Peak-agnostic high-resolution cis-regulatory circuitry mapping using single cell multiome data.

Single same cell RNAseq/ATACseq multiome data provide unparalleled potential to develop high resolution maps of the cell-type specific transcriptional regulatory circuitry underlying gene expression. We present CREMA, a framework that recovers the full cis-regulatory circuitry by modeling gene expression and chromatin activity in individual cells without peak-calling or cell type labeling constraints. We demonstrate that CREMA overcomes the limitations of existing methods that fail to identify about half of functional regulatory elements which are outside the called chromatin 'peaks'. These circuit sites outside called peaks are shown to be important cell type specific functional regulatory loci, sufficient to distinguish individual cell types. Analysis of mouse pituitary data identifies a Gata2-circuit for the gonadotrope-enriched disease-associated Pcsk1 gene, which is experimentally validated by reduced gonadotrope expression in a gonadotrope conditional Gata2-knockout model. We present a web accessible human immune cell regulatory circuit resource, and provide CREMA as an R package.

Activin E is a transforming growth factor ß ligand that signals specifically through activin receptor-like kinase 7.

Activins are one of the three distinct subclasses within the greater Transforming growth factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, like ActC. Collectively, our results establish ActE as a specific signaling ligand which activates the type I receptor, ALK7.

Transcription factor GATA2 may potentiate follicle-stimulating hormone production in mice via induction of the BMP antagonist gremlin in gonadotrope cells.

Mammalian reproduction depends on the gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone, which are secreted by pituitary gonadotrope cells. The zinc-finger transcription factor GATA2 was previously implicated in FSH production in male mice; however, its mechanisms of action and role in females were not determined. To directly address GATA2 function in gonadotropes, we generated and analyzed gonadotrope-specific Gata2 KO mice using the Cre-lox system. We found that while conditional KO (cKO) males exhibited ∼50% reductions in serum FSH levels and pituitary FSHß subunit (Fshb) expression relative to controls, FSH production was apparently normal in cKO females. In addition, RNA-seq analysis of purified gonadotropes from control and cKO males revealed a profound decrease in expression of gremlin (Grem1), a bone morphogenetic protein (BMP) antagonist. We show Grem1 was expressed in gonadotropes, but not other cell lineages, in the adult male mouse pituitary. Furthermore, Gata2, Grem1, and Fshb mRNA levels were significantly higher in the pituitaries of WT males relative to females but decreased in males treated with estradiol and increased following ovariectomy in control but not cKO females. Finally, we found that recombinant gremlin stimulated Fshb expression in pituitary cultures from WT mice. Collectively, the data suggest that GATA2 promotes Grem1 expression in gonadotropes and that the gremlin protein potentiates FSH production. The mechanisms of gremlin action have not yet been established but may involve attenuation of BMP binding to activin type II receptors in gonadotropes, facilitating induction of Fshb transcription by activins or related ligands.

Ablation of TGFBR3 (betaglycan) in oocytes does not affect fertility in female mice.

Ovarian follicle development is regulated by locally produced TGFß superfamily members. The TGFß type III receptor (TGFBR3, or betaglycan), which regulates the actions of diverse TGFß ligands, including inhibins, is expressed in different ovarian cell types. However, its functional roles in the ovary have not been investigated in vivo. Here, we ablated Tgfbr3 in murine oocytes using the Cre-loxP system. Oocyte-specific Tgfbr3 knockout (cKO) females were fertile, producing litters of similar size and frequency as controls. Their ovarian weights and histology were also normal. Though we confirmed efficient recombination of the floxed alleles, we did not detect Tgfbr3 mRNA in purified oocytes from superovulated cKO or control mice. These results challenge earlier observations of betaglycan protein expression in this cell type. Regardless, Tgfbr3 in the murine oocyte is clearly dispensable for female fertility.

Gonadotrope-specific deletion of the BMP type 2 receptor does not affect reproductive physiology in mice†‡.

