RESUMO
Neurogenesis is now known to play a role in adult hypothalamic function, yet the cell-cell mechanisms regulating this neurogenesis remain poorly understood. Here, we show that Hedgehog (Hh)/Gli signaling positively regulates hypothalamic neurogenesis in both larval and adult zebrafish and is necessary and sufficient for normal hypothalamic proliferation rates. Hh-responsive radial glia represent a relatively highly proliferative precursor population that gives rise to dopaminergic, serotonergic, and GABAergic neurons. In situ and transgenic reporter analyses revealed substantial heterogeneity in cell-cell signaling within the hypothalamic niche, with slow cycling Nestin-expressing cells residing among distinct and overlapping populations of Sonic Hh (Shh)-expressing, Hh-responsive, Notch-responsive, and Wnt-responsive radial glia. This work shows for the first time that Hh/Gli signaling is a key component of the complex cell-cell signaling environment that regulates hypothalamic neurogenesis throughout life.
Assuntos
Proteínas Hedgehog , Peixe-Zebra , Animais , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Hipotálamo/metabolismo , Larva/metabolismo , Neurogênese , Transdução de Sinais , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Hedgehog (Hh) signaling is required for embryogenesis and continues to play key roles postembryonically in many tissues, influencing growth, stem cell proliferation, and tumorigenesis. Systems for conditional regulation of Hh signaling facilitate the study of these postembryonic Hh functions. RESULTS: We used the hsp70l promoter to generated three heat-shock-inducible transgenic lines that activate Hh signaling and one line that represses Hh signaling. Heat-shock activation of these transgenes appropriately recapitulates early embryonic loss or gain of Hh function phenotypes. Hh signaling remains activated 24 hr after heat shock in the Tg(hsp70l:shha-EGFP) and Tg(hsp70l:dnPKA-BGFP) lines, while a single heat shock of the Tg(hsp70l:gli1-EGFP) or Tg(hsp70l:gli2aDR-EGFP) lines results in a 6- to 12-hr pulse of Hh signal activation or inactivation, respectively. Using both in situ hybridization and quantitative polymerase chain reaction, we show that these lines can be used to manipulate Hh signaling through larval and juvenile stages. A ptch2 promoter element was used to generate new reporter lines that allow clear visualization of Hh responding cells throughout the life cycle, including graded Hh responses in the embryonic central nervous system. CONCLUSIONS: These zebrafish transgenic lines provide important new experimental tools to study the embryonic and postembryonic roles of Hh signaling.
Assuntos
Resposta ao Choque Térmico/fisiologia , Proteínas Hedgehog/genética , Proteínas Oncogênicas/genética , Transativadores/genética , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico/genética , Proteínas Hedgehog/metabolismo , Proteínas Oncogênicas/metabolismo , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética , Transativadores/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
The vertebrate adenohypophysis forms as a placode at the anterior margin of the neural plate, requiring both hedgehog (Hh) and fibroblast growth factor (Fgf) mediated cell-cell signaling for induction and survival of endocrine cell types. Using small molecule inhibitors to modulate signaling levels during zebrafish development we show that graded Hh and Fgf signaling independently help establish the two subdomains of the adenohypophysis, the anteriorly located pars distalis (PD) and the posterior pars intermedia (PI). High levels of Hh signaling are required for formation of the PD and differentiation of anterior endocrine cell types, whereas lower levels of Hh signaling are required for formation of the PI and differentiation of posterior endocrine cell types. In contrast, high Fgf signaling levels are required for formation of the PI and posterior endocrine cell differentiation, whereas anterior regions require lower levels of Fgf signaling. Based on live observations and marker analyses, we show that the PD forms first at the midline closest to the central nervous system source of Sonic hedgehog. In contrast the PI appears to form from more lateral/posterior cells close to a central nervous system source of Fgf3. Together our data show that graded Hh and Fgf signaling independently direct induction of the PD and PI and help establish endocrine cell fates along the anterior/posterior axis of the zebrafish adenohypophysis. These data suggest that there are distinct origins and signaling requirements for the PD and PI.
