Single-cell RNA sequencing reveals PDGFRα+ stromal cell subpopulations that promote proacinar cell differentiation in embryonic salivary gland organoids.

Stromal cells can direct the differentiation of epithelial progenitor cells during organ development. Fibroblast growth factor (FGF) signaling is essential for submandibular salivary gland development. Through stromal fibroblast cells, FGF2 can indirectly regulate proacinar cell differentiation in organoids, but the mechanisms are not understood. We performed single-cell RNA-sequencing and identified multiple stromal cell subsets, including Pdgfra+ stromal subsets expressing both Fgf2 and Fgf10. When combined with epithelial progenitor cells in organoids, magnetic-activated cell-sorted PDGFRα+ cells promoted proacinar cell differentiation similarly to total stroma. Gene expression analysis revealed that FGF2 increased the expression of multiple stromal genes, including Bmp2 and Bmp7. Both BMP2 and BMP7 synergized with FGF2, stimulating proacinar cell differentiation but not branching. However, stromal cells grown without FGF2 did not support proacinar organoid differentiation and instead differentiated into myofibroblasts. In organoids, TGFß1 treatment stimulated myofibroblast differentiation and inhibited the proacinar cell differentiation of epithelial progenitor cells. Conversely, FGF2 reversed the effects of TGFß1. We also demonstrated that adult salivary stromal cells were FGF2 responsive and could promote proacinar cell differentiation. These FGF2 signaling pathways may have applications in future regenerative therapies.

Notch signaling determines cell-fate specification of the two main types of vomeronasal neurons of rodents.

The ability of terrestrial vertebrates to find food and mating partners, and to avoid predators, relies on the detection of chemosensory information. Semiochemicals responsible for social and sexual behaviors are detected by chemosensory neurons of the vomeronasal organ (VNO), which transmits information to the accessory olfactory bulb. The vomeronasal sensory epithelium of most mammalian species contains a uniform vomeronasal system; however, rodents and marsupials have developed a more complex binary vomeronasal system, containing vomeronasal sensory neurons (VSNs) expressing receptors of either the V1R or V2R family. In rodents, V1R/apical and V2R/basal VSNs originate from a common pool of progenitors. Using single cell RNA-sequencing, we identified differential expression of Notch1 receptor and Dll4 ligand between the neuronal precursors at the VSN differentiation dichotomy. Our experiments show that Notch signaling is required for effective differentiation of V2R/basal VSNs. In fact, Notch1 loss of function in neuronal progenitors diverts them to the V1R/apical fate, whereas Notch1 gain of function redirects precursors to V2R/basal. Our results indicate that Notch signaling plays a pivotal role in triggering the binary differentiation dichotomy in the VNO of rodents.

Msl3 promotes germline stem cell differentiation in female Drosophila.

Gamete formation from germline stem cells (GSCs) is essential for sexual reproduction. However, the regulation of GSC differentiation is incompletely understood. Set2, which deposits H3K36me3 modifications, is required for GSC differentiation during Drosophila oogenesis. We discovered that the H3K36me3 reader Male-specific lethal 3 (Msl3) and histone acetyltransferase complex Ada2a-containing (ATAC) cooperate with Set2 to regulate GSC differentiation in female Drosophila. Msl3, acting independently of the rest of the male-specific lethal complex, promotes transcription of genes, including a germline-enriched ribosomal protein S19 paralog RpS19b. RpS19b upregulation is required for translation of RNA-binding Fox protein 1 (Rbfox1), a known meiotic cell cycle entry factor. Thus, Msl3 regulates GSC differentiation by modulating translation of a key factor that promotes transition to an oocyte fate.

Following the p63/Keratin5 basal cells in the sensory and non-sensory epithelia of the vomeronasal organ.