Activins selectively stimulate follicle-stimulating hormone (FSH) secretion by pituitary gonadotrope cells. More recently, other members of the TGFbeta superfamily, the bone morphogenetic proteins (BMPs), were reported to regulate FSH synthesis. Activins and BMPs independently and synergistically stimulate transcription of the FSHbeta subunit (Fshb) gene in immortalized gonadotrope-like cells. Both ligands can signal via the activin receptor type IIA (ACVR2A) to regulate FSH synthesis in vitro. In vivo, global Acvr2a knockout mice exhibit a 60% reduction in circulating FSH relative to wild-type animals, suggesting that activins, BMPs, or related ligands might signal through additional type II receptors to regulate FSH in vivo. Although the leading candidates are ACVR2B and the BMP type II receptor (BMPR2), only the latter mediates activin or BMP2 induction of Fshb transcription in vitro. Here, we generated mice carrying a loss of function mutation in Bmpr2 specifically in gonadotropes. Puberty onset, estrous cyclicity, and reproductive organ weights were similar between control and conditional knockout females. Serum FSH and luteinizing hormone (LH) and pituitary expression of Fshb and the LHbeta subunit (Lhb) were similarly unaffected by the gene deletion in both sexes. These results suggest that BMPR2 might not play a necessary role in FSH synthesis or secretion in vivo or that another type II receptor, such as ACVR2A, can fully compensate for its absence. These data also further contribute to the emerging concept that BMPs may not be physiological regulators of FSH in vivo.

Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type heterogeneity.

Immediate-early response genes (IEGs) are rapidly and transiently induced following an extracellular signal. Elucidating the IEG response patterns in single cells (SCs) requires assaying large numbers of timed samples at high accuracy while minimizing handling effects. To achieve this, we developed and validated RNA stabilization Buffer for Examination of Single-cell Transcriptomes (RNA-Best), a versatile single-step cell and tissue preservation protocol that stabilizes RNA in intact SCs without perturbing transcription patterns. We characterize for the first time SC heterogeneity in IEG responses to pulsatile gonadotropin-releasing hormone (GnRH) stimuli in pituitary gonadotrope cells. Our study identifies a gene-specific hierarchical pattern of all-or-none transcript induction elicited by increasing concentrations of GnRH. This quantal pattern of gene activation raises the possibility that IEG activation, when accurately resolved at the SC level, may be mediated by gene bits that behave as pure binary switches.

SMAD3 Regulates Follicle-stimulating Hormone Synthesis by Pituitary Gonadotrope Cells in Vivo.

Pituitary follicle-stimulating hormone (FSH) is an essential regulator of fertility in females and of quantitatively normal spermatogenesis in males. Pituitary-derived activins are thought to act as major stimulators of FSH synthesis by gonadotrope cells. In vitro, activins signal via SMAD3, SMAD4, and forkhead box L2 (FOXL2) to regulate transcription of the FSHß subunit gene (Fshb). Consistent with this model, gonadotrope-specific Smad4 or Foxl2 knock-out mice have greatly reduced FSH and are subfertile. The role of SMAD3 in vivo is unresolved; however, residual FSH production in Smad4 conditional knock-out mice may derive from partial compensation by SMAD3 and its ability to bind DNA in the absence of SMAD4. To test this hypothesis and determine the role of SMAD3 in FSH biosynthesis, we generated mice lacking both the SMAD3 DNA binding domain and SMAD4 specifically in gonadotropes. Conditional knock-out females were hypogonadal, acyclic, and sterile and had thread-like uteri; their ovaries lacked antral follicles and corpora lutea. Knock-out males were fertile but had reduced testis weights and epididymal sperm counts. These phenotypes were consistent with those of Fshb knock-out mice. Indeed, pituitary Fshb mRNA levels were nearly undetectable in both male and female knock-outs. In contrast, gonadotropin-releasing hormone receptor mRNA levels were significantly elevated in knock-outs in both sexes. Interestingly, luteinizing hormone production was altered in a sex-specific fashion. Overall, our analyses demonstrate that SMAD3 is required for FSH synthesis in vivo.

A novel IGSF1 mutation in a large Irish kindred highlights the need for familial screening in the IGSF1 deficiency syndrome.

