Jessica Tollkuhn

Homeodomain-Mediated β-Catenin-Dependent Switching Events Dictate Cell-Lineage Determination

While the biological roles of canonical Wnt/beta-catenin signaling in development and disease are well documented, understanding the molecular logic underlying the functionally distinct nuclear transcriptional programs mediating the diverse functions of beta-catenin remains a major challenge. Here, we report an unexpected strategy for beta-catenin-dependent regulation of cell-lineage determination based on interactions between beta-catenin and a specific homeodomain factor, Prop1, rather than Lef/Tcfs. beta-catenin acts as a binary switch to simultaneously activate expression of the critical lineage-determining transcription factor, Pit1, and to repress the gene encoding the lineage-inhibiting transcription factor, Hesx1, acting via TLE/Reptin/HDAC1 corepressor complexes. The strategy of functionally distinct actions of a homeodomain factor in response to Wnt signaling is suggested to be prototypic of a widely used mechanism for generating diverse cell types from pluripotent precursor cells in response to common signaling pathways during organogenesis.

Estrogen Masculinizes Neural Pathways and Sex-Specific Behaviors

Sex hormones are essential for neural circuit development and sex-specific behaviors. Male behaviors require both testosterone and estrogen, but it is unclear how the two hormonal pathways intersect. Circulating testosterone activates the androgen receptor (AR) and is also converted into estrogen in the brain via aromatase. We demonstrate extensive sexual dimorphism in the number and projections of aromatase-expressing neurons. The masculinization of these cells is independent of AR but can be induced in females by either testosterone or estrogen, indicating a role for aromatase in sexual differentiation of these neurons. We provide evidence suggesting that aromatase is also important in activating male-specific aggression and urine marking because these behaviors can be elicited by testosterone in males mutant for AR and in females subjected to neonatal estrogen exposure. Our results suggest that aromatization of testosterone into estrogen is important for the development and activation of neural circuits that control male territorial behaviors.

The androgen receptor governs the execution, but not programming, of male sexual and territorial behaviors

Testosterone and estrogen are essential for male behaviors in vertebrates. How these two signaling pathways interact to control masculinization of the brain and behavior remains to be established. Circulating testosterone activates the androgen receptor (AR) and also serves as the source of estrogen in the brain. We have used a genetic strategy to delete AR specifically in the mouse nervous system. This approach permits us to determine the function of AR in sexually dimorphic behaviors in males while maintaining circulating testosterone levels within the normal range. We find that AR mutant males exhibit masculine sexual and territorial displays, but they have striking deficits in specific components of these behaviors. Taken together with the surprisingly limited expression of AR in the developing brain, our findings indicate that testosterone acts as a precursor to estrogen to masculinize the brain and behavior, and signals via AR to control the levels of male behavioral displays.

Required enhancer-matrin-3 network interactions for a homeodomain transcription program

Homeodomain proteins, described 30 years ago, exert essential roles in development as regulators of target gene expression; however, the molecular mechanisms underlying transcriptional activity of homeodomain factors remain poorly understood. Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program. Pit1 association with Satb1 (ref. 8) and β-catenin is required for this tethering event. A naturally occurring, dominant negative, point mutation in human PIT1(R271W), causing combined pituitary hormone deficiency, results in loss of Pit1 association with β-catenin and Satb1 and therefore the matrin-3-rich network, blocking Pit1-dependent enhancer/coding target gene activation. This defective activation can be rescued by artificial tethering of the mutant R271W Pit1 protein to the matrin-3 network, bypassing the pre-requisite association with β-catenin and Satb1 otherwise required. The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation. These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.

Notch-Dependent Pituitary SOX2+ Stem Cells Exhibit a Timed Functional Extinction in Regulation of the Postnatal Gland

Summary Although SOX2+ stem cells are present in the postnatal pituitary gland, how they are regulated molecularly and whether they are required for pituitary functions remain unresolved questions. Using a conditional knockout animal model, here we demonstrate that ablation of the canonical Notch signaling in the embryonic pituitary gland leads to progressive depletion of the SOX2+ stem cells and hypoplastic gland. Furthermore, we show that the SOX2+ stem cells initially play a significant role in contributing to postnatal pituitary gland expansion by self-renewal and differentiating into distinct lineages in the immediate postnatal period. However, we found that within several weeks postpartum, the SOX2+ stem cells switch to an essentially dormant state and are no longer required for homeostasis/tissue adaptation. Our results present a dynamic tissue homeostatic model in which stem cells provide an initial contribution to the growth of the neonatal pituitary gland, whereas the mature gland can be maintained in a stem cell-independent fashion.

A custody battle for the mind: evidence for extensive imprinting in the brain.

Relatively few genes (approximately 100) have previously been shown to be imprinted such that their expression in progeny derives from either the maternal or paternal copy. Two recent studies by Gregg et al. (2010a, 2010b) in Science expand this list by an order of magnitude, revealing complex patterns of parent-of-origin bias in gene expression in the brain that are developmentally and regionally restricted, and in many cases, sexually dimorphic.