The vomeronasal organ (VNO) is a part of the accessory olfactory system, which detects pheromones and chemical factors that trigger a spectrum of sexual and social behaviors. The vomeronasal epithelium (VNE) shares several features with the epithelium of the main olfactory epithelium (MOE). However, it is a distinct neuroepithelium populated by chemosensory neurons that differ from the olfactory sensory neurons in cellular structure, receptor expression, and connectivity. The vomeronasal organ of rodents comprises a sensory epithelium (SE) and a thin non-sensory epithelium (NSE) that morphologically resembles the respiratory epithelium. Sox2-positive cells have been previously identified as the stem cell population that gives rise to neuronal progenitors in MOE and VNE. In addition, the MOE also comprises p63 positive horizontal basal cells, a second pool of quiescent stem cells that become active in response to injury. Immunolabeling against the transcription factor p63, Keratin-5 (Krt5), Krt14, NrCAM, and Krt5Cre tracing experiments highlighted the existence of horizontal basal cells distributed along the basal lamina of SE of the VNO. Single cell sequencing and genetic lineage tracing suggest that the vomeronasal horizontal basal cells arise from basal progenitors at the boundary between the SE and NSE proximal to the marginal zones. Moreover, our experiments revealed that the NSE of rodents is, like the respiratory epithelium, a stratified epithelium where the p63/Krt5+ basal progenitor cells self-replicate and give rise to the apical columnar cells facing the lumen of the VNO.

Smad4-dependent morphogenic signals control the maturation and axonal targeting of basal vomeronasal sensory neurons to the accessory olfactory bulb.

The vomeronasal organ (VNO) contains two main types of vomeronasal sensory neurons (VSNs) that express distinct vomeronasal receptor (VR) genes and localize to specific regions of the neuroepithelium. Morphogenic signals are crucial in defining neuronal identity and network formation; however, if and what signals control maturation and homeostasis of VSNs is largely unexplored. Here, we found transforming growth factor ß (TGFß) and bone morphogenetic protein (BMP) signal transduction in postnatal mice, with BMP signaling being restricted to basal VSNs and at the marginal zones of the VNO: the site of neurogenesis. Using different Smad4 conditional knockout mouse models, we disrupted canonical TGFß/BMP signaling in either maturing basal VSNs (bVSNs) or all mature VSNs. Smad4 loss of function in immature bVSNs compromises dendritic knob formation, pheromone induced activation, correct glomeruli formation in the accessory olfactory bulb (AOB) and survival. However, Smad4 loss of function in all mature VSNs only compromises correct glomeruli formation in the posterior AOB. Our results indicate that Smad4-mediated signaling drives the functional maturation and connectivity of basal VSNs.

Mechanisms underlying pre- and postnatal development of the vomeronasal organ.

The vomeronasal organ (VNO) is sensory organ located in the ventral region of the nasal cavity in rodents. The VNO develops from the olfactory placode during the secondary invagination of olfactory pit. The embryonic vomeronasal structure appears as a neurogenic area where migratory neuronal populations like endocrine gonadotropin-releasing hormone-1 (GnRH-1) neurons form. Even though embryonic vomeronasal structures are conserved across most vertebrate species, many species including humans do not have a functional VNO after birth. The vomeronasal epithelium (VNE) of rodents is composed of two major types of vomeronasal sensory neurons (VSNs): (1) VSNs distributed in the apical VNE regions that express vomeronasal type-1 receptors (V1Rs) and the G protein subunit Gαi2, and (2) VSNs in the basal territories of the VNE that express vomeronasal type-2 receptors (V2Rs) and the G subunit Gαo. Recent studies identified a third subclass of Gαi2 and Gαo VSNs that express the formyl peptide receptor family. VSNs expressing V1Rs or V2Rs send their axons to distinct regions of the accessory olfactory bulb (AOB). Together, VNO and AOB form the accessory olfactory system (AOS), an olfactory subsystem that coordinates the social and sexual behaviors of many vertebrate species. In this review, we summarize our current understanding of cellular and molecular mechanisms that underlie VNO development. We also discuss open questions for study, which we suggest will further enhance our understanding of VNO morphogenesis at embryonic and postnatal stages.

Gli3 Regulates Vomeronasal Neurogenesis, Olfactory Ensheathing Cell Formation, and GnRH-1 Neuronal Migration.