OBJECTIVE: Loss-of-function mutations in IGSF1 result in X-linked central congenital hypothyroidism (CeCH), occurring in isolation or associated with additional pituitary hormone deficits. Intrafamilial penetrance is highly variable and a minority of heterozygous females are also affected. We identified and characterized a novel IGSF1 mutation and investigated its associated phenotypes in a large Irish kindred. DESIGN, PATIENTS AND MEASUREMENTS: A novel hemizygous IGSF1 mutation was identified by direct sequencing in two brothers with CeCH, and its functional consequences were characterized in vitro. Genotype-phenotype correlations were investigated in the wider kindred. RESULTS: The mutant IGSF1 protein (c.2318T > C, p.L773P) exhibited decreased plasma membrane expression in vitro due to impaired trafficking from the endoplasmic reticulum. Ten hemizygous males and 11 heterozygous females exhibited characteristic endocrine deficits. Ireland operates a TSH-based CH screening programme, which does not detect CeCH; therefore, genetic ascertainment preceded biochemical diagnosis of moderate CH in five of seven boys as well as their 75-year-old grandfather. Clinical features potentially attributable to hypothyroidism were variable; normal free T3 (FT3) and low/low normal reverse T3 (rT3) concentrations suggested that preferential deiodination of FT4 to FT3 may help maintain tissue euthyroidism in some individuals. However, neonatal jaundice, delayed speech or growth, and obesity were observed in seven subjects in whom diagnosis was delayed. CONCLUSIONS: As observed with other IGSF1 mutations, p.L773P results in variably penetrant IGSF1 deficiency syndrome. Our observations emphasize the need for multi-generation genetic ascertainment in affected families, especially where TSH-based CH screening programmes may fail to detect CeCH at birth.

Structural basis for potency differences between GDF8 and GDF11.

BACKGROUND: Growth/differentiation factor 8 (GDF8) and GDF11 are two highly similar members of the transforming growth factor ß (TGFß) family. While GDF8 has been recognized as a negative regulator of muscle growth and differentiation, there are conflicting studies on the function of GDF11 and whether GDF11 has beneficial effects on age-related dysfunction. To address whether GDF8 and GDF11 are functionally identical, we compared their signaling and structural properties. RESULTS: Here we show that, despite their high similarity, GDF11 is a more potent activator of SMAD2/3 and signals more effectively through the type I activin-like receptor kinase receptors ALK4/5/7 than GDF8. Resolution of the GDF11:FS288 complex, apo-GDF8, and apo-GDF11 crystal structures reveals unique properties of both ligands, specifically in the type I receptor binding site. Lastly, substitution of GDF11 residues into GDF8 confers enhanced activity to GDF8. CONCLUSIONS: These studies identify distinctive structural features of GDF11 that enhance its potency, relative to GDF8; however, the biological consequences of these differences remain to be determined.

Pituitary Hormone Secretion Profiles in IGSF1 Deficiency Syndrome.

BACKGROUND: Loss-of-function mutations in immunoglobulin superfamily member 1 (IGSF1) cause an X-linked syndrome of central hypothyroidism, macroorchidism, delayed pubertal testosterone rise, variable prolactin deficiency and variable partial GH deficiency in childhood. The clinical features and gene expression pattern suggest a pivotal role for IGSF1 in the pituitary, but detailed knowledge on pituitary hormone secretion in this syndrome is lacking. We therefore aimed to study the 24-hour pituitary hormone secretion in male patients with IGSF1 deficiency. METHODS: We collected blood samples every 10 min for 24 h in eight adult male IGSF1-deficient patients and measured circulating TSH, prolactin and gonadotropins. Deconvolution, modified cosinor and approximate entropy analyses were applied to quantify secretion rates, diurnal rhythmicity and regularity of hormone release. Results were compared to healthy controls matched for age and body mass index. RESULTS: Compared to healthy controls, IGSF1-deficient patients showed decreased pulsatile secretion of TSH with decreased disorderliness and reduced diurnal variation. Basal and pulsatile secretion of FSH was increased by over 200%, while LH secretion did not differ from healthy controls. We observed a bimodal distribution of prolactin secretion, i.e. severe deficiency in three and increased basal and total secretion in the other five patients. CONCLUSION: The altered TSH secretion pattern is consistent with the previously hypothesized defect in thyrotropin-releasing hormone signaling in IGSF1 deficiency. However, the phenotype is more extensive and includes increased FSH secretion without altered LH secretion as well as either undetectable or increased prolactin secretion.

Follicle-stimulating hormone synthesis and fertility are intact in mice lacking SMAD3 DNA binding activity and SMAD2 in gonadotrope cells.

