SOX2 is required independently in both stem and differentiated cells for pituitary tumorigenesis in p27-null mice.

P27, a cell cycle inhibitor, is also able to drive repression of Sox2 This interaction plays a crucial role during development of p27-/- pituitary tumors because loss of one copy of Sox2 impairs tumorigenesis [H. Li et al., Cell Stem Cell 11, 845-852 (2012)]. However, SOX2 is expressed in both endocrine and stem cells (SCs), and its contribution to tumorigenesis in either cell type is unknown. We have thus explored the cellular origin and mechanisms underlying endocrine tumorigenesis in p27-/- pituitaries. We found that pituitary hyperplasia is associated with reduced cellular differentiation, in parallel with increased levels of SOX2 in stem and endocrine cells. Using conditional loss-of-function and lineage tracing approaches, we show that SOX2 is required cell autonomously in p27-/- endocrine cells for these to give rise to tumors, and in SCs for promotion of tumorigenesis. This is supported by studies deleting the Sox2 regulatory region 2 (Srr2), the target of P27 repressive action. Single cell transcriptomic analysis further reveals that activation of a SOX2-dependent MAPK pathway in SCs is important for tumorigenesis. Altogether, our data highlight different aspects of the role of SOX2 following loss of p27, according to cellular context, and uncover an unexpected SOX2-dependent tumor-promoting role for SCs. Our results imply that targeting SCs, in addition to tumor cells, may represent an efficient antitumoral strategy in certain contexts.

Pathogenic variants in RNPC3 are associated with hypopituitarism and primary ovarian insufficiency.

PURPOSE: We aimed to investigate the molecular basis underlying a novel phenotype including hypopituitarism associated with primary ovarian insufficiency. METHODS: We used next-generation sequencing to identify variants in all pedigrees. Expression of Rnpc3/RNPC3 was analyzed by in situ hybridization on murine/human embryonic sections. CRISPR/Cas9 was used to generate mice carrying the p.Leu483Phe pathogenic variant in the conserved murine Rnpc3 RRM2 domain. RESULTS: We described 15 patients from 9 pedigrees with biallelic pathogenic variants in RNPC3, encoding a specific protein component of the minor spliceosome, which is associated with a hypopituitary phenotype, including severe growth hormone (GH) deficiency, hypoprolactinemia, variable thyrotropin (also known as thyroid-stimulating hormone) deficiency, and anterior pituitary hypoplasia. Primary ovarian insufficiency was diagnosed in 8 of 9 affected females, whereas males had normal gonadal function. In addition, 2 affected males displayed normal growth when off GH treatment despite severe biochemical GH deficiency. In both mouse and human embryos, Rnpc3/RNPC3 was expressed in the developing forebrain, including the hypothalamus and Rathke's pouch. Female Rnpc3 mutant mice displayed a reduction in pituitary GH content but with no reproductive impairment in young mice. Male mice exhibited no obvious phenotype. CONCLUSION: Our findings suggest novel insights into the role of RNPC3 in female-specific gonadal function and emphasize a critical role for the minor spliceosome in pituitary and ovarian development and function.

A homozygous Y443C variant in the RNPC3 is associated with severe syndromic congenital hypopituitarism and diffuse brain atrophy.

Biallelic RNPC3 variants have been reported in a few patients with growth hormone deficiency, either in isolation or in association with central hypothyroidism, congenital cataract, neuropathy, developmental delay/intellectual disability, hypogonadism, and pituitary hypoplasia. To describe a new patient with syndromic congenital hypopituitarism and diffuse brain atrophy due to RNPC3 mutations and to compare her clinical and molecular characteristics and pituitary functions with previously published patients. A 20-year-old female presented with severe growth, neuromotor, and developmental delay. Her weight, height, and head circumference were 5135 gr (-25.81 SDS), 68 cm (-16.17 SDS), and 34 cm (-17.03 SDS), respectively. She was prepubertal, and had dysmorphic facies, contractures, and spasticity in the extremities, and severe truncal hypotonia. There were no radiological signs of a skeletal dysplasia. The bone age was extremely delayed at 2 years. Investigation of pituitary function revealed growth hormone, prolactin, and thyroid-stimulating hormone deficiencies. Whole-exome sequencing revealed a novel homozygous missense (c.1328A > G; Y443C) variant in RNPC3. Cranial MRI revealed a hypoplastic anterior pituitary with diffuse cerebral and cerebellar atrophy. The Y443C variant in RNPC3 associated with syndromic congenital hypopituitarism and abnormal brain development. This report extends the RNPC3-related hypopituitarism phenotype with a severe neurodegenerative presentation.

