is aberrantly overexpressed in AML. Moreover, its increased expression correlates with poor prognosis in AML patients. We demonstrate that ALKBH5 is required for the development and maintenance of AML and self-renewal of leukemia stem/initiating cells (LSCs/LICs) but not essential for normal hematopoiesis. Mechanistically, ALKBH5 exerts tumor-promoting effects in AML by post-transcriptional regulation of its critical targets such as TACC3, a prognosis-associated oncogene in various cancers. Collectively, our findings reveal crucial functions of ALKBH5 in leukemogenesis and LSC/LIC self-renewal/maintenance and highlight the therapeutic potential of targeting the ALKBH5/m6A axis.

Functional role of kallikrein 5 and proteinase-activated receptor 2 in eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic, food antigen-driven, inflammatory disease of the esophagus and is associated with impaired barrier function. Evidence is emerging that loss of esophageal expression of the serine peptidase inhibitor, kazal type 7 (SPINK7), is an upstream event in EoE pathogenesis. Here, we provide evidence that loss of SPINK7 mediates its pro-EoE effects via kallikrein 5 (KLK5) and its substrate, protease-activated receptor 2 (PAR2). Overexpression of KLK5 in differentiated esophageal epithelial cells recapitulated the effect of SPINK7 gene silencing, including barrier impairment and loss of desmoglein-1 expression. Conversely, KLK5 deficiency attenuated allergen-induced esophageal protease activity, modified commensal microbiome composition, and attenuated eosinophilia in a murine model of EoE. Inhibition of PAR2 blunted the cytokine production associated with loss of SPINK7 in epithelial cells and attenuated the allergen-induced esophageal eosinophilia in vivo. Clinical samples substantiated dysregulated PAR2 expression in the esophagus of patients with EoE, and delivery of the clinically approved drug α1 antitrypsin (A1AT, a protease inhibitor) inhibited experimental EoE. These findings demonstrate a role for the balance between KLK5 and protease inhibitors in the esophagus and highlight EoE as a protease-mediated disease. We suggest that antagonizing KLK5 and/or PAR2 has potential to be therapeutic for EoE.

Generation and characterization of Six2 conditional mice.

Heterozygous deletion of Six2, which encodes a member of sine oculis homeobox family transcription factors, has recently been associated with the frontonasal dysplasia syndrome FND4. Previous studies showed that Six2 is expressed in multiple tissues during craniofacial development in mice, including embryonic head mesoderm, postmigratory frontonasal neural crest cells, and epithelial and mesenchymal cells of the developing palate and nasal structures. Whereas Six2 -/- mice exhibited cranial base defects but did not recapitulate frontonasal phenotypes of FND4 patients, Six1 -/- Six2 -/- double mutant mice showed severe craniofacial defects including midline facial clefting. The complex phenotypes of FND4 patients and of Six1 -/- Six2 -/- mutant mice indicate that Six2 plays crucial roles in distinct cell types at multiple stages of craniofacial morphogenesis. Here we report generation of mice carrying insertions of a pair of loxP sites flanking exon-1 of the Six2 gene (Six2 f allele) using CRISPR/Cas9-mediated genome editing. We show that the Six2 f allele functions normally and is effectively inactivated by Cre-mediated recombination in vivo. Furthermore, we show that Six2 f/f ;Wnt1-Cre mice recapitulated cranial base defects but not neonatal lethality of Six2 -/- mice. These results indicate that Six2 f/f mice enable systematic investigation of cell type- and stage-specific Six2 function in development and disease.

Systemic administration of AAV-Slc25a46 mitigates mitochondrial neuropathy in Slc25a46-/- mice.

Mitochondrial disorders are the result of nuclear and mitochondrial DNA mutations that affect multiple organs, with the central and peripheral nervous system often affected. Currently, there is no cure for mitochondrial disorders. Currently, gene therapy offers a novel approach for treating monogenetic disorders, including nuclear genes associated with mitochondrial disorders. We utilized a mouse model carrying a knockout of the mitochondrial fusion-fission-related gene solute carrier family 25 member 46 (Slc25a46) and treated them with neurotrophic AAV-PHP.B vector carrying the mouse Slc25a46 coding sequence. Thereafter, we used immunofluorescence staining and western blot to test the transduction efficiency of this vector. Toluidine blue staining and electronic microscopy were utilized to assess the morphology of optic and sciatic nerves following treatment, and the morphology and respiratory chain activity of mitochondria within these tissues were determined as well. The adeno-associated virus (AAV) vector effectively transduced in the cerebrum, cerebellum, heart, liver and sciatic nerves. AAV-Slc25a46 treatment was able to rescue the premature death in the mutant mice (Slc25a46-/-). The treatment-improved electronic conductivity of the peripheral nerves increased mobility and restored mitochondrial complex activities. Most notably, mitochondrial morphology inside the tissues of both the central and peripheral nervous systems was normalized, and the neurodegeneration, chronic neuroinflammation and loss of Purkinje cell dendrites observed within the mutant mice were alleviated. Overall, our study shows that AAV-PHP.B's neurotrophic properties are plausible for treating conditions where the central nervous system is affected, such as many mitochondrial diseases, and that AAV-Slc25a46 could be a novel approach for treating SLC25A46-related mitochondrial disorders.

