Six3 Expression Research Articles

#2200 Kidney stem/progenitor cells can be isolated from the urine of neonates independently of their gestational age at birth

Abstract Background and Aims In the human kidney, nephron structures derive from a population of SIX2 positive kidney stem/progenitor cells. These cells are only present during kidney development, which is reported to terminate at approximately 36 weeks of gestation. We have previously described a non-invasive strategy to isolate the native SIX2 positive kidney stem/progenitor cells from the urine of neonates born before 36 weeks of gestation, named the neonatal kidney/stem progenitor cells (nKSPC) [1]. In preterm neonates, nephrogenesis is still ongoing at the time of birth and continues postnatally, enabling isolation of kidney stem/progenitor cells from the voided urine. In this study, we aimed to determine the efficiency of nKSPC isolation from the urine of neonates and which gestational age (GA) results in the highest yield of nKSPC. We hypothesized that the lower the gestational age, the higher the probability of isolating nKSPC. Method 37 fresh urine samples were obtained from 36 neonates (9 female, 27 male) at day 1 after birth at the Neonatology department of University Hospitals Leuven. Five urine samples were collected from extreme preterm (< 28 weeks GA), 6 samples from very preterm (28–32 weeks GA), 18 samples from moderate to late preterm (32-37 weeks GA) and 8 samples from term neonates (>37 weeks). When a sample yielded cell growth, cell colonies were subcultured to achieve clonal expansion. Cell lines were characterized for the SIX2 stem cell marker using RT-qPCR and immunofluorescence (IF) staining. SIX2 positive cell lines were further evaluated for their potential to differentiate into kidney epithelial cells (i.e. proximal tubular epithelial cells (PTEC) and podocytes) in 2D cultures using our previously established protocols [1]. Results From the 37 urine samples collected, 28 samples yielded cell growth (76%). After subcloning, 147 cell lines were characterized for the expression of SIX2, of which 42 were SIX2 positive. Four SIX2 positive cell lines were derived from extreme preterm, 12 from very preterm, 13 from moderate to late preterm and 13 from term neonates. SIX2 positive cell lines isolated from term neonates exhibited similar cells growth and differentiation potential compared to those isolated from extreme preterm neonates. Additionally, we observed a dose-response effect with regard to expression levels of SIX2 and the differentiation potential: cell lines with higher levels of SIX2 maintained their undifferentiated, uninduced state while lower levels of SIX2 enabled successful differentiation to PTEC and/or podocytes. Conclusion This study demonstrates that SIX2 positive nKSPC can be isolated from the urine of neonates, independently of their GA at birth. This could indicate that nephrogenesis persists longer than what has previously been reported (i.e. 36 weeks). Furthermore, the nKSPC exhibit a dose-response effect with regard to levels of SIX2 and induction of differentiation. This effect has previously been observed in an in vivo mice model [2]. To further investigate the timing of nephrogenesis cessation, we aim to perform a SIX2 staining on human fetal kidney tissue across different GA. Furthermore, we aim to further characterize in detail the isolated nKSPC using single cell RNA sequencing.