SOX2 is sequentially required for progenitor proliferation and lineage specification in the developing pituitary.

Sox2 mutations are associated with pituitary hormone deficiencies and the protein is required for pituitary progenitor proliferation, but its function has not been well characterized in this context. SOX2 is known to activate expression of Six6, encoding a homeodomain transcription factor, in the ventral diencephalon. Here, we find that the same relationship likely exists in the pituitary. Moreover, because Six6 deletion is associated with a similar phenotype as described here for loss of Sox2, Six6 appears to be an essential downstream target of SOX2 in the gland. We also uncover a second role for SOX2. Whereas cell differentiation is reduced in Sox2 mutants, some endocrine cells are generated, such as POMC-positive cells in the intermediate lobe. However, loss of SOX2 here results in complete downregulation of the melanotroph pioneer factor PAX7, and subsequently a switch of identity from melanotrophs to ectopic corticotrophs. Rescuing proliferation by ablating the cell cycle negative regulator p27 (also known as Cdkn1b) in Sox2 mutants does not restore melanotroph emergence. Therefore, SOX2 has two independent roles during pituitary morphogenesis; firstly, promotion of progenitor proliferation, and subsequently, acquisition of melanotroph identity.

Genomic code for Sox2 binding uncovers its regulatory role in Six3 activation in the forebrain.

The SRY-related HMG box transcription factor Sox2 plays critical roles throughout embryogenesis. Haploinsufficiency for SOX2 results in human developmental defects including anophthalmia, microphthalmia and septo-optic dysplasia, a congenital forebrain defect. To understand how Sox2 plays a role in neurogenesis, we combined genomic and in vivo transgenic approaches to characterize genomic regions occupied by Sox2 in the developing forebrain. Six3, a homeobox gene associated with holoprosencephaly, a forebrain midline defect, was identified as a Sox2 transcriptional target. This study shows that Sox2 directly regulates a previously unidentified long-range forebrain enhancer to activate Six3 expression in the rostral diencephalon. Further biochemical and genetic evidences indicated a direct regulatory link between Sox2 and Six3 during forebrain development, providing a better understanding of a common molecular mechanism underlying these forebrain defects.

Direct transcriptional regulation of Six6 is controlled by SoxB1 binding to a remote forebrain enhancer.

Six6, a sine oculis homeobox protein, plays a crucial and conserved role in the development of the forebrain and eye. To understand how the expression of Six6 is regulated during embryogenesis, we screened ~250 kb of genomic DNA encompassing the Six6 locus for cis-regulatory elements capable of directing reporter gene expression to sites of Six6 transcription in transgenic mouse embryos. Here, we describe two novel enhancer elements, that are highly conserved in vertebrate species and whose activities recapitulate Six6 expression in the ventral forebrain and eye, respectively. Cross-species comparisons of the Six6 forebrain enhancer sequences revealed highly conserved binding sites matching the consensus for homeodomain and SoxB1 transcription factors. Deletion of either of the binding sites resulted in loss of the forebrain enhancer activity in the ventral forebrain. Moreover, our studies show that members of the SoxB1 family, including Sox2 and Sox3, are expressed in the overlapping region of the ventral forebrain with Six6 and can bind to the Six6 forebrain enhancer. Loss of function of SoxB1 genes in vivo further emphasizes their role in regulating Six6 forebrain enhancer activity. Thus, our data strongly suggest that SoxB1 transcription factors are direct activators of Six6 expression in the ventral forebrain.

SOX3 is required during the formation of the hypothalamo-pituitary axis.

The pituitary develops from the interaction of the infundibulum, a region of the ventral diencephalon, and Rathke's pouch, a derivative of oral ectoderm. Postnatally, its secretory functions are controlled by hypothalamic neurons, which also derive from the ventral diencephalon. In humans, mutations affecting the X-linked transcription factor SOX3 are associated with hypopituitarism and mental retardation, but nothing is known of their etiology. We find that deletion of Sox3 in mice leads to defects of pituitary function and of specific central nervous system (CNS) midline structures. Cells in the ventral diencephalon, where Sox3 is usually highly expressed, have altered properties in mutant embryos, leading to abnormal development of Rathke's pouch, which does not express the gene. Pituitary and hypothalamic defects persist postnatally, and SOX3 may also function in a subset of hypothalamic neurons. This study shows how sensitive the pituitary is to subtle developmental defects and how one gene can act at several levels in the hypothalamic-pituitary axis.

SOX9-positive pituitary stem cells differ according to their position in the gland and maintenance of their progeny depends on context.

