Obesity disrupts the pituitary-hepatic UPR communication leading to NAFLD progression.

Obesity alters levels of pituitary hormones that govern hepatic immune-metabolic homeostasis, dysregulation of which leads to nonalcoholic fatty liver disease (NAFLD). However, the impact of obesity on intra-pituitary homeostasis is largely unknown. Here, we uncovered a blunted unfolded protein response (UPR) but elevated inflammatory signatures in pituitary glands of obese mice and humans. Furthermore, we found that obesity inflames the pituitary gland, leading to impaired pituitary inositol-requiring enzyme 1α (IRE1α)-X-box-binding protein 1 (XBP1) UPR branch, which is essential for protecting against pituitary endocrine defects and NAFLD progression. Intriguingly, pituitary IRE1-deletion resulted in hypothyroidism and suppressed the thyroid hormone receptor B (THRB)-mediated activation of Xbp1 in the liver. Conversely, activation of the hepatic THRB-XBP1 axis improved NAFLD in mice with pituitary UPR defect. Our study provides the first evidence and mechanism of obesity-induced intra-pituitary cellular defects and the pathophysiological role of pituitary-liver UPR communication in NAFLD progression.

Genetic Analysis of the Stereotypic Phenotype in Peromyscus maniculatus (deer mice).

Peromyscus maniculatus, including the laboratory stock BW, have been used as a model organism for autism spectrum disorder and obsessive-compulsive disorder because of the high occurrence of stereotypy. Several studies have identified neurological and environmental components of the phenotype; however, the heritability of the phenotype has not been examined. This study characterizes the incidence and heritability of vertical jumping stereotypy (VS) and backflipping (BF) behavior in the BW stock of the Peromyscus Genetic Stock Center, which are indicative of autism spectrum disorders. In addition, interspecies crosses between P. maniculatus and P. polionotus were also performed to further dissect genetically stereotypic behavior. The inheritance pattern of VS suggests that multiple genes result in a quantitative trait with low VS being dominant over high VS. The inheritance pattern of BF suggests that fewer genes are involved, with one allele causing BF in a dominant fashion. An association analysis in BW could reveal the underlying genetic loci associated with stereotypy in P. maniculatus, especially for the BF behavior.

Flexible copula model for integrating correlated multi-omics data from single-cell experiments.

With recent advances in technologies to profile multi-omics data at the single-cell level, integrative multi-omics data analysis has been increasingly popular. It is increasingly common that information such as methylation changes, chromatin accessibility, and gene expression are jointly collected in a single-cell experiment. In biomedical studies, it is often of interest to study the associations between various data types and to examine how these associations might change according to other factors such as cell types and gene regulatory components. However, since each data type usually has a distinct marginal distribution, joint analysis of these changes of associations using multi-omics data is statistically challenging. In this paper, we propose a flexible copula-based framework to model covariate-dependent correlation structures independent of their marginals. In addition, the proposed approach could jointly combine a wide variety of univariate marginal distributions, either discrete or continuous, including the class of zero-inflated distributions. The performance of the proposed framework is demonstrated through a series of simulation studies. Finally, it is applied to a set of experimental data to investigate the dynamic relationship between single-cell RNA sequencing, chromatin accessibility, and DNA methylation at different germ layers during mouse gastrulation.

Counteracting epigenetic mechanisms regulate the structural development of neuronal circuitry in human neurons.

Autism spectrum disorders (ASD) are associated with defects in neuronal connectivity and are highly heritable. Genetic findings suggest that there is an overrepresentation of chromatin regulatory genes among the genes associated with ASD. ASH1 like histone lysine methyltransferase (ASH1L) was identified as a major risk factor for ASD. ASH1L methylates Histone H3 on Lysine 36, which is proposed to result primarily in transcriptional activation. However, how mutations in ASH1L lead to deficits in neuronal connectivity associated with ASD pathogenesis is not known. We report that ASH1L regulates neuronal morphogenesis by counteracting the catalytic activity of Polycomb Repressive complex 2 group (PRC2) in stem cell-derived human neurons. Depletion of ASH1L decreases neurite outgrowth and decreases expression of the gene encoding the neurotrophin receptor TrkB whose signaling pathway is linked to neuronal morphogenesis. The neuronal morphogenesis defect is overcome by inhibition of PRC2 activity, indicating that a balance between the Trithorax group protein ASH1L and PRC2 activity determines neuronal morphology. Thus, our work suggests that ASH1L may epigenetically regulate neuronal morphogenesis by modulating pathways like the BDNF-TrkB signaling pathway. Defects in neuronal morphogenesis could potentially impair the establishment of neuronal connections which could contribute to the neurodevelopmental pathogenesis associated with ASD in patients with ASH1L mutations.