Characterizing post-branching nephrogenesis in the neonatal rabbit

Human nephrogenesis ends prior to birth in term infants (34–36 week gestation), with most (60%) nephrons forming in late gestation in two post-branching nephrogenesis (PBN) periods: arcading and lateral branch nephrogenesis. Preterm infants, however, must execute PBN postnatally. Extreme prematurity is associated with low nephron counts. Identifying additional model(s) that undergo PBN postnatally will help support postnatal PBN in preterm infants. The rabbit exhibits longer postnatal nephrogenesis than the mouse but whether it forms nephrons through PBN has not been determined. We performed morphologic and immunohistological assessments of rabbit nephrogenesis from birth (post-conceptual day 31 or 32) to PC49 using H&E and antibodies against SIX1, SIX2, WT1, ZO-1, and JAG1 in the postnatal period. We performed 3D rendering of the nephrogenic niche to assess for PBN, and supplemented the staining with RNAScope to map the expression of Six1, Six2 (nephron progenitors, NPC), and Ret (ureteric bud tip) transcripts to determine the nephrogenic niche postnatal lifespan. Unlike the mouse, rabbit SIX2 disappeared from NPC before SIX1, resembling the human niche. Active nephrogenesis as defined by the presence of SIX1 + naïve NPC/tip population persisted only until PC35–36 (3–5 postnatal days). 3D morphologic assessments of the cortical nephrons identified an elongated tubule with attached glomeruli extending below the UB tip, consistent with PBN arcades, but not with lateral branch nephrogenesis. We conclude that the rabbit shows morphologic and molecular evidence of PBN arcades continuing postnatally for a shorter period than previously thought. The rabbit is the first non-primate expressing SIX1 in the progenitor population. Our findings suggest that studies of arcading in postnatal nephrogenic niche should be performed within the first 5 days of life in the rabbit.

Prenatal prednisone exposure disturbs fetal kidney development and its characteristics

Prednisone is a synthetic glucocorticoid that is commonly used in both human and veterinary medication. Now, it is also recognized as an emerging environmental contaminant. Pregnant women may be exposed to prednisone actively or passively through multiple pathways and cause developmental toxicity to the fetus. However, the impact of prenatal prednisone exposure (PPE) on fetal kidney development remains unclear. In this study, pregnant mice were administered prednisone intragastrically during full-term pregnancy with different doses (0.25, 0.5, or 1 mg/(kg·day)), or at the dose of 1 mg/(kg·day) in different gestational days (GD) (GD0-9, GD10-18, or GD0-18). The pregnant mice were euthanized on GD18. HE staining revealed fetal kidney dysplasia, with an enlarged glomerular Bowman's capsule space and a reduced capillary network in the PPE groups. The expression of the podocyte and the mesangial cell marker genes was significantly reduced in the PPE groups. However, overall gene expression in renal tubules and collecting ducts were markedly increased. All of the above effects were more pronounced in high-dose, full-term pregnancy, and female fetuses. Studies on the mechanism of the female fetal kidney have revealed that PPE reduced the expression of Six2, increased the expression of Hnf1β, Hnf4α, and Wnt9b, and inhibited the expression of glial cell line-derived neurotrophic factor (GDNF) and Notch signaling pathways. In conclusion, this study demonstrated that there is a sex difference in the developmental toxicity of PPE to the fetal kidney, and the time effect is manifested as full-term pregnancy > early pregnancy > mid-late pregnancy.

349-OR: Selected DYRK1A Inhibitors Simultaneously Drive Both Human Beta-Cell Proliferation and Differentiation

Small molecule DYRK1A inhibitors induce adult human beta cell proliferation and expand human beta cell mass. Harmine also enhances differentiation and function in human beta cells in vitro and in vivo, increasing expression of key beta cell genes. It is unknown whether this “pro-differentiation effect” is a feature of all DYRK1A inhibitors. We compared harmine, 2-2c, 5-Iodotubericidin (5-IT), GNF4877, INDY, CC-401 and Leucettine-41 at doses that maximally drive human beta cell proliferation. As anticipated, harmine, 2-2c and 5-IT all increased expression of PDX1, MAFA, NKX6.1, SLC2A2, PCSK1, MAFB, SIX2, SLC30A8, and ENTPD3. These effects were observed in normal human islets as well as in islets from donors with T2D. Surprisingly, in simultaneous experiments, the DYRK1A inhibitors GNF4877, INDY, CC-401 and Leucettine failed to induce these beta cell differentiation markers. The pro-differentiation effect of harmine was independent of DYRK1A inhibition: silencing DYRK1A in human islets induced beta cell proliferation, but had no effect on differentiation; harmine treatment in DYRK1A-silenced islets restored beta cell pro-differentiation effects. The pro-differentiation effect was also independent of NFAT signaling and DREAM Complex effects, since NFAT overexpression, or p130, p107, E2F4 and E2F5 silencing had no effect on beta cell differentiation. Similarly, silencing other known harmine targets (DYRK2, 3, 4, the CLK and HIPK family) had no effect on either human beta cell differentiation nor proliferation. In summary, harmine, 2-2c and 5-IT are unique among the DYRK1A inhibitor family in their simultaneous ability to enhance both beta cell proliferation and differentiation. While the beta cell proliferation is mediated by DYRK1A inhibition, the pro-differentiation effects of harmine, 2-2c and 5-IT are distinct, and unexplained in mechanistic terms. These considerations have important implications for DYRK1A inhibitor drug development. Disclosure P. Wang: None. K. Kumar: None. H. Liu: None. O. Wood: None. E. Karakose: None. L. Choleva: None. L. Lambertini: None. A. Garcia-Ocana: Consultant; Sun Pharmaceutical Industries Ltd. R.J. DeVita: None. A.F. Stewart: None.