Stem cell (SC) differentiation and maintenance of resultant progeny underlie cell turnover in many organs, but it is difficult to pinpoint the contribution of either process. In the pituitary, a central regulator of endocrine axes, adult SCs undergo activation after target organ ablation, providing a well-characterized paradigm to study an adaptative response in a multi-organ system. Here, we used single-cell technologies to characterize SC heterogeneity and mobilization together with lineage tracing. We show that SC differentiation occurs more frequently than thought previously. In adaptative conditions, differentiation increases and is more diverse than demonstrated by the lineage tracing experiments. Detailed examination of SC progeny suggests that maintenance of selected nascent cells underlies SC output, highlighting a trophic role for the microenvironment. Analyses of cell trajectories further predict pathways and potential regulators. Our model provides a valuable system to study the influence of evolving states on the mechanisms of SC mobilization.

Neural plate progenitors give rise to both anterior and posterior pituitary cells.

The pituitary is the master neuroendocrine gland, which regulates body homeostasis. It consists of the anterior pituitary/adenohypophysis harboring hormones producing cells and the posterior pituitary/neurohypophysis, which relays the passage of hormones from the brain to the periphery. It is accepted that the adenohypophysis originates from the oral ectoderm (Rathke's pouch), whereas the neural ectoderm contributes to the neurohypophysis. Single-cell transcriptomics of the zebrafish pituitary showed that cyp26b1-positive astroglial pituicytes of the neurohypophysis and prop1-positive adenohypophyseal progenitors expressed common markers implying lineage relatedness. Genetic tracing identifies that, in contrast to the prevailing dogma, neural plate precursors of zebrafish (her4.3+) and mouse (Sox1+) contribute to both neurohypophyseal and a subset of adenohypophyseal cells. Pituicyte-derived retinoic-acid-degrading enzyme Cyp26b1 fine-tunes differentiation of prop1+ progenitors into hormone-producing cells. These results challenge the notion that adenohypophyseal cells are exclusively derived from non-neural ectoderm and demonstrate that crosstalk between neuro- and adeno-hypophyseal cells affects differentiation of pituitary cells.

Novel Candidate Regulators and Developmental Trajectory of Pituitary Thyrotropes.

The pituitary gland regulates growth, metabolism, reproduction, the stress response, uterine contractions, lactation, and water retention. It secretes hormones in response to hypothalamic input, end organ feedback, and diurnal cues. The mechanisms by which pituitary stem cells are recruited to proliferate, maintain quiescence, or differentiate into specific cell types, especially thyrotropes, are not well understood. We used single-cell RNA sequencing in juvenile P7 mouse pituitary cells to identify novel factors in pituitary cell populations, with a focus on thyrotropes and rare subtypes. We first observed cells coexpressing markers of both thyrotropes and gonadotropes, such as Pou1f1 and Nr5a1. This was validated in vivo by both immunohistochemistry and lineage tracing of thyrotropes derived from Nr5a1-Cre; mTmG mice and demonstrates that Nr5a1-progenitors give rise to a proportion of thyrotropes during development. Our data set also identifies novel factors expressed in pars distalis and pars tuberalis thyrotropes, including the Shox2b isoform in all thyrotropes and Sox14 specifically in Pou1f1-negative pars tuberalis thyrotropes. We have therefore used single-cell transcriptomics to determine a novel developmental trajectory for thyrotropes and potential novel regulators of thyrotrope populations.

The cortical hem lacks stem cell potential despite expressing SOX9 and HOPX.

The adult dentate gyrus (DG) of rodents hosts a neural stem cell (NSC) niche capable of generating new neurons throughout life. The embryonic origin and molecular mechanisms underlying formation of DG NSCs are still being investigated. We performed a bulk transcriptomic analysis on mouse developing archicortex conditionally deleted for Sox9, a SoxE transcription factor controlling both gliogenesis and NSC formation, and identified Hopx, a recently identified marker of both prospective adult DG NSCs and astrocytic progenitors, as being downregulated. We confirm SOX9 is required for HOPX expression in the embryonic archicortex. In particular, we found that both NSC markers are highly expressed in the cortical hem (CH), while only weakly in the adjacent dentate neuroepithelium (DNE), suggesting a potential CH embryonic origin for DG NSCs. However, we demonstrate both in vitro and in vivo that the embryonic CH, as well as its adult derivatives, lacks stem cell potential. Instead, deletion of Sox9 in the DNE affects both HOPX expression and NSC formation in the adult DG. We conclude that HOPX expression in the CH is involved in astrocytic differentiation downstream of SOX9, which we previously showed regulates DG development by inducing formation of a CH-derived astrocytic scaffold. Altogether, these results suggest that both proteins work in a dose-dependent manner to drive either astrocytic differentiation in CH or NSC formation in DNE.