During mammalian development, gonadotropin-releasing-hormone-1 neurons (GnRH-1ns) migrate from the developing vomeronasal organ (VNO) into the brain asserting control of pubertal onset and fertility. Recent data suggest that correct development of the olfactory ensheathing cells (OEC) is imperative for normal GnRH-1 neuronal migration. However, the full ensemble of molecular pathways that regulate OEC development remains to be fully deciphered. Loss-of-function of the transcription factor Gli3 is known to disrupt olfactory development, however, if Gli3 plays a role in GnRH-1 neuronal development is unclear. By analyzing Gli3 extra-toe mutants (Gli3Xt/Xt), we found that Gli3 loss-of-function compromises the onset of achaete-scute family bHLH transcription factor 1 (Ascl-1)+ vomeronasal progenitors and the formation of OEC in the nasal mucosa. Surprisingly, GnRH-1 neurogenesis was intact in Gli3Xt/Xt mice but they displayed significant defects in GnRH-1 neuronal migration. In contrast, Ascl-1null mutants showed reduced neurogenesis for both vomeronasal and GnRH-1ns but less severe defects in OEC development. These observations suggest that Gli3 is critical for OEC development in the nasal mucosa and subsequent GnRH-1 neuronal migration. However, the nonoverlapping phenotypes between Ascl-1 and Gli3 mutants indicate that Ascl-1, while crucial for GnRH-1 neurogenesis, is not required for normal OEC development. Because Kallmann syndrome (KS) is characterized by abnormal GnRH-1ns migration, we examined whole-exome sequencing data from KS subjects. We identified and validated a GLI3 loss-of-function variant in a KS individual. These findings provide new insights into GnRH-1 and OECs development and demonstrate that human GLI3 mutations contribute to KS etiology.SIGNIFICANCE STATEMENT The transcription factor Gli3 is necessary for correct development of the olfactory system. However, if Gli3 plays a role in controlling GnRH-1 neuronal development has not been addressed. We found that Gli3 loss-of-function compromises the onset of Ascl-1+ vomeronasal progenitors, formation of olfactory ensheathing cells in the nasal mucosa, and impairs GnRH-1 neuronal migration to the brain. By analyzing Ascl-1null mutants we dissociated the neurogenic defects observed in Gli3 mutants from lack of olfactory ensheathing cells in the nasal mucosa, moreover, we discovered that Ascl-1 is necessary for GnRH-1 ontogeny. Analyzing human whole-exome sequencing data, we identified a GLI3 loss-of-function variant in a KS individual. Our data suggest that GLI3 is a candidate gene contributing to KS etiology.

Automated quantification of vomeronasal glomeruli number, size, and color composition after immunofluorescent staining.

Glomeruli are neuropil-rich regions of the main or accessory olfactory bulbs (AOB) where the axons of olfactory or vomeronasal neurons and dendrites of mitral/tufted cells form synaptic connections. In the main olfactory system, olfactory sensory neurons (OSNs) expressing the same receptor innervate 1 or 2 glomeruli. However, in the accessory olfactory system, vomeronasal sensory neurons (VSNs) expressing the same receptor can innervate up to 30 different glomeruli in the AOB. Genetic mutation disrupting genes with a role in defining the identity/diversity of olfactory and vomeronasal neurons can alter the number and size of glomeruli. Interestingly, 2 cell surface molecules, Kirrel2 and Kirrel3, have been indicated as playing a critical role in the organization of axons into glomeruli in the AOB. Being able to quantify differences in glomeruli features, such as number, size, or immunoreactivity for specific markers, is an important experimental approach to validate the role of specific genes in controlling neuronal connectivity and circuit formation in either control or mutant animals. Since the manual recognition and quantification of glomeruli on digital images is a challenging and time-consuming task, we generated a program in Python able to identify glomeruli in digital images and quantify their properties, such as size, number, and pixel intensity. Validation of our program indicates that our script is a fast and suitable tool for high-throughput quantification of glomerular features of mouse lines with different genetic makeup.

The transcription factor Tfap2e/AP-2ε plays a pivotal role in maintaining the identity of basal vomeronasal sensory neurons.

The identity of individual neuronal cell types is defined and maintained by the expression of specific combinations of transcriptional regulators that control cell type-specific genetic programs. The epithelium of the vomeronasal organ of mice contains two major types of vomeronasal sensory neurons (VSNs): 1) the apical VSNs which express vomeronasal 1 receptors (V1r) and the G-protein subunit Gαi2 and; 2) the basal VSNs which express vomeronasal 2 receptors (V2r) and the G-protein subunit Gαo. Both cell types originate from a common pool of progenitors and eventually acquire apical or basal identity through largely unknown mechanisms. The transcription factor AP-2ε, encoded by the Tfap2e gene, plays a role in controlling the development of GABAergic interneurons in the main and accessory olfactory bulb (AOB), moreover AP-2ε has been previously described to be expressed in the basal VSNs. Here we show that AP-2ε is expressed in post-mitotic VSNs after they commit to the basal differentiation program. Loss of AP-2ε function resulted in reduced number of basal VSNs and in an increased number of neurons expressing markers of the apical lineage. Our work suggests that AP-2ε, which is expressed in late phases of differentiation, is not needed to initiate the apical-basal differentiation dichotomy but for maintaining the basal VSNs' identity. In AP-2ε mutants we observed a large number of cells that entered the basal program can express apical genes, our data suggest that differentiated VSNs of mice retain a notable level of plasticity.