Estrogen receptor alpha is required in GABAergic, but not glutamatergic, neurons to masculinize behavior

ABSTRACT Masculinization of the altricial rodent brain is driven by estrogen signaling during a perinatal critical period. Genetic deletion of estrogen receptor alpha (Esr1/ER&agr;) results in altered hypothalamic‐pituitary‐gonadal (HPG) axis signaling and a dramatic reduction of male sexual and territorial behaviors. However, the role of ER&agr; in masculinizing distinct classes of neurons remains unexplored. We deleted ER&agr; in excitatory or inhibitory neurons using either a Vglut2 or Vgat driver and assessed male behaviors. We find that Vglut2‐Cre;Esr1lox/lox mutant males lack ER&agr; in the ventrolateral region of the ventromedial hypothalamus (VMHvl) and posterior ventral portion of the medial amygdala (MePV). These mutants recapitulate the increased serum testosterone levels seen with constitutive ER&agr; deletion, but have none of the behavioral deficits. In contrast, Vgat‐Cre;Esr1lox/lox males with substantial ER&agr; deletion in inhibitory neurons, including those of the principal nucleus of the bed nucleus of the stria terminalis (BNSTpr), posterior dorsal MeA (MePD), and medial preoptic area (MPOA) have normal testosterone levels, but display alterations in mating and territorial behaviors. These mutants also show dysmasculinized expression of androgen receptor (AR) and estrogen receptor beta (Esr2). Our results demonstrate that ER&agr; masculinizes GABAergic neurons that gate the display of male‐typical behaviors. HIGHLIGHTSMales do not require estrogen receptor alpha in vGlut2+ excitatory neurons for display of sexual and territorial behaviorsMales lacking ER&agr; in vGAT+ inhibitory neurons have deficits in sexual and territorial behaviorsMales lacking ER&agr; in vGAT+ neurons display alterations in gene expression in select vGAT+ brain areas

A Central Extended Amygdala Circuit That Modulates Anxiety

Both the amygdala and the bed nucleus of the stria terminalis (BNST) have been implicated in maladaptive anxiety characteristics of anxiety disorders. However, the underlying circuit and cellular mechanisms have remained elusive. Here we show that mice with Erbb4 gene deficiency in somatostatin-expressing (SOM+) neurons exhibit heightened anxiety as measured in the elevated plus maze test and the open field test, two assays commonly used to assess anxiety-related behaviors in rodents. Using a combination of electrophysiological, molecular, genetic, and pharmacological techniques, we demonstrate that the abnormal anxiety in the mutant mice is caused by enhanced excitatory synaptic inputs onto SOM+ neurons in the central amygdala (CeA), and the resulting reduction in inhibition onto downstream SOM+ neurons in the BNST. Notably, our results indicate that an increase in dynorphin signaling in SOM+ CeA neurons mediates the paradoxical reduction in inhibition onto SOM+ BNST neurons, and that the consequent enhanced activity of SOM+ BNST neurons is both necessary for and sufficient to drive the elevated anxiety. Finally, we show that the elevated anxiety and the associated synaptic dysfunctions and increased dynorphin signaling in the CeA–BNST circuit of the Erbb4 mutant mice can be recapitulated by stress in wild-type mice. Together, our results unravel previously unknown circuit and cellular processes in the central extended amygdala that can cause maladaptive anxiety. SIGNIFICANCE STATEMENT The central extended amygdala has been implicated in anxiety-related behaviors, but the underlying mechanisms are unclear. Here we found that somatostatin-expressing neurons in the central amygdala (CeA) controls anxiety through modulation of the stria terminalis, a process that is mediated by an increase in dynorphin signaling in the CeA. Our results reveal circuit and cellular dysfunctions that may account for maladaptive anxiety.

Epithelial cell integrin β1 is required for developmental angiogenesis in the pituitary gland

Significance During embryogenesis, a dense vascular network develops in the pituitary gland through the process of angiogenesis. In tandem, pituitary gland precursor cells differentiate into hormone-producing cells that will rely on the vasculature to carry out regulated endocrine function. Our data show that expression of the cell surface adhesion molecule, integrin β1, in the epithelial-derived precursor cells is required for development of the vasculature and coordinated terminal differentiation of endocrine cells. As a key component of the vertebrate neuroendocrine system, the pituitary gland relies on the progressive and coordinated development of distinct hormone-producing cell types and an invading vascular network. The molecular mechanisms that drive formation of the pituitary vasculature, which is necessary for regulated synthesis and secretion of hormones that maintain homeostasis, metabolism, and endocrine function, remain poorly understood. Here, we report that expression of integrin β1 in embryonic pituitary epithelial cells is required for angiogenesis in the developing mouse pituitary gland. Deletion of pituitary epithelial integrin β1 before the onset of angiogenesis resulted in failure of invading endothelial cells to recruit pericytes efficiently, whereas deletion later in embryogenesis led to decreased vascular density and lumen formation. In both cases, lack of epithelial integrin β1 was associated with a complete absence of vasculature in the pituitary gland at birth. Within pituitary epithelial cells, integrin β1 directs a large transcriptional program that includes components of the extracellular matrix and associated signaling factors that are linked to the observed non–cell-autonomous effects on angiogenesis. We conclude that epithelial integrin β1 functions as a critical and canonical regulator of developmental angiogenesis in the pituitary gland, thus providing insight into the long-standing systems biology conundrum of how vascular invasion is coordinated with tissue development.

Gene regulatory mechanisms underlying sex differences in brain development and psychiatric disease

The sexual differentiation of the mammalian nervous system requires the precise coordination of the temporal and spatial regulation of gene expression in diverse cell types. Sex hormones act at multiple developmental time points to specify sex‐typical differentiation during embryonic and early development and to coordinate subsequent responses to gonadal hormones later in life by establishing sex‐typical patterns of epigenetic modifications across the genome. Thus, mutations associated with neuropsychiatric conditions may result in sexually dimorphic symptoms by acting on different neural substrates or chromatin landscapes in males and females. Finally, as stress hormone signaling may directly alter the molecular machinery that interacts with sex hormone receptors to regulate gene expression, the contribution of chronic stress to the pathogenesis or presentation of mental illness may be additionally different between the sexes. Here, we review the mechanisms that contribute to sexual differentiation in the mammalian nervous system and consider some of the implications of these processes for sex differences in neuropsychiatric conditions.