SOX2-expressing progenitor cells generate all of the major cell types in the adult mouse pituitary gland.

The pituitary gland adapts the proportion of each of its endocrine cell types to meet differing hormonal demands throughout life. There is circumstantial evidence that multipotent adult progenitor cells contribute to this plasticity, but these cells have not been identified. Here, we describe a small (<0.05%) population of progenitor cells in the adult pituitary gland. We show that these cells express SOX2, a marker of several early embryonic progenitor and stem cell types, and form "pituispheres" in culture, which can grow, form secondary spheres, and differentiate to all of the pituitary endocrine cell types, as well as folliculostellate cells. Differentiation of cells in the pituispheres was associated with the expression of nestin, SOX9, and S100. Cells expressing SOX2 and E-cadherin are found throughout Rathke's pouch (RP) in embryos but persist in the adult gland, mostly in a narrow zone lining the pituitary cleft, but also are scattered throughout the pituitary. However, unlike in embryonic RP, most of these SOX2(+) cells in the adult gland also express SOX9 and S100. We suggest that this SOX2(+)/SOX9(+) population represents transit-amplifying cells, whereas the SOX2(+)/SOX9(-) cells we identify are multipotent progenitor/stem cells persisting in the adult pituitary.

Dentate gyrus development requires a cortical hem-derived astrocytic scaffold.

During embryonic development, radial glial cells give rise to neurons, then to astrocytes following the gliogenic switch. Timely regulation of the switch, operated by several transcription factors, is fundamental for allowing coordinated interactions between neurons and glia. We deleted the gene for one such factor, SOX9, early during mouse brain development and observed a significantly compromised dentate gyrus (DG). We dissected the origin of the defect, targeting embryonic Sox9 deletion to either the DG neuronal progenitor domain or the adjacent cortical hem (CH). We identified in the latter previously uncharacterized ALDH1L1+ astrocytic progenitors, which form a fimbrial-specific glial scaffold necessary for neuronal progenitor migration toward the developing DG. Our results highlight an early crucial role of SOX9 for DG development through regulation of astroglial potential acquisition in the CH. Moreover, we illustrate how formation of a local network, amidst astrocytic and neuronal progenitors originating from adjacent domains, underlays brain morphogenesis.

CRISPR/Cas9 Deletion of SOX2 Regulatory Region 2 (SRR2) Decreases SOX2 Malignant Activity in Glioblastoma.

SOX2 is a transcription factor associated with stem cell activity in several tissues. In cancer, SOX2 expression is increased in samples from several malignancies, including glioblastoma, and high SOX2 levels are associated with the population of tumor-initiating cells and with poor patient outcome. Therefore, understanding how SOX2 is regulated in cancer cells is relevant to tackle tumorigenesis. The SOX2 regulatory region 2(SRR2) is located downstream of the SOX2 coding region and mediates SOX2 expression in embryonic and adult stem cells. In this study, we deleted SRR2 using CRISPR/Cas9 in glioblastoma cells. Importantly, SRR2-deleted glioblastoma cells presented reduced SOX2 expression and decreased proliferative activity and self-renewal capacity in vitro. In line with these results, SRR2-deleted glioblastoma cells displayed decreased tumor initiation and growth in vivo. These effects correlated with an elevation of p21CIP1 cell cycle and p27KIP1 quiescence regulators. In conclusion, our data reveal that SRR2 deletion halts malignant activity of SOX2 and confirms that the SRR2 enhancer regulates SOX2 expression in cancer.

Adult pituitary progenitors/stem cells: from in vitro characterization to in vivo function.

Stem cells/progenitors are being discovered in a growing number of adult tissues. They have been hypothesized for a long time to exist in the pituitary, especially because this gland is characterized by its plasticity as it constantly adapts its hormonal response to evolving needs, under the control of the hypothalamus. Recently, five labs have reported the presence of adult progenitors in the gland and shown their endocrine differentiation potential, using different in vitro assays, selection methods and markers to purify and characterize these similar cell populations. These will be discussed here, highlighting common points, and also differences. Thanks to these recent developments it is now possible to integrate progenitors into the physiology of the gland, and uncover their participation in normal but also pathological situations. Moreover, experimental situations inducing generation of new endocrine cells can now be re-visited in light of the involvement of progenitors, and also used to better understand their role. Some of these aspects will also be developed in this review.