GnRH, anosmia and hypogonadotropic hypogonadism--where are we?

Gonadotropin releasing hormone (GnRH) neurons originate the nasal placode and migrate into the brain during prenatal development. Once within the brain, these cells become integral components of the hypothalamic-pituitary-gonadal axis, essential for reproductive function. Disruption of this system causes hypogonadotropic hypogonadism (HH). HH associated with anosmia is clinically defined as Kallman syndrome (KS). Recent work examining the developing nasal region has shed new light on cellular composition, cell interactions and molecular cues responsible for the development of this system in different species. This review discusses some developmental aspects, animal models and current advancements in our understanding of pathologies affecting GnRH. In addition we discuss how development of neural crest derivatives such as the glia of the olfactory system and craniofacial structures control GnRH development and reproductive function.

Illuminating the terminal nerve: Uncovering the link between GnRH-1 neuron and olfactory development.

During embryonic development, the olfactory placode (OP) generates migratory neurons, including olfactory pioneer neurons, cells of the terminal nerve (TN), gonadotropin-releasing hormone-1 (GnRH-1) neurons, and other uncharacterized neurons. Pioneer neurons from the OP induce olfactory bulb (OB) morphogenesis. In mice, GnRH-1 neurons appear in the olfactory system around mid-gestation and migrate via the TN axons to different brain regions. The GnRH-1 neurons are crucial in controlling the hypothalamic-pituitary-gonadal axis. Kallmann syndrome is characterized by impaired olfactory system development, defective OBs, secretion of GnRH-1, and infertility. The precise mechanistic link between the olfactory system and GnRH-1 development remains unclear. Studies in humans and mice highlight the importance of the prokineticin-2/prokineticin-receptor-2 (Prokr2) signaling pathway in OB morphogenesis and GnRH-1 neuronal migration. Prokr2 loss-of-function mutations can cause Kallmann syndrome (KS), and hence the Prokr2 signaling pathway represents a unique model to decipher the olfactory/GnRH-1 connection. We discovered that Prokr2 is expressed in the TN neurons during the critical period of GnRH-1 neuron formation, migration, and induction of OB morphogenesis. Single-cell RNA sequencing identified that the TN is formed by neurons distinct from the olfactory neurons. The TN neurons express multiple genes associated with KS. Our study suggests that the aberrant development of pioneer/TN neurons might cause the KS spectrum.

A revised conceptual framework for mouse vomeronasal pumping and stimulus sampling.

The physiological performance of any sensory organ is determined by its anatomy and physical properties. Consequently, complex sensory structures with elaborate features have evolved to optimize stimulus detection. Understanding these structures and their physical nature forms the basis for mechanistic insights into sensory function. Despite its crucial role as a sensor for pheromones and other behaviorally instructive chemical cues, the vomeronasal organ (VNO) remains a poorly characterized mammalian sensory structure. Fundamental principles of its physico-mechanical function, including basic aspects of stimulus sampling, remain poorly explored. Here, we revisit the classical vasomotor pump hypothesis of vomeronasal stimulus uptake. Using advanced anatomical, histological, and physiological methods, we demonstrate that large parts of the lateral mouse VNO are composed of smooth muscle. Vomeronasal smooth muscle tissue comprises two subsets of fibers with distinct topography, structure, excitation-contraction coupling, and, ultimately, contractile properties. Specifically, contractions of a large population of noradrenaline-sensitive cells mediate both transverse and longitudinal lumen expansion, whereas cholinergic stimulation targets an adluminal group of smooth muscle fibers. The latter run parallel to the VNO's rostro-caudal axis and are ideally situated to mediate antagonistic longitudinal constriction of the lumen. This newly discovered arrangement implies a novel mode of function. Single-cell transcriptomics and pharmacological profiling reveal the receptor subtypes involved. Finally, 2D/3D tomography provides non-invasive insight into the intact VNO's anatomy and mechanics, enables measurement of luminal fluid volume, and allows an assessment of relative volume change upon noradrenergic stimulation. Together, we propose a revised conceptual framework for mouse vomeronasal pumping and, thus, stimulus sampling.