Mammalian germ cells are determined after PGC colonization of the nascent gonad.

Mammalian primordial germ cells (PGCs) are induced in the embryonic epiblast, before migrating to the nascent gonads. In fish, frogs, and birds, the germline segregates even earlier, through the action of maternally inherited germ plasm. Across vertebrates, migrating PGCs retain a broad developmental potential, regardless of whether they were induced or maternally segregated. In mammals, this potential is indicated by expression of pluripotency factors, and the ability to generate teratomas and pluripotent cell lines. How the germline loses this developmental potential remains unknown. Our genome-wide analyses of embryonic human and mouse germlines reveal a conserved transcriptional program, initiated in PGCs after gonadal colonization, that differentiates germ cells from their germline precursors and from somatic lineages. Through genetic studies in mice and pigs, we demonstrate that one such gonad-induced factor, the RNA-binding protein DAZL, is necessary in vivo to restrict the developmental potential of the germline; DAZL's absence prolongs expression of a Nanog pluripotency reporter, facilitates derivation of pluripotent cell lines, and causes spontaneous gonadal teratomas. Based on these observations in humans, mice, and pigs, we propose that germ cells are determined after gonadal colonization in mammals. We suggest that germ cell determination was induced late in embryogenesis-after organogenesis has begun-in the common ancestor of all vertebrates, as in modern mammals, where this transition is induced by somatic cells of the gonad. We suggest that failure of this process of germ cell determination likely accounts for the origin of human testis cancer.

The S52F FOXF1 Mutation Inhibits STAT3 Signaling and Causes Alveolar Capillary Dysplasia.

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal congenital disorder causing respiratory failure and pulmonary hypertension shortly after birth. There are no effective treatments for ACDMPV other than lung transplant, and new therapeutic approaches are urgently needed. Although ACDMPV is linked to mutations in the FOXF1 gene, molecular mechanisms through which FOXF1 mutations cause ACDMPV are unknown.Objectives: To identify molecular mechanisms by which S52F FOXF1 mutations cause ACDMPV.Methods: We generated a clinically relevant mouse model of ACDMPV by introducing the S52F FOXF1 mutation into the mouse Foxf1 gene locus using CRISPR/Cas9 technology. Immunohistochemistry, whole-lung imaging, and biochemical methods were used to examine vasculature in Foxf1WT/S52F lungs and identify molecular mechanisms regulated by FOXF1.Measurements and Main Results: FOXF1 mutations were identified in 28 subjects with ACDMPV. Foxf1WT/S52F knock-in mice recapitulated histopathologic findings in ACDMPV infants. The S52F FOXF1 mutation disrupted STAT3-FOXF1 protein-protein interactions and inhibited transcription of Stat3, a critical transcriptional regulator of angiogenesis. STAT3 signaling and endothelial proliferation were reduced in Foxf1WT/S52F mice and human ACDMPV lungs. S52F FOXF1 mutant protein did not bind chromatin and was transcriptionally inactive. Furthermore, we have developed a novel formulation of highly efficient nanoparticles and demonstrated that nanoparticle delivery of STAT3 cDNA into the neonatal circulation restored endothelial proliferation and stimulated lung angiogenesis in Foxf1WT/S52F mice.Conclusions: FOXF1 acts through STAT3 to stimulate neonatal lung angiogenesis. Nanoparticle delivery of STAT3 is a promising strategy to treat ACDMPV associated with decreased STAT3 signaling.

Histone H3 trimethylation at lysine 36 guides m6A RNA modification co-transcriptionally.

DNA and histone modifications have notable effects on gene expression1. Being the most prevalent internal modification in mRNA, the N6-methyladenosine (m6A) mRNA modification is as an important post-transcriptional mechanism of gene regulation2-4 and has crucial roles in various normal and pathological processes5-12. However, it is unclear how m6A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally. We show that m6A modifications are enriched in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m6A methyltransferase complex (MTC), which in turn facilitates the binding of the m6A MTC to adjacent RNA polymerase II, thereby delivering the m6A MTC to actively transcribed nascent RNAs to deposit m6A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m6A abundance transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the important roles of H3K36me3 and METTL14 in determining specific and dynamic deposition of m6A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation.