The activin/inhibin system regulates follicle-stimulating hormone (FSH) synthesis and release by pituitary gonadotrope cells in mammals. In vitro cell line data suggest that activins stimulate FSH ß-subunit (Fshb) transcription via complexes containing the receptor-regulated SMAD proteins SMAD2 and SMAD3. Here, we used a Cre-loxP approach to determine the necessity for SMAD2 and/or SMAD3 in FSH synthesis in vivo. Surprisingly, mice with conditional mutations in both Smad2 and Smad3 specifically in gonadotrope cells are fertile and produce FSH at quantitatively normal levels. Notably, however, we discovered that the recombined Smad3 allele produces a transcript that encodes the entirety of the SMAD3 C-terminal Mad homology 2 (MH2) domain. This protein behaves similarly to full-length SMAD3 in Fshb transcriptional assays. As the truncated protein lacks the N-terminal Mad homology 1 (MH1) domain, these results show that SMAD3 DNA-binding activity as well as SMAD2 are dispensable for normal FSH synthesis in vivo. Furthermore, the observation that deletion of proximal exons does not remove all SMAD3 function may facilitate interpretation of divergent phenotypes previously described in different Smad3 knockout mouse lines.

Follicle-stimulating hormone synthesis and fertility depend on SMAD4 and FOXL2.

Follicle-stimulating hormone (FSH) is an essential regulator of gonadal function and fertility. Loss-of-function mutations in the FSHB/Fshb gene cause hypogonadotropic hypogonadism in humans and mice. Both gonadotropin-releasing hormone (GnRH) and activins, members of the transforming growth factor ß (TGFß) superfamily, stimulate FSH synthesis; yet, their relative roles and mechanisms of action in vivo are unknown. Here, using conditional gene-targeting, we show that the canonical mediator of TGFß superfamily signaling, SMAD4, is absolutely required for normal FSH synthesis in both male and female mice. Moreover, when the Smad4 gene is ablated in combination with its DNA binding cofactor Foxl2 in gonadotrope cells, mice make essentially no FSH and females are sterile. Indeed, the phenotype of these animals is remarkably similar to that of Fshb-knockout mice. Not only do these results establish SMAD4 and FOXL2 as essential master regulators of Fshb transcription in vivo, they also suggest that activins, or related ligands, could play more important roles in FSH synthesis than GnRH.

IGSF1 variants in boys with familial delayed puberty.

UNLABELLED: The immunoglobulin superfamily member 1 (IGSF1) gene encodes a plasma membrane glycoprotein mainly expressed in pituitary and testes. Loss-of-function mutations in IGSF1 cause an X-linked syndrome of central hypothyroidism (CeH), macroorchidism, and delayed puberty (delayed rise of testosterone, but normal timing of testicular growth). As this syndrome was discovered in patients with CeH, it is unknown whether IGSF1 mutations might also cause delayed puberty without CeH. We therefore determined the prevalence of IGSF1 sequence variants in 30 patients with an apparent X-linked form of constitutional delay of growth and puberty (CDGP). In four families, we discovered three novel variants of unknown clinical significance (VUCSs), with possible pathogenicity predicted by in silico analysis. However, the genotype did not fully cosegregate with CDGP, all three VUCSs showed normal plasma membrane expression in transfected HEK293 cells, and no other features of the IGSF1 deficiency syndrome were observed in family members carrying the VUCSs. The observation of hyperprolactinemia in two carriers remains unexplained. CONCLUSION: There is insufficient evidence to conclude that the three observed VUCSs in IGSF1 are associated with CDGP, making it unlikely that IGSF1 mutations are a prevalent cause of CDGP.

The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding.

Myostatin is a member of the transforming growth factor-beta (TGF-beta) family and a strong negative regulator of muscle growth. Here, we present the crystal structure of myostatin in complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of myostatin very closely resembles that of TGF-beta class members and that this region alone can be swapped into activin A to confer signalling through the non-canonical type I receptor Alk5. Furthermore, the N-terminal domain of Fst288 undergoes conformational rearrangements to bind myostatin and likely acts as a site of specificity for the antagonist. In addition, a unique continuous electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. Overall, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle-wasting disorders.

Mechanisms of activin-stimulated FSH synthesis: the story of a pig and a FOX.