Illuminating the Terminal Nerve: Uncovering the Link between GnRH-1 and Olfactory Development.

During embryonic development, the olfactory placode (OP) generates migratory neurons, including olfactory pioneer neurons, cells of the terminal nerve (TN), Gonadotropin-releasing hormone-1 (GnRH-1) neurons, and other uncharacterized neurons. Pioneer neurons from the olfactory placode induce olfactory bulb morphogenesis. In mice, GnRH-1 neurons appear in the olfactory system around mid-gestation and migrate via the terminal nerve axons to different brain regions. The GnRH-1 neurons are crucial in controlling the hypothalamic-pituitary-gonadal (HPG) axis. Kallmann syndrome is characterized by impaired olfactory system development, defective olfactory bulbs, defective secretion of GnRH-1, and infertility. The precise mechanistic link between the olfactory system and GnRH-1 development remains unclear. Studies in humans and mice highlight the importance of the Prokineticin-2/Prokineticin-Receptor-2 (Prokr2) signaling pathway in olfactory bulb morphogenesis and GnRH-1 neuronal migration. Prokr2 loss-of-function mutations can cause Kallmann syndrome, and hence the Prokr2 signaling pathway represents a unique model to decipher the olfactory/GnRH-1 connection. We discovered that Prokr2 is expressed in the TN neurons during the critical period of GnRH-1 neuron formation, migration, and induction of olfactory bulb morphogenesis. Single-cell RNA sequencing identified that the TN is formed by neurons that are distinct from the olfactory neurons. The TN neurons express multiple genes associated with KS. Our study suggests that the aberrant development of pioneer/TN neurons might cause the KS spectrum. Key Points: 1) Pioneer or terminal nerve neurons play a crucial role in initiating the development of the olfactory bulbs. We found that the Prokineticin Receptor-2 gene, associated with Kallmann syndrome, is expressed by the olfactory pioneer/terminal nerve neurons.2) We genetically traced, isolated, and conducted Single-cell RNA sequencing on terminal nerve neurons of rodents. This analysis revealed a significant enrichment of gene expression related to Kallmann syndrome.3) Our study indicates that the investigation of Pioneer/terminal nerve neurons should be a pivotal focal point for comprehending developmental defects affecting olfactory and GnRH-1 systems.

Loss Of Chromodomain of Male-Specific Lethal 3 (MSL3) Does Not Affect Spermatogenesis In Rodents.

Msl3 is a member of the chromatin-associated male-specific lethal MSL complex which is responsible for the transcriptional upregulation of genes on the X chromosome in males Drosophila. Although the dosage complex operates differently in mammals, the Msl3 gene is conserved from flies to humans. Msl3 is required for meiotic entry during Drosophila oogenesis. Recent reports indicate that also in primates, Msl3 is expressed in undifferentiated germline cells before meiotic entry. However, if Msl3 plays a role in the meiotic entry of mammals has yet to be explored. To study this, we used mouse spermatogenesis as a study model. Analyses of single cells RNA-seq data revealed that, in mice, Msl3 is mostly expressed in meiotic cells. To test the role of Msl3 in meiosis, we used a male germline-specific Stra8-iCre driver and a newly generated Msl3flox conditional knock-out mouse line. Msl3 conditional loss-of-function in spermatogonia did not cause spermatogenesis defects or changes in the expression of genes related to meiosis. Our data suggest that, in mice, Msl3 exhibits delayed expression compared to Drosophila and primates, and loss-of-function mutations disrupting the chromodomain of Msl3 alone do not impede meiotic entry in rodents.

A role for FE65 in controlling GnRH-1 neurogenesis.