Activating mutations in BRAF disrupt the hypothalamo-pituitary axis leading to hypopituitarism in mice and humans.

Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans.

Using genomic resources for linkage analysis in Peromyscus with an application for characterizing Dominant Spot.

BACKGROUND: Peromyscus are the most common mammalian species in North America and are widely used in both laboratory and field studies. The deer mouse, P. maniculatus and the old-field mouse, P. polionotus, are closely related and can generate viable and fertile hybrid offspring. The ability to generate hybrid offspring, coupled with developing genomic resources, enables researchers to conduct linkage analysis studies to identify genomic loci associated with specific traits. RESULTS: We used available genomic data to identify DNA polymorphisms between P. maniculatus and P. polionotus and used the polymorphic data to identify the range of genetic complexity that underlies physiological and behavioral differences between the species, including cholesterol metabolism and genes associated with autism. In addition, we used the polymorphic data to conduct a candidate gene linkage analysis for the Dominant spot trait and determined that Dominant spot is linked to a region of chromosome 20 that contains a strong candidate gene, Sox10. During the linkage analysis, we found that the spot size varied quantitively in affected Peromyscus based on genetic background. CONCLUSIONS: The expanding genomic resources for Peromyscus facilitate their use in linkage analysis studies, enabling the identification of loci associated with specific traits. More specifically, we have linked a coat color spotting phenotype, Dominant spot, with Sox10, a member the neural crest gene regulatory network, and that there are likely two genetic modifiers that interact with Dominant spot. These results establish Peromyscus as a model system for identifying new alleles of the neural crest gene regulatory network.

Microglia morphology and proinflammatory signaling in the nucleus accumbens during nicotine withdrawal.

Smoking is the largest preventable cause of death and disease in the United States. However, <5% of quit attempts are successful, underscoring the urgent need for novel therapeutics. Microglia are one untapped therapeutic target. While previous studies have shown that microglia mediate both inflammatory responses in the brain and brain plasticity, little is known regarding their role in nicotine dependence and withdrawal phenotypes. Here, we examined microglial changes in the striatum-a mesolimbic region implicated in the rewarding effects of drugs and the affective disruptions occurring during withdrawal. We show that both nicotine and withdrawal induce microglial morphological changes; however, proinflammatory effects and anxiogenic behaviors were observed only during nicotine withdrawal. Pharmacological microglial depletion during withdrawal prevented these effects. These results define differential effects of nicotine and withdrawal on inflammatory signaling in the brain, laying the groundwork for development of future smoking cessation therapeutics.

All Together Now: Modeling the Interaction of Neural With Non-neural Systems Using Organoid Models.

The complex development of the human nervous system has been traditionally studied using a combination of animal models, human post-mortem brain tissue, and human genetics studies. However, there has been a lack of experimental human cellular models that would allow for a more precise elucidation of the intricate dynamics of early human brain development. The development of stem cell technologies, both embryonic and induced pluripotent stem cells (iPSCs), has given neuroscientists access to the previously inaccessible early stages of human brain development. In particular, the recent development of three-dimensional culturing methodologies provides a platform to study the differentiation of stem cells in both normal development and disease states in a more in vivo like context. Three-dimensional neural models or cerebral organoids possess an innate advantage over two-dimensional neural cultures as they can recapitulate tissue organization and cell type diversity that resemble the developing brain. Brain organoids also provide the exciting opportunity to model the integration of different brain regions in vitro. Furthermore, recent advances in the differentiation of non-neuronal tissue from stem cells provides the opportunity to study the interaction between the developing nervous system and other non-neuronal systems that impact neuronal function. In this review, we discuss the potential and limitations of the organoid system to study in vitro neurological diseases that arise in the neuroendocrine and the enteric nervous system or from interactions with the immune system.

Regulation of Pituitary Progenitor Differentiation by ß-Catenin.