Cell population characterization and discovery using single-cell technologies in endocrine systems.

In the last 15 years, single-cell technologies have become robust and indispensable tools to investigate cell heterogeneity. Beyond transcriptomic, genomic and epigenome analyses, technologies are constantly evolving, in particular toward multi-omics, where analyses of different source materials from a single cell are combined, and spatial transcriptomics, where resolution of cellular heterogeneity can be detected in situ. While some of these techniques are still being optimized, single-cell RNAseq has commonly been used because the examination of transcriptomes allows characterization of cell identity and, therefore, unravel previously uncharacterized diversity within cell populations. Most endocrine organs have now been investigated using this technique, and this has given new insights into organ embryonic development, characterization of rare cell types, and disease mechanisms. Here, we highlight recent studies, particularly on the hypothalamus and pituitary, and examine recent findings on the pancreas and reproductive organs where many single-cell experiments have been performed.

Studies of mice deleted for Sox3 and uc482: relevance to X-linked hypoparathyroidism.

Hypoparathyroidism is genetically heterogeneous and characterized by low plasma calcium and parathyroid hormone (PTH) concentrations. X-linked hypoparathyroidism (XLHPT) in two American families, is associated with interstitial deletion-insertions involving deletions of chromosome Xq27.1 downstream of SOX3 and insertions of predominantly non-coding DNA from chromosome 2p25.3. These could result in loss, gain, or movement of regulatory elements, which include ultraconserved element uc482, that could alter SOX3 expression,. To investigate this, we analysed SOX3 expression in EBV-transformed lymphoblastoid cells from 3 affected males, 3 unaffected males, and 4 carrier females from one XLHPT family. SOX3 expression was similar in all individuals, indicating that the spatiotemporal effect of the interstitial deletion-insertion on SOX3 expression postulated to occur in developing parathyroids did not manifest in lymphoblastoids. Expression of SNTG2, which is duplicated and inserted into the X chromosome, and ATP11C, which is moved telomerically, were also similarly expressed in all individuals. Investigation of male hemizygous (Sox3-/Y and uc482-/Y) and female heterozygous (Sox3+/- and uc482+/-) knock-out mice, together with wild-type littermates (male Sox3+/Y and uc482+/Y, and female Sox3+/+ and uc482+/+), revealed Sox3-/Y, Sox3+/-, uc482-/Y, and uc482+/- mice to have normal plasma biochemistry, compared to their respective wild-type littermates. When challenged with a low calcium diet, all mice had hypocalcaemia, and elevated plasma PTH concentrations and alkaline phosphatase activities, and Sox3-/Y, Sox3+/-, uc482-/Y, and uc482+/- mice had similar plasma biochemistry, compared to wild-type littermates. Thus, these results indicate that absence of Sox3 or uc482 does not cause hypoparathyroidism, and that XLHPT likely reflects a more complex mechanism.

Mutations within Sox2/SOX2 are associated with abnormalities in the hypothalamo-pituitary-gonadal axis in mice and humans.

The transcription factor SOX2 is expressed most notably in the developing CNS and placodes, where it plays critical roles in embryogenesis. Heterozygous de novo mutations in SOX2 have previously been associated with bilateral anophthalmia/microphthalmia, developmental delay, short stature, and male genital tract abnormalities. Here we investigated the role of Sox2 in murine pituitary development. Mice heterozygous for a targeted disruption of Sox2 did not manifest eye defects, but showed abnormal anterior pituitary development with reduced levels of growth hormone, luteinizing hormone, and thyroid-stimulating hormone. Consequently, we identified 8 individuals (from a cohort of 235 patients) with heterozygous sequence variations in SOX2. Six of these were de novo mutations, predicted to result in truncated protein products, that exhibited partial or complete loss of function (DNA binding, nuclear translocation, or transactivation). Clinical evaluation revealed that, in addition to bilateral eye defects, SOX2 mutations were associated with anterior pituitary hypoplasia and hypogonadotropic hypogonadism, variable defects affecting the corpus callosum and mesial temporal structures, hypothalamic hamartoma, sensorineural hearing loss, and esophageal atresia. Our data show that SOX2 is necessary for the normal development and function of the hypothalamo-pituitary and reproductive axes in both humans and mice.

SOX2 plays a critical role in the pituitary, forebrain, and eye during human embryonic development.

CONTEXT: Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus. OBJECTIVE: We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues. RESULTS: All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit beta-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathke's pouch, the developing anterior pituitary, and the eye. CONCLUSIONS: Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-beta-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.