Chd5 Regulates the Transcription Factor Six3 to Promote Neuronal Differentiation.

Chromodomain helicase DNA-binding protein 5 (Chd5) is an ATP-dependent chromatin remodeler that promotes neuronal differentiation. However, the mechanism behind the action of Chd5 during neurogenesis is not clearly understood. Here we use transcriptional profiling of cells obtained from Chd5 deficient mice at early and late stages of neuronal differentiation to show that Chd5 regulates neurogenesis by directing stepwise transcriptional changes. During early stages of neurogenesis, Chd5 promotes expression of the proneural transcription factor Six3 to repress Wnt5a, a non-canonical Wnt ligand essential for the maturation of neurons. This previously unappreciated ability of Chd5 to transcriptionally repress neuronal maturation factors is critical for both lineage specification and maturation. Thus, Chd5 facilitates early transcriptional changes in neural stem cells, thereby initiating transcriptional programs essential for neuronal fate specification.

De novo SIX2 activation in human kidneys treated with neonatal kidney stem/progenitor cells

During development, nephron structures are derived from a SIX2+ stem cell population. After 36 weeks of gestation, these cells are exhausted, and no new nephrons are formed. We have previously described a non-invasive strategy to isolate and expand the native SIX2+ kidney stem cells from the urine of preterm neonates, named neonatal kidney stem/progenitor cells (nKSPC). Here, we investigated the safety and feasibility of administering nKSPC into human kidneys discarded for transplantation during normothermic machine perfusion (NMP) and evaluated the regenerative and immunomodulatory potential of nKSPC treatment. We found that nKSPC administration during NMP is safe and feasible. Interestingly, nKSPC induced the de novo expression of SIX2 in proximal tubular cells of the donor kidneys and upregulated regenerative markers such as SOX9 and VEGF. This is the first time that SIX2 re-expression is observed in adult human kidneys. Moreover, nKSPC administration significantly lowered levels of kidney injury biomarkers and reduced inflammatory cytokine levels via the tryptophan-IDO-kynurenine pathway. In conclusion, nKSPC is a novel cell type to be applied in kidney-targeted cell therapy, with the potential to induce an endogenous regenerative process and immunomodulation.

Heterozygous variants in SIX3 and POU1F1 cause pituitary hormone deficiency in mouse and man.