The pituitary gland is a critical organ that is necessary for many physiological processes, including growth, reproduction, and stress response. The secretion of pituitary hormones from specific cell types regulates these essential processes. Pituitary hormone cell types arise from a common pool of pituitary progenitors, and mutations that disrupt the formation and differentiation of pituitary progenitors result in hypopituitarism. Canonical WNT signaling through CTNNB1 (ß-catenin) is known to regulate the formation of the POU1F1 lineage of pituitary cell types. When ß-catenin is deleted during the initial formation of the pituitary progenitors, Pou1f1 is not transcribed, which leads to the loss of the POU1F1 lineage. However, when ß-catenin is deleted after lineage specification, there is no observable effect. Similarly, the generation of a ß-catenin gain-of-function allele in early pituitary progenitors or stem cells results in the formation of craniopharyngiomas, whereas stimulating ß-catenin in differentiated cell types has no effect. PROP1 is a pituitary-specific transcription factor, and the peak of PROP1 expression coincides with a critical time point in pituitary organogenesis-that is, after pituitary progenitor formation but before lineage specification. We used a Prop1-cre to conduct both loss- and gain-of-function studies on ß-catenin during this critical time point. Our results demonstrate that pituitary progenitors remain sensitive to both loss and gain of ß-catenin at this time point, and that either manipulation results in hypopituitarism.

Canonical WNT Signaling Regulates the Pituitary Organizer and Pituitary Gland Formation.

The pituitary organizer is a domain within the ventral diencephalon that expresses Bmp4, Fgf8, and Fgf10, which induce the formation of the pituitary precursor, Rathke's pouch, from the oral ectoderm. The WNT signaling pathway regulates this pituitary organizer such that loss of Wnt5a leads to an expansion of the pituitary organizer and an enlargement of Rathke's pouch. WNT signaling is classified into canonical signaling, which is mediated by ß-CATENIN, and noncanonical signaling, which operates independently of ß-CATENIN. WNT5A is typically classified as a noncanonical WNT; however, other WNT family members are expressed in the ventral diencephalon and nuclear localized ß-CATENIN is observed in the ventral diencephalon. Therefore, we sought to determine whether canonical WNT signaling is necessary for regulation of pituitary organizer function. Using a conditional loss-of-function approach, we deleted ß-catenin within the mouse ventral diencephalon. Mutant embryos have a smaller Rathke's pouch, resulting from a reduced pituitary organizer, especially Fgf8. This result suggests that canonical WNT signaling promotes pituitary organizer function, instead of inhibiting it. To test this hypothesis, we stimulated canonical WNT signaling in the ventral diencephalon using an inducible gain-of-function allele of ß-catenin and found that stimulating canonical WNT signaling expands the domain of Fgf8 and results in a dysmorphic Rathke's pouch. These results demonstrate that canonical WNT signaling in the ventral diencephalon is necessary for proper expression of pituitary organizer genes and suggests that a balance of both canonical and noncanonical WNT signaling is necessary to ensure proper formation of Rathke's pouch.

Regulation of pituitary stem cells by epithelial to mesenchymal transition events and signaling pathways.

The anterior pituitary gland is comprised of specialized cell-types that produce and secrete polypeptide hormones in response to hypothalamic input and feedback from target organs. These specialized cells arise from stem cells that express SOX2 and the pituitary transcription factor PROP1, which is necessary to establish the stem cell pool and promote an epithelial to mesenchymal-like transition, releasing progenitors from the niche. The adult anterior pituitary responds to physiological challenge by mobilizing the SOX2-expressing progenitor pool and producing additional hormone-producing cells. Knowledge of the role of signaling pathways and extracellular matrix components in these processes may lead to improvements in the efficiency of differentiation of embryonic stem cells or induced pluripotent stem cells into hormone producing cells in vitro. Advances in our basic understanding of pituitary stem cell regulation and differentiation may lead to improved diagnosis and treatment for patients with hypopituitarism.

ß-catenin is required in the neural crest and mesencephalon for pituitary gland organogenesis.

BACKGROUND: The pituitary gland is a highly vascularized tissue that requires coordinated interactions between the neural ectoderm, oral ectoderm, and head mesenchyme during development for proper physiological function. The interactions between the neural ectoderm and oral ectoderm, especially the role of the pituitary organizer in shaping the pituitary precursor, Rathke's pouch, are well described. However, less is known about the role of head mesenchyme in pituitary organogenesis. The head mesenchyme is derived from definitive mesoderm and neural crest, but the relative contributions of these tissues to the mesenchyme adjacent to the pituitary are not known. RESULTS: We carried out lineage tracing experiments using two neural crest-specific mouse cre lines, Wnt1-cre and P0-cre, and determined that the head mesenchyme rostral to the pituitary gland is neural crest derived. To assess the role of the neural crest in pituitary development we ablated it, using Wnt1-cre to delete Ctnnb1 (ß-catenin), which is required for neural crest development. The Wnt1-cre is active in the neural ectoderm, principally in the mesencephalon, but also in the posterior diencephalon. Loss of ß-catenin in this domain causes a rostral shift in the ventral diencephalon, including the pituitary organizer, resulting in pituitary dysmorphology. The neural crest deficient embryos have abnormally dilated pituitary vasculature due to a loss of neural crest derived pericytes. CONCLUSIONS: ß-catenin in the Wnt1 expression domain, including the neural crest, plays a critical role in regulation of pituitary gland growth, development, and vascularization.