Gonadotropin-releasing hormone-1 (GnRH-1) neurons migrate from the nasal placode to the forebrain where they control gonadal function via the hypothalamic-pituitary-gonadal axis. The birth of GnRH-1-expressing neurons is one of the first neurogenic events in the developing nasal placode. By gene expression screening on single GnRH-1 neurons, amyloid precursor binding protein-1 (FE65) was identified in migratory GnRH-1 neurons. FE65 has been shown to modulate ß1-integrin dynamics, actin cytoskeleton, cell motility, and FE65/amyloid precursor protein signaling has been described in neuro/glial cell fate determination as well as in modulating neurogenesis. Analysis of two mouse lines, one deficient for the 97 kDa FE65 isoform and a second deficient for the 97 and 60 kDa forms of FE65, showed overlapping phenotypes. In both lines, no migratory defects of the GnRH-1 neurons were observed, but a 25% increase in GnRH-1 neuronal number during embryonic development was found. Bromodeoxyuridine birth tracing and spatiotemporal tracking of GnRH-1 cell precursors demonstrated that the lack of the N-terminal portion of FE65, which includes part of the functional nuclear translocation/gene transcription domain of FE65 (WW domain), extends the timing of GnRH-1 neurogenesis in the developing nasal placode without affecting proliferation of GnRH-1 neuronal progenitors or cell death. The observed changes in the dynamics of GnRH-1 neurogenesis highlight a unique role for the 97 kDa isoform of FE65 and suggest that GnRH-1 cells, which have a short neurogenic window, originate from multipotent progenitors able to generate distinct cell types as GnRH-1 neurogenesis declines in response to environmental changes.

Development of the gonadotropin-releasing hormone system.

This review summarizes the current understanding of the development of the neuroendocrine gonadotropin-releasing hormone (GnRH) system, including discussion on open questions regarding (1) transcriptional regulation of the Gnrh1 gene; (2) prenatal development of the GnRH1 system in rodents and humans; and (3) paracrine and synaptic communication during migration of the GnRH cells.

Androgen Regulation of Corticotropin Releasing Factor Receptor 1 in the Mouse Brain.

Stress-related mood disorders like anxiety and depression are more prevalent in women than men and are often associated with hypothalamic-pituitary-adrenal (HPA) axis dysregulation. Androgen actions through androgen receptors (ARs) decrease HPA axis responses and stress-associated behaviors. Corticotropin releasing factor (CRF) and its binding to CRF receptor 1 (CRFR1) is also critical for regulation of the HPA axis, anxiety, and depression. We first determined CRFR1/AR co-localization patterns in male and female CRFR1-GFP mice. High co-localization was found within the paraventricular nucleus (PVN), dorsolateral and anteroventral subdivisions of the bed nucleus of the stria terminalis (BSTdl and BSTav), medial preoptic area (MPOA), and posterodorsal medial amygdala (MePD). We next determined whether the non-aromatizable androgen dihydrotestosterone (DHT) regulates CRFR1 expression and stress-induced activation of CRFR1-expressing cells. In the PVN, CRFR1-GFP cell number decreased following gonadectomy (GDX), but DHT treatment reversed this effect. GDX-DHT treated mice also had a decreased CRFR1-GFP cell number within the BSTdl compared to gonad intact and GDX-untreated groups. Following restraint stress GDX-blank mice showed fewer c-Fos/CRFR1 co-localized neurons in the MePD compared to gonad intact and GDX-DHT groups indicating decreased stress-induced activation of CRFR1 neurons following GDX. Higher plasma corticosterone (CORT) was found in GDX males compared to GDX-DHT and sham males following restraint stress, with a negative correlation between PVN CRFR1+ neurons and corticosterone levels 30- and 90-min following restraint. Together these findings show androgens can directly alter CRFR1 levels in the brain which may have implications for sex differences in regulation of the HPA axis and stress-related behaviors.

Identifying Isl1 Genetic Lineage in the Developing Olfactory System and in GnRH-1 Neurons.