Congenital hypopituitarism is a genetically heterogeneous condition that is part of a spectrum disorder that can include holoprosencephaly. Heterozygous mutations in SIX3 cause variable holoprosencephaly in humans and mice. We identified two children with neonatal hypopituitarism and thin pituitary stalk who were doubly heterozygous for rare, likely deleterious variants in the transcription factors SIX3 and POU1F1. We used genetically engineered mice to understand the disease pathophysiology. Pou1f1 loss of function heterozygotes are unaffected; Six3 heterozygotes have pituitary gland dysmorphology and incompletely ossified palate; and the Six3+/-; Pou1f1+/dw double; heterozygote mice have a pronounced phenotype, including pituitary growth through the palate. The interaction of Pou1f1 and Six3 in mice supports the possibility of digenic pituitary disease in children. Disruption of Six3 expression in the oral ectoderm completely ablated anterior pituitary development, and deletion of Six3 in the neural ectoderm blocked development of the pituitary stalk and both anterior and posterior pituitary lobes. Six3 is required in both oral and neural ectodermal tissues for activation of signaling pathways and transcription factors necessary for pituitary cell fate. These studies clarify the mechanism of SIX3 action in pituitary development and provide support for a digenic basis for hypopituitarism.

SIX3 function in cancer: progression and comprehensive analysis.

The homeobox gene family encodes transcription factors that are essential for cell growth, proliferation, and differentiation, and its dysfunction is linked to tumor initiation and progression. Sine oculis homeobox (SIX) belongs to the homeobox gene family, with SIX3 being a core member. Recent studies indicate that SXI3 functions as a cancer suppressor or promoter, which is mainly dependent on SIX3's influence on the signal pathways that promote or inhibit cancer in cells. The low expression of SIX3 in most malignant tumors was confirmed by detailed studies, which could promote the cell cycle, proliferation, migration, and angiogenesis. The recovery or upregulation of SIX3 expression to suppress cancer is closely related to the direct or indirect inhibition of the Wnt pathway. However, in some malignancies, such as esophageal cancer and gastric cancer, SIX3 is a tumor-promoting factor, and repressing SIX3 improves patients' prognosis. This review introduces the research progress of SIX3 in tumors and gives a comprehensive analysis, intending to explain why SIX3 plays different roles in different cancers and provide new cancer therapy strategies.

Osteogenic Commitment of Human Periodontal Ligament Cells Is Predetermined by Methylation, Chromatin Accessibility and Expression of Key Transcription Factors.

Periodontal ligament stem cells (PDLCs) can be used as a valuable source in cell therapies to regenerate bone tissue. However, the potential therapeutic outcomes are unpredictable due to PDLCs’ heterogeneity regarding the capacity for osteoblast differentiation and mineral nodules production. Here, we identify epigenetic (DNA (hydroxy)methylation), chromatin (ATAC-seq) and transcriptional (RNA-seq) differences between PDLCs presenting with low (l) and high (h) osteogenic potential. The primary cell populations were investigated at basal state (cultured in DMEM) and after 10 days of osteogenic stimulation (OM). At a basal state, the expression of transcription factors (TFs) and the presence of gene regulatory regions related to osteogenesis were detected in h-PDLCs in contrast to neuronal differentiation prevalent in l-PDLCs. These differences were also observed under stimulated conditions, with genes and biological processes associated with osteoblast phenotype activated more in h-PDLCs. Importantly, even after the induction, l-PDLCs showed hypermethylation and low expression of genes related to bone development. Furthermore, the analysis of TFs motifs combined with TFs expression suggested the relevance of SP1, SP7 and DLX4 regulation in h-PDLCs, while motifs for SIX and OLIG2 TFs were uniquely enriched in l-PDLCs. Additional analysis including a second l-PDLC population indicated that the high expression of OCT4, SIX3 and PPARG TFs could be predictive of low osteogenic commitment. In summary, several biological processes related to osteoblast commitment were activated in h-PDLCs from the onset, while l-PDLCs showed delay in the activation of the osteoblastic program, restricted by the persistent methylation of gene related to bone development. These processes are pre-determined by distinguishable epigenetic and transcriptional patterns, the recognition of which could help in selection of PDLCs with pre-osteoblastic phenotype.