Embryonic Development of the Deer Mouse, Peromyscus maniculatus.

Deer mice, or Peromyscus maniculatus, are an emerging model system for use in biomedicine. P. maniculatus are similar in appearance to laboratory mice, Mus musculus, but are more closely related to hamsters than to Mus. The laboratory strains of Peromyscus have captured a high degree of the genetic variability observed in wild populations, and are more similar to the genetic variability observed in humans than are laboratory strains of Mus. The Peromyscus Genetic Stock Center at the University of South Carolina maintains several lines of Peromyscus harboring mutations that result in developmental defects. We present here a description of P. maniculatus development from gastrulation to late gestation to serve as a guide for researchers interested in pursuing developmental questions in Peromyscus.

All Hormone-Producing Cell Types of the Pituitary Intermediate and Anterior Lobes Derive From Prop1-Expressing Progenitors.

Mutations in PROP1, the most common known cause of combined pituitary hormone deficiency in humans, can result in the progressive loss of all hormones of the pituitary anterior lobe. In mice, Prop1 mutations result in the failure to initiate transcription of Pou1f1 (also known as Pit1) and lack somatotropins, lactotropins, and thyrotropins. The basis for this species difference is unknown. We hypothesized that Prop1 is expressed in a progenitor cell that can develop into all anterior lobe cell types, and not just the somatotropes, thyrotropes, and lactotropes, which are collectively known as the PIT1 lineage. To test this idea, we produced a transgenic Prop1-cre mouse line and conducted lineage-tracing experiments of Prop1-expressing cells. The results reveal that all hormone-secreting cell types of both the anterior and intermediate lobes are descended from Prop1-expressing progenitors. The Prop1-cre mice also provide a valuable genetic reagent with a unique spatial and temporal expression for generating tissue-specific gene rearrangements early in pituitary gland development. We also determined that the minimal essential sequences for reliable Prop1 expression lie within 10 kilobases of the mouse gene and demonstrated that human PROP1 can substitute functionally for mouse Prop1. These studies enhance our understanding of the pathophysiology of disease in patients with PROP1 mutations.

Homeodomain Proteins SIX3 and SIX6 Regulate Gonadotrope-specific Genes During Pituitary Development.

Sine oculis-related homeobox 3 (SIX3) and SIX6, 2 closely related homeodomain transcription factors, are involved in development of the mammalian neuroendocrine system and mutations of Six6 adversely affect fertility in mice. We show that both small interfering RNA knockdown in gonadotrope cell lines and knockout of Six6 in both embryonic and adult male mice (Six6 knockout) support roles for SIX3 and SIX6 in transcriptional regulation in gonadotrope gene expression and that SIX3 and SIX6 can functionally compensate for each other. Six3 and Six6 expression patterns in gonadotrope cell lines reflect the timing of the expression of pituitary markers they regulate. Six3 is expressed in an immature gonadotrope cell line and represses transcription of the early lineage-specific pituitary genes, GnRH receptor (GnRHR) and the common α-subunit (Cga), whereas Six6 is expressed in a mature gonadotrope cell line and represses the specific ß-subunits of LH and FSH (LHb and FSHb) that are expressed later in development. We show that SIX6 repression requires interaction with transducin-like enhancer of split corepressor proteins and competition for DNA-binding sites with the transcriptional activator pituitary homeobox 1. Our studies also suggest that estradiol and circadian rhythm regulate pituitary expression of Six6 and Six3 in adult females but not in males. In summary, SIX3 and SIX6 play distinct but compensatory roles in regulating transcription of gonadotrope-specific genes as gonadotrope cells differentiate.

Pituitary gland development and disease: from stem cell to hormone production.