Recognition of RNA N6-methyladenosine by IGF2BP proteins enhances mRNA stability and translation.

N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) and is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate. Here, we report the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) as a distinct family of m6A readers that target thousands of mRNA transcripts through recognizing the consensus GG(m6A)C sequence. In contrast to the mRNA-decay-promoting function of YTH domain-containing family protein 2, IGF2BPs promote the stability and storage of their target mRNAs (for example, MYC) in an m6A-dependent manner under normal and stress conditions and therefore affect gene expression output. Moreover, the K homology domains of IGF2BPs are required for their recognition of m6A and are critical for their oncogenic functions. Thus, our work reveals a different facet of the m6A-reading process that promotes mRNA stability and translation, and highlights the functional importance of IGF2BPs as m6A readers in post-transcriptional gene regulation and cancer biology.

Tumor suppressor gene Rb is required for self-renewal of spermatogonial stem cells in mice.

The retinoblastoma tumor suppressor gene Rb is essential for maintaining the quiescence and for regulating the differentiation of somatic stem cells. Inactivation of Rb in somatic stem cells typically leads to their overexpansion, often followed by increased apoptosis, defective terminal differentiation, and tumor formation. However, Rb's roles in germ-line stem cells have not been explored. We conditionally disrupted the Rb gene in mouse germ cells in vivo and discovered unanticipated consequences for GFRa1-protein-expressing A(single) (GFRa1(+) A(s)) spermatogonia, the major source of male germ-line stem cells. Rb-deficient GFRa1(+) A(s) spermatogonia were present at normal density in testes 5 d after birth, but they lacked the capacity for self-renewal, resulting in germ cell depletion by 2 mo of age. Rb deficiency did not affect the proliferative activity of GFRa1(+) A(s) spermatogonia, but their progeny were exclusively transit-amplifying progenitor spermatogonia and did not include GFRa1(+) A(s) spermatogonia. In addition, Rb deficiency caused prolonged proliferation of progenitor spermatogonia, transiently enlarging this population. Despite these defects, Rb deficiency did not block terminal differentiation into functional sperm; offspring were readily obtained from young males whose germ cell pool was not yet depleted. We conclude that Rb is required for self-renewal of germ-line stem cells, but contrary to its critical roles in somatic stem cells, it is dispensable for their proliferative activity and terminal differentiation. Thus, this study identifies an unexpected function for Rb in maintaining the stem cell pool in the male germ line.

Direct reprogramming of fibroblasts into embryonic Sertoli-like cells by defined factors.

Sertoli cells are considered the "supporting cells" of the testis that play an essential role in sex determination during embryogenesis and in spermatogenesis during adulthood. Their essential roles in male fertility along with their immunosuppressive and neurotrophic properties make them an attractive cell type for therapeutic applications. Here we demonstrate the generation of induced embryonic Sertoli-like cells (ieSCs) by ectopic expression of five transcription factors. We characterize the role of specific transcription factor combinations in the transition from fibroblasts to ieSCs and identify key steps in the process. Initially, transduced fibroblasts underwent a mesenchymal to epithelial transition and then acquired the ability to aggregate, formed tubular-like structures, and expressed embryonic Sertoli-specific markers. These Sertoli-like cells facilitated neuronal differentiation and self-renewal of neural progenitor cells (NPCs), supported the survival of germ cells in culture, and cooperated with endogenous embryonic Sertoli and primordial germ cells in the generation of testicular cords in the fetal gonad.

Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development.

The Tet family of enzymes (Tet1/2/3) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Mouse embryonic stem cells (mESCs) highly express Tet1 and have an elevated level of 5hmC. Tet1 has been implicated in ESC maintenance and lineage specification in vitro but its precise function in development is not well defined. To establish the role of Tet1 in pluripotency and development, we have generated Tet1 mutant mESCs and mice. Tet1(-/-) ESCs have reduced levels of 5hmC and subtle changes in global gene expression, and are pluripotent and support development of live-born mice in tetraploid complementation assay, but display skewed differentiation toward trophectoderm in vitro. Tet1 mutant mice are viable, fertile, and grossly normal, though some mutant mice have a slightly smaller body size at birth. Our data suggest that Tet1 loss leading to a partial reduction in 5hmC levels does not affect pluripotency in ESCs and is compatible with embryonic and postnatal development.