An organ-derived extracellular matrix triggers in situ kidney regeneration in a preclinical model

It has not been considered that nephrons regenerate in adult mammals. We present that an organ-derived extracellular matrix in situ induces nephron regeneration in a preclinical model. A porcine kidney-derived extracellular matrix was sutured onto the surface of partial nephrectomy (PN)-treated kidney. Twenty-eight days after implantation, glomeruli, vessels, and renal tubules, characteristic of nephrons, were histologically observed within the matrix. No fibrillogenesis was observed in the matrix nor the matrix-sutured kidney, although this occurred in a PN kidney without the matrix, indicating the structures were newly induced by the matrix. The expression of renal progenitor markers, including Sall1, Six2, and WT-1, within the matrix supported the induction of nephron regeneration by the matrix. Furthermore, active blood flow was observed inside the matrix using computed tomography. The matrix provides structural and functional foundations for the development of cell-free scaffolds with a remarkably low risk of immune rejection and cancerization.

Deletion of the homeodomain gene Six3 from kisspeptin neurons causes subfertility in female mice

The homeodomain transcription factor SIX3 is a known regulator of eye, nose, and forebrain development, and has recently been implicated in female reproduction. Germline heterozygosity of SIX3 is sufficient to cause subfertility, but the cell populations that mediate this role are unknown. The neuropeptide kisspeptin is a critical component of the reproductive axis and plays roles in sexual maturation, ovulation, and the maintenance of gonadotropin secretion. We used Cre-Lox technology to remove Six3 specifically from kisspeptin neurons in mice to test the hypothesis that SIX3 in kisspeptin neurons is required for reproduction. We found that loss of Six3 in kisspeptin neurons causes subfertility and estrous cycle irregularities in females, but no effect in males. Overall, we find that SIX3 expression in kisspeptin neurons is an important contributor to female fertility.

Six1 and Six2 of the Sine Oculis Homeobox Subfamily are Not Functionally Interchangeable in Mouse Nephron Formation.

The vertebrate Six1 and Six2 arose by gene duplication from the Drosophila sine oculis and have since diverged in their developmental expression patterns. Both genes are expressed in nephron progenitors of human fetal kidneys, and mutations in SIX1 or SIX2 cause branchio-oto-renal syndrome or renal hypodysplasia respectively. Since ∼80% of SIX1 target sites are shared by SIX2, it is speculated that SIX1 and SIX2 may be functionally interchangeable by targeting common downstream genes. In contrast, in mouse kidneys, Six1 expression in the metanephric mesenchyme lineage overlaps with Six2 only transiently, while Six2 expression is maintained in the nephron progenitors throughout development. This non-overlapping expression between Six1 and Six2 in mouse nephron progenitors promoted us to examine if Six1 can replace Six2. Surprisingly, forced expression of Six1 failed to rescue Six2-deficient kidney phenotype. We found that Six1 mediated Eya1 nuclear translocation and inhibited premature epithelialization of the progenitors but failed to rescue the proliferation defects and cell death caused by Six2-knockout. Genome-wide binding analyses showed that Six1 selectively occupied a small subset of Six2 target sites, but many Six2-bound loci crucial to the renewal and differentiation of nephron progenitors lacked Six1 occupancy. Altogether, these data indicate that Six1 cannot substitute Six2 to drive nephrogenesis in mouse kidneys, thus demonstrating that the difference in physiological roles of Six1 and Six2 in kidney development stems from both transcriptional regulations of the genes and divergent biochemical properties of the proteins.

The Transcriptional and Immunological Roles of Six2 in Clear Cell Renal Cell Carcinoma