During embryonic development, symmetric ectodermal thickenings [olfactory placodes (OP)] give rise to several cell types that comprise the olfactory system, such as those that form the terminal nerve ganglion (TN), gonadotropin releasing hormone-1 neurons (GnRH-1ns), and other migratory neurons in rodents. Even though the genetic heterogeneity among these cell types is documented, unidentified cell populations arising from the OP remain. One candidate to identify placodal derived neurons in the developing nasal area is the transcription factor Isl1, which was recently identified in GnRH-3 neurons of the terminal nerve in fish, as well as expression in neurons of the nasal migratory mass (MM). Here, we analyzed the Isl1 genetic lineage in chemosensory neuronal populations in the nasal area and migratory GnRH-1ns in mice using in situ hybridization, immunolabeling a Tamoxifen inducible Isl1CreERT and a constitutive Isl1Cre knock-in mouse lines. In addition, we also performed conditional Isl1 ablation in developing GnRH neurons. We found Isl1 lineage across non-sensory cells of the respiratory epithelium and sustentacular cells of OE and VNO. We identified a population of transient embryonic Isl1 + neurons in the olfactory epithelium and sparse Isl1 + neurons in postnatal VNO. Isl1 is expressed in almost all GnRH neurons and in approximately half of the other neuron populations in the MM. However, Isl1 conditional ablation alone does not significantly compromise GnRH-1 neuronal migration or GnRH-1 expression, suggesting compensatory mechanisms. Further studies will elucidate the functional and mechanistic role of Isl1 in development of migratory endocrine neurons.

Sex-dependent effects of chronic variable stress on discrete corticotropin-releasing factor receptor 1 cell populations.

Anxiety and depression are strikingly more prevalent in women compared with men. Dysregulation of corticotropin-releasing factor (CRF) binding to its cognate receptor (CRFR1) is thought to play a critical role in the etiology of these disorders. In the present study, we investigated whether there were sex differences in the effects of chronic variable stress (CVS) on CRFR1 cells using CRFR1-GFP reporter mice experiencing a 9-day CVS paradigm. Brains were collected from CVS and stress naïve female and male mice following exposure to the open field test. This CVS paradigm effectively increased anxiety-like behavior in female and male mice. In addition, we assessed changes in activation of CRFR1 cells (co-localization with c-Fos and phosphorylated CREB (pCREB)) in stress associated brain structures, including two sexually dimorphic CRFR1 cell groups in the anteroventral periventricular nucleus (AVPV/PeN; F>M) and paraventricular hypothalamus (PVN; M>F). CVS increased CRFR1-GFP cell number as well as the number of CRFR1/pCREB co-expressing cells in the female but not male AVPV/PeN. In the PVN, the number of CRFR1/pCREB co-expressing cells was overall greater in males regardless of treatment and CVS resulted in a male-specific reduction of CRFR1/c-Fos cells. In addition, CVS induced a female-specific reduction in CRFR1/c-Fos cells within the anteroventral bed nucleus of the stria terminalis and both sexes exhibited a reduction in CRFR1/c-Fos co-expressing cells following CVS within the ventral basolateral amygdala. Overall, these sex-specific effects of CVS on CRFR1 populations may have implications for sex differences in stress-induction of mood disorders.

An in vivo model of Met-driven lymphoma as a tool to explore the therapeutic potential of Met inhibitors.

PURPOSE: Met, the tyrosine kinase receptor for hepatocyte growth factor, is frequently deregulated in human cancer. Recent evidence indicates that Met amplification may confer resistance to treatments directed toward other receptor tyrosine kinases. Thus, there is a need to develop Met inhibitors into therapeutic tools, to be used alone or in combination with other molecularly targeted drugs. Preclinical validation of Met inhibitors has thus far been done in nude mice bearing cancer cells xenografts. A far superior model would be a transgenic line developing spontaneous Met-driven tumors with high penetrance and short latency. EXPERIMENTAL DESIGN: To this end, we introduced into the mouse genome TPR-MET, the oncogenic form of MET. The Tpr-Met protein ensures deregulation of Met signaling because dimerization motifs in the Tpr moiety cause ligand-independent activation of the Met kinase. RESULTS: Here, we describe a TPR-MET transgenic line that develops thymic T-cell lymphoma with full penetrance and very short latency. In the tumors, Tpr-Met and its effectors were phosphorylated. Treatment of tumor-derived T lymphocytes with the selective Met inhibitor PHA-665752 at nanomolar concentrations abolished phosphorylation of Met and downstream effectors and led to caspase-mediated apoptosis. I.v. administration of PHA-665752 to transgenic mice bearing lymphomas in exponential growth phase led to a significant decrease in tumor growth and, in some cases, to tumor regression. CONCLUSIONS: Our transgenic line, which within 2 months reliably develops Tpr-Met-driven T-cell lymphoma, represents a valuable tool to explore the efficacy and therapeutic potential of Met kinase inhibitors as anticancer drugs.