In the developing hypothalamus, a variety of neurons are generated adjacent to each other in a highly coordinated, but poorly understood process. A critical question that remains unanswered is how coordinated development of multiple neuronal types is achieved in this relatively narrow anatomical region. We focus on dopaminergic (DA) and oxytocinergic (OT) neurons as a paradigm for development of two prominent hypothalamic cell types. We report that the development of DA and OT-like neurons in the zebrafish is orchestrated by two novel pathways that regulate the expression of the homeodomain-containing protein Orthopedia (Otp), a key determinant of hypothalamic neural differentiation. Genetic analysis showed that the G-protein-coupled receptor PAC1 and the zinc finger-containing transcription factor Fezl act upstream to Otp. In vivo and in vitro experiments demonstrated that Fezl and PAC1 regulate Otp at the transcriptional and the post-transcriptional levels, respectively. Our data reveal a new genetic network controlling the specification of hypothalamic neurons in vertebrates, and places Otp as a critical determinant underlying Fezl-and PAC1-mediated differentiation.

SUMMARY Regulation of corticotropin-releasing hormone (CRH) activity is critical for the animal’s adaptation to stressful challenges, and its dysregulation is associated with psychiatric disorders in humans. However, the molecular mechanism underlying this transcriptional response to stress is not well understood. Using various stress paradigms in mouse and zebrafish, we show that the hypothalamic transcription factor Orthopedia modulates the expression of CRH as well as the splicing factor Ataxin 2-Binding Protein-1 (A2BP1/Rbfox-1). We further show that the G protein coupled receptor PAC1, which is a known A2BP1/Rbfox-1 splicing target and an important mediator of CRH activity, is alternatively spliced in response to a stressful challenge. The generation of PAC1-hop messenger RNA isoform by alternative splicing is required for termination of CRH transcription, normal activation of the hypothalamic-pituitary-adrenal axis and adaptive anxiety-like behavior. Our study identifies an evolutionarily conserved biochemical pathway that modulates the neuronal adaptation to stress through transcriptional activation and alternative splicing.

Chemokines are key regulators of hematopoiesis and host defense. We report here that functional expression of the chemokine receptor CXCR4 on human immature CD34 ؉ hematopoietic progenitors was increased as a result of sustained elevation in cellular cAMP by dbcAMP and prostaglandin E2. This effect of cAMP was specifically mediated by PKC activity. CXCR4 expression and PKC activation by cAMP were decreased after the inhibition of cAMP effector-Rap1 by Spa1 overexpression. Interference with the activation of Rac1, a downstream target of Rap1, prevented the cAMPinduced increase in PKC activity and CXCR4 levels. Functional manifestation of the effects of cAMP-elevating agents revealed an increased ability of human CD34 ؉ cells to transmigrate the bone marrow (BM) endothelial layer and adhere to BM stroma in vitro, and it augmented the homing potential to the BM and spleens of immunodeficient mice in a Rac1-and a PKC-dependent manner. cAMP-and TNF␣-stimulated pathways converged in PKC-activated CXCR4 expression and MMP-2/MMP-9 secretion. cAMP treatment had a beneficial effect on CD34 ؉ cell survival in a PKC-mediated fashion. Taken IntroductionConsiderable effort has been invested in recent years toward understanding the mechanisms that govern hematopoietic stem/ progenitor cell trafficking and development. Chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) is the only known powerful chemoattractant for defined populations of primitive human (CD34 ϩ CD38 Ϫ/low ) and murine (Sca-1 ϩ Thy-1 low c-kit ϩ Lin Ϫ ) hematopoietic progenitor cells. [1][2][3] The primary receptor for SDF-1, CXCR4, is expressed on a variety of immature and mature hematopoietic cells and on neuronal, endothelial, and epithelial cells. [4][5][6] Multiple studies have demonstrated major roles for SDF-1/CXCR4 interaction in human stem cell migration in vivo. Mice lacking either SDF-1 or CXCR4 exhibit several lethal defects, including impairment in hematopoiesis and stem cell seeding of the fetal BM. [7][8][9] We have previously shown that homing and engraftment of human CD34 ϩ CD38 Ϫ/low hematopoietic progenitors transplanted into immunodeficient mice are regulated by cell surface human CXCR4 expression and BMproduced murine SDF-1. 2,[10][11][12][13] The in vitro migratory capacity of enriched CD34 ϩ progenitors, particularly the expression and functionality of CXCR4 on these cells, directly correlates with clinical hematopoietic recovery after autologous stem cell transplantation. [14][15][16] Thus, understanding the mechanisms and molecular pathways that affect CXCR4 expression and cellular signaling might have important implications for clinical stem/ progenitor cell transplantation.Recently, we identified a specific isoform of the PKC family, atypical PKC, as a key regulator of SDF-1/CXCR4-activated signaling in human hematopoietic progenitors, demonstrating that ectopic PKC expression increases SDF-1-induced motility, whereas the inhibition of PKC activity impairs survival, proliferation, adhesion and, engraftment of immature CD34 ϩ proge...

Proper response to stress and social stimuli depends on orchestrated development of hypothalamic neuronal circuits. Here we address the effects of the developmental transcription factor orthopedia (Otp) on hypothalamic development and function. We show that developmental mutations in the zebrafish paralogous gene otpa but not otpb affect both stress response and social preference. These behavioral phenotypes were associated with developmental alterations in oxytocinergic (OXT) neurons. Thus, otpa and otpb differentially regulate neuropeptide switching in a newly identified subset of OXT neurons that co-express the corticotropin-releasing hormone (CRH). Single-cell analysis revealed that these neurons project mostly to the hindbrain and spinal cord. Ablation of this neuronal subset specifically reduced adult social preference without affecting stress behavior, thereby uncoupling the contribution of a specific OXT cluster to social behavior from the general otpa−/− deficits. Our findings reveal a new role for Otp in controlling developmental neuropeptide balance in a discrete OXT circuit whose disrupted development affects social behavior.DOI: http://dx.doi.org/10.7554/eLife.22170.001