Background: Six2, a transcription factor, exerts an oncogenic role in clear cell renal cell carcinoma (ccRCC). Increased Six2 expression could enhance cancer metastasis. However, the regulatory mechanism of Six2 in promoting metastasis remains unclear. The purpose of this study is to analyze the regulatory pattern of Six2 and the potential role of Six2 in the tumor immune microenvironment. Materials and Methods: Firstly, transcriptional data in TCGA-KIRC cohorts was used to analyze the relationship between Six2 expression and clinical information. Secondly, we detect the association between Six2 and the tumor immune microenvironment in ccRCC. Then, we analyzed Six2-related differentially expressed genes (DEGs) and constructed a prognostic model using the Lasso-Cox algorithm by integrating Six2 ChIP data and co-expressed genes. Next, we analyzed the clinical significance and immunotherapy sensitivity of this model. Results: Six2 was overexpressed in RCC cells compared with normal kidney cells and upregulated Six2 was positively linked with clinical stage, grade, T stage, M stage, and poor survival. And Six2 was correlated with the remodeling of the tumor microenvironment. Potential downstream effectors and biological functions regulated by Six2 were identified using in silico analysis. Meanwhile, a risk model based on 8 Six2 target genes was established to classify ccRCC patients into high-and-low risk groups. This risk model showed a reliable ability to forecast the overall survival of ccRCC patients and predict the susceptibility to immunotherapy. Conclusions: Our findings provide a promising prognostic indicator for ccRCC patients and help better understand the transcriptional and immunological role of Six2 in ccRCC.

Comprehensive analysis of EMT-related genes and lncRNAs in the prognosis, immunity, and drug treatment of colorectal cancer

BackgroundEMT is an important biological process in the mechanism of tumor invasion and metastasis. However, there are still many unknowns about the specific mechanism of EMT in tumor. At present, a comprehensive analysis of EMT-related genes in colorectal cancer (CRC) is still lacking.MethodsAll the data were downloaded from public databases including TCGA database (488 tumor samples and 52 normal samples) as the training set and the GEO database (GSE40967 including 566 tumor samples and 19 normal samples, GSE12945 including 62 tumor samples, GSE17536 including 177 tumor samples, GSE17537 including 55 tumor samples) as the validation sets. One hundred and sixty-six EMT-related genes (EMT-RDGs) were selected from the Molecular Signatures Database. Bioinformatics methods were used to analyze the correlation between EMT-RDGs and CRC prognosis, metastasis, drug efficacy, and immunity.ResultsWe finally obtained nine prognostic-related EMT-RDGs (FGF8, NOG, PHLDB2, SIX2, SNAI1, TBX5, TIAM1, TWIST1, TCF15) through differential expression analysis, Unicox and Lasso regression analysis, and then constructed a risk prognosis model. There were significant differences in clinical characteristics, 22 immune cells, and immune functions between the high-risk and low-risk groups and the different states of the nine prognostic-related EMT-RDGs. The methylation level and mutation status of nine prognostic-related EMT-RDGs all affect their regulation of EMT. The Cox proportional hazards regression model was also constructed by the methylation sites of nine prognostic-related EMT-RDGs. In addition, the expression of FGF8, PHLDB2, SIX2, and SNAIL was higher and the expression level of NOG and TWIST1 was lower in the non-metastasis CRC group. Nine prognostic-related EMT-RDGs also affected the drug treatment response of CRC.ConclusionsTargeting these nine prognostic-related EMT-RDGs can regulate CRC metastasis and immune, which is beneficial for the prognosis of CRC patients, improve drug sensitivity in CRC patients.

MiR-4306 Suppresses Proliferation of Esophageal Squamous Cell Carcinoma Cell by Targeting SIX3.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers and the primary cause of cancer-related mortality in China. micoRNA plays a vital role during tumor initiation and malignant progression. miR-4306 has been reported to negatively regulate aggressive cell phenotypes in triple-negative breast cancer (TNBC). Nevertheless, the function of miR-4306 in ESCC was still not clear. In this study, we detected miR-4306 expression by quantitative real-time reverse transcription-PCR (qRT-PCR) and found that miR-4306 expression was downregulated in human ESCC tissue samples and cell lines. Moreover, miR-4306 overexpression could restrain ESCC cell proliferation, migratory and invasive ability and epithelial-mesenchymal transition (EMT), promote cell apoptosis after treatment with or without cisplatin. In contrast, inhibiting the expression of miR-4306 showed the opposing results. Furthermore, we explored the molecular mechanism of effects of miR-4306 and found that miR-4306 inhibited the expression of SIX3 by interaction with SIX3 3'UTR in ESCC cells, and SIX3 overexpression significantly reversed the effect of miR-4306-mediated ESCC cells proliferation. The current study provided evidence of miR-4306 as a tumor suppression gene in ESCC.

