Requirement of Fgf pathway in the development of gnrh3 neurons.

<p><b>A</b>, Decreased GFP cell numbers after injection of <i>fgfr1</i>-MO as compared with embryos injected with control MO2 (ctrl-MO2). <b>B–E</b>, Decreased <i>gnrh3</i>-expressing cells as detected by (B, D) GFP- or (C, E) <i>in situ</i> hybridization. The anterior is to the left in all panels. Twenty embryos were counted for each data point. **<i>P</i><0.01.</p

Hedgehog-PKA Signaling and gnrh3 Regulate the Development of Zebrafish gnrh3 Neurons

<div><p>GnRH neurons secrete GnRH that controls the development of the reproduction system. Despite many studies, the signals controlling the development of GnRH neurons from its progenitors have not been fully established. To understand the development of GnRH neurons, we examined the development of <i>gnrh3</i>-expressing cells using a transgenic zebrafish line that expresses green fluorescent protein (GFP) and LacZ driven by the <i>gnrh3</i> promoter. GFP and LacZ expression recapitulated that of <i>gnrh3</i> in the olfactory region, olfactory bulb and telencephalon. Depletion of <i>gnrh3</i> by morpholinos led to a reduction of GFP- and gnrh3-expressing cells, while over-expression of <i>gnrh3</i> mRNA increased the number of these cells. This result indicates a positive feed-forward regulation of gnrh3 cells by gnrh3. The gnrh3 cells were absent in embryos that lack