Many aspects of pituitary development have become better understood in the past two decades. The signaling pathways regulating pituitary growth and shape have emerged, and the balancing interactions between the pathways are now appreciated. Markers for multipotent progenitor cells are being identified, and signature transcription factors have been discovered for most hormone-producing cell types. We now realize that pulsatile hormone secretion involves a 3D integration of cellular networks. About a dozen genes are known to cause pituitary hypoplasia when mutated due to their essential roles in pituitary development. Similarly, a few genes are known that predispose to familial endocrine neoplasia, and several genes mutated in sporadic pituitary adenomas are documented. In the next decade, we anticipate gleaning a deeper appreciation of these processes at the molecular level, insight into the development of the hypophyseal portal blood system, and evolution of better therapeutics for congenital and acquired hormone deficiencies and for common craniopharyngiomas and pituitary adenomas.

Efficient, specific, developmentally appropriate cre-mediated recombination in anterior pituitary gonadotropes and thyrotropes.

Tissue-specific expression of cre recombinase is a well-established genetic tool to analyze gene function, and it is limited only by the efficiency and specificity of available cre mouse strains. Here, we report the generation of a transgenic line containing a cre cassette with codon usage optimized for mammalian cells (iCre) under the control of a mouse glycoprotein hormone α-subunit (αGSU) regulatory sequences in a bacterial artificial chromosome genomic clone. Initial analysis of this transgenic line, Tg(αGSU-iCre), with cre reporter strains reveals onset of cre activity in the differentiating cells of the developing anterior pituitary gland at embryonic day 12.5, with a pattern characteristic of endogenous αGSU. In adult mice, αGSU-iCre was active in the anterior lobe of the pituitary gland and in the cells that produce αGSU (gonadotropes and thyrotropes) with high penetrance. Little or no activity was observed in other tissues, including skeletal and cardiac muscle, brain, kidney, lungs, testis, ovary, and liver. This αGSU-iCre line is suitable for efficient, specific, and developmentally regulated deletion of floxed alleles in anterior pituitary gonadotropes and thyrotropes.

Pituitary stem cell update and potential implications for treating hypopituitarism.

Stem cells have been identified in organs with both low and high cell turnover rates. They are characterized by the expression of key marker genes for undifferentiated cells, the ability to self-renew, and the ability to regenerate tissue after cell loss. Several recent reports present evidence for the presence of pituitary stem cells. Here we offer a critical review of the field and suggest additional studies that could resolve points of debate. Recent reports have relied on different markers, including SOX2, nestin, GFRa2, and SCA1, to identify pituitary stem cells and progenitors. Future studies will be needed to resolve the relationships between cells expressing these markers. Members of the Sox family of transcription factors are likely involved in the earliest steps of pituitary stem cell proliferation and the earliest transitions to differentiation. The transcription factor PROP1 and the NOTCH signaling pathway may regulate the transition to differentiation. Identification of the stem cell niche is an important step in understanding organ development. The niche may be the marginal zone around the lumen of Rathke's pouch, between the anterior and intermediate lobes of mouse pituitary, because cells in this region apparently give birth to all six pituitary hormone cell lineages. Stem cells have been shown to play a role in recurrent malignancies in some tissues, and their role in pituitary hyperplasia, pituitary adenomas, and tumors is an important area for future investigation. From a therapeutic viewpoint, the ability to cultivate and grow stem cells in a pituitary predifferentiation state might also be helpful for the long-term treatment of pituitary deficiencies.

Birthdating studies reshape models for pituitary gland cell specification.

The intermediate and anterior lobes of the pituitary gland are derived from an invagination of oral ectoderm that forms Rathke's pouch. During gestation proliferating cells are enriched around the pouch lumen, and they appear to delaminate as they exit the cell cycle and differentiate. During late mouse gestation and the postnatal period, anterior lobe progenitors re-enter the cell cycle and expand the populations of specialized, hormone-producing cells. At birth, all cell types are present, and their localization appears stratified based on cell type. We conducted a birth dating study of Rathke's pouch derivatives to determine whether the location of specialized cells at birth is correlated with the timing of cell cycle exit. We find that all of the anterior lobe cell types initiate differentiation concurrently with a peak between e11.5 and e13.5. Differentiation of intermediate lobe melanotropes is delayed relative to anterior lobe cell types. We discovered that specialized cell types are not grouped together based on birth date and are dispersed throughout the anterior lobe. Thus, the apparent stratification of specialized cells at birth is not correlated with cell cycle exit. Thus, the currently popular model of cell specification, dependent upon timing of extrinsic, directional gradients of signaling molecules, needs revision. We propose that signals intrinsic to Rathke's pouch are necessary for cell specification between e11.5 and e13.5 and that cell-cell communication likely plays an important role in regulating this process.