Glucocorticoid signaling in pancreatic islets modulates gene regulatory programs and genetic risk of type 2 diabetes.

Glucocorticoids are key regulators of glucose homeostasis and pancreatic islet function, but the gene regulatory programs driving responses to glucocorticoid signaling in islets and the contribution of these programs to diabetes risk are unknown. In this study we used ATAC-seq and RNA-seq to map chromatin accessibility and gene expression from eleven primary human islet samples cultured in vitro with the glucocorticoid dexamethasone at multiple doses and durations. We identified thousands of accessible chromatin sites and genes with significant changes in activity in response to glucocorticoids. Chromatin sites up-regulated in glucocorticoid signaling were prominently enriched for glucocorticoid receptor binding sites and up-regulated genes were enriched for ion transport and lipid metabolism, whereas down-regulated chromatin sites and genes were enriched for inflammatory, stress response and proliferative processes. Genetic variants associated with glucose levels and T2D risk were enriched in glucocorticoid-responsive chromatin sites, including fine-mapped variants at 51 known signals. Among fine-mapped variants in glucocorticoid-responsive chromatin, a likely casual variant at the 2p21 locus had glucocorticoid-dependent allelic effects on beta cell enhancer activity and affected SIX2 and SIX3 expression. Our results provide a comprehensive map of islet regulatory programs in response to glucocorticoids through which we uncover a role for islet glucocorticoid signaling in mediating genetic risk of T2D.

SIX3 Variant Causes Pituitary Stalk Interruption Syndrome and Combined Pituitary Hormone Deficiency

The genetic basis for congenital hypopituitarism and related disorders is beginning to emerge, and over 30 causal genes have been identified. Mutations in some of these genes can also cause holoprosencephaly (HPE) or septo-optic dysplasia. SIX3 is a homeodomain protein expressed in the developing brain, pituitary gland, and eye. Heterozygous mutations in SIX3 cause variable HPE in humans and mice. We identified two children with neonatal GH and TSH deficiency and stalk interruption who were doubly heterozygous for rare, likely deleterious variants in SIX3 and POU1F1. Functional studies demonstrated that both variants are disruptive. We used Six3 and Pou1f1 loss of function mice to assess the genetic interaction between Six3 and Pou1f1. Six3 heterozygotes have variable pituitary gland dysmorphology, while Pou1f1 heterozygotes are normal. A significant portion of the Six3+/-; Pou1f1+/dw doubly heterozygous mice have a more pronounced pituitary phenotype than Six3+/-, supporting the possibility of digenic pituitary disease. To understand the role of SIX3 in pituitary and hypothalamic development, we used Prop1-cre and Nkx2.1-cre to delete Six3. Disruption of Six3 expression in Rathke’s pouch caused poor activation of Lhx3 expression and arrested anterior pituitary development. The Nkx2.1-cre, Six3flox/flox embryos had no evidence of infundibulum evagination and failed to induce FGF and BMP signaling, which normally drive expansion of Rathke’s pouch. By E11.5 cells in Rathke’s pouch underwent apoptosis. The Nkx2.1-cre, Six3flox/flox embryos failed to activate expression of Lhx2 and Tbx3 in the neural ectoderm. These embryos had elevated CCND1, MYCN, and Axin2 expression in the area of the presumptive infundibulum. This indicates that SIX3 is necessary to repress cell proliferation and Wnt/beta-catenin signals to promote formation of the pituitary stalk. Thus, Six3 has essential roles in both the neural and oral ectoderm for hypothalamic and pituitary development, respectively. Heterozygous loss of function variants in SIX3 could be a contributor to multiple pituitary hormone deficiencies in children, especially if there are associated craniofacial abnormalities or PSIS.