Activins were discovered and, in fact, named more than a quarter century ago based on their abilities to stimulate pituitary follicle-stimulating hormone (FSH) synthesis and secretion. However, it is only in the last decade that we have finally come to understand their underlying mechanisms of action in gonadotroph cells. In this minireview, we chronicle the research that led to the recent discovery of forkhead box L2 (FOXL2) as an essential mediator of activin-regulated FSH beta subunit (Fshb) transcription in vitro and in vivo.

Photoperiod-dependent regulation of gonadotropin-releasing hormone 1 messenger ribonucleic acid levels in the songbird brain.

Annual changes in day length induce marked changes in reproductive function in temperate zone vertebrates. In many avian species, in contrast to other seasonally breeding animals, plasticity in hypothalamic gonadotropin-releasing hormone - 1 (GnRH1) expression rather than (or in addition to) release governs changes in pituitary-gonadal activity. Investigations of the cellular and molecular mechanisms that govern GnRH1 plasticity were previously hindered by a collective inability of scientists in the field to characterize the gnrh1 cDNA in songbirds. We finally overcame this roadblock after data from the zebra finch (Taeniopygia guttata) genome project enabled us to rapidly clone the gnrh1 cDNA from hypothalamic RNA of zebra finches and European starlings (Sturnus vulgaris). Here, we review the original data that identified GnRH1 protein plasticity in the songbird brain and discuss earlier failed attempts to clone gnrh1 in these animals. Then, we present recent efforts, including our own, that successfully characterized gnrh1 in zebra finch and starling, and demonstrated dynamic regulation of gnrh1 mRNA expression, particularly in sub-populations of preoptic area neurons, in the latter. Overall, this paper highlights GnRH1 plasticity in the avian brain, and weaves into the narrative the previously untold story of the challenges to sequencing gnrh1 in songbirds.

Prerequisite endocardial-mesenchymal transition for murine cardiac trabecular angiogenesis.

Coronary heart disease damages the trabecular myocardium, and the regeneration of trabecular vessels may alleviate ischemic injury. However, the origins and developmental mechanisms of trabecular vessels remain unknown. Here, we show that murine ventricular endocardial cells generate trabecular vessels through an "angioEMT" mechanism. Time course fate mapping defined a specific wave of trabecular vascularization by ventricular endocardial cells. Single-cell transcriptomics and immunofluorescence identified a subpopulation of ventricular endocardial cells that underwent endocardial-mesenchymal transition (EMT) before these cells generated trabecular vessels. Ex vivo pharmacological activation and in vivo genetic inactivation experiments identified an EMT signal in ventricular endocardial cells involving SNAI2-TGFB2/TGFBR3, which was a prerequisite for later trabecular-vessel formation. Additional loss- and gain-of-function genetic studies showed that VEGFA-NOTCH1 signaling regulated post-EMT trabecular angiogenesis by ventricular endocardial cells. Our finding that trabecular vessels originate from ventricular endocardial cells through a two-step angioEMT mechanism could inform better regeneration medicine for coronary heart disease.

Automated single-cell omics end-to-end framework with data-driven batch inference.

To facilitate single cell multi-omics analysis and improve reproducibility, we present SPEEDI (Single-cell Pipeline for End to End Data Integration), a fully automated end-to-end framework for batch inference, data integration, and cell type labeling. SPEEDI introduces data-driven batch inference and transforms the often heterogeneous data matrices obtained from different samples into a uniformly annotated and integrated dataset. Without requiring user input, it automatically selects parameters and executes pre-processing, sample integration, and cell type mapping. It can also perform downstream analyses of differential signals between treatment conditions and gene functional modules. SPEEDI's data-driven batch inference method works with widely used integration and cell-typing tools. By developing data-driven batch inference, providing full end-to-end automation, and eliminating parameter selection, SPEEDI improves reproducibility and lowers the barrier to obtaining biological insight from these valuable single-cell datasets. The SPEEDI interactive web application can be accessed at https://speedi.princeton.edu/.

Activin E is a TGFß ligand that signals specifically through activin receptor-like kinase 7.

Activins are one of the three distinct subclasses within the greater Transforming Growth Factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, similar to ActC. Collectively, our results establish ActE as an ALK7 ligand, thereby providing a link between genetic and in vivo studies of ActE as a regulator of adipose tissue.