CRISPR-based genome editing in primary human pancreatic islet cells

Gene targeting studies in primary human islets could advance our understanding of mechanisms driving diabetes pathogenesis. Here, we demonstrate successful genome editing in primary human islets using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). CRISPR-based targeting efficiently mutated protein-coding exons, resulting in acute loss of islet β-cell regulators, like the transcription factor PDX1 and the KATP channel subunit KIR6.2, accompanied by impaired β-cell regulation and function. CRISPR targeting of non-coding DNA harboring type 2 diabetes (T2D) risk variants revealed changes in ABCC8, SIX2 and SIX3 expression, and impaired β-cell function, thereby linking regulatory elements in these target genes to T2D genetic susceptibility. Advances here establish a paradigm for genetic studies in human islet cells, and reveal regulatory and genetic mechanisms linking non-coding variants to human diabetes risk.

Linking Penetrance and Transcription in DYT-THAP1: Insights From a Human iPSC-Derived Cortical Model.

The THAP1 gene encodes a transcription factor, and pathogenic variants cause a form of autosomal dominant, isolated dystonia (DYT-THAP1) with reduced penetrance. Factors underlying both reduced penetrance and the disease mechanism of DYT-THAP1 are largely unknown. We performed transcriptome analysis on 29 cortical neuronal precursors derived from human-induced pluripotent stem cell lines generated from manifesting and nonmanifesting THAP1 mutation carriers and control individuals. Whole transcriptome analysis showed a penetrance-linked signature with expressional changes more pronounced in the group of manifesting (MMCs) than in nonmanifesting mutation carriers (NMCs) when compared to controls. A direct comparison of the transcriptomes in MMCs versus NMCs showed significant upregulation of the DRD4 gene in MMCs. A gene set enrichment analysis demonstrated alterations in various neurotransmitter release cycle pathways, extracellular matrix organization, and deoxyribonucleic acid methylation between MMCs and NMCs. When specifically considering transcription factors, the expression of YY1 and SIX2 differed in MMCs versus NMCs. Further, THAP1 was upregulated in the group of MMCs. To our knowledge, this is the first report systematically analyzing reduced penetrance in DYT-THAP1 in a human model using transcriptomes. Our findings indicate that transcriptional alterations during cortical development influence DYT-THAP1 pathogenesis and penetrance. We reinforce previously linked pathways including dopamine and eukaryotic translation initiation factor 2 alpha signaling in the pathogenesis of dystonia including DYT-THAP1 and suggest extracellular matrix organization and deoxyribonucleic acid methylation as mediators of disease protection. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

SIX2 and SIX3 coordinately regulate functional maturity and fate of human pancreatic β cells.

The physiological functions of many vital tissues and organs continue to mature after birth, but the genetic mechanisms governing this postnatal maturation remain an unsolved mystery. Human pancreatic β cells produce and secrete insulin in response to physiological cues like glucose, and these hallmark functions improve in the years after birth. This coincides with expression of the transcription factors SIX2 and SIX3, whose functions in native human β cells remain unknown. Here, we show that shRNA-mediated SIX2 or SIX3 suppression in human pancreatic adult islets impairs insulin secretion. However, transcriptome studies revealed that SIX2 and SIX3 regulate distinct targets. Loss of SIX2 markedly impaired expression of genes governing β-cell insulin processing and output, glucose sensing, and electrophysiology, while SIX3 loss led to inappropriate expression of genes normally expressed in fetal β cells, adult α cells, and other non-β cells. Chromatin accessibility studies identified genes directly regulated by SIX2. Moreover, β cells from diabetic humans with impaired insulin secretion also had reduced SIX2 transcript levels. Revealing how SIX2 and SIX3 govern functional maturation and maintain developmental fate in native human β cells should advance β-cell replacement and other therapeutic strategies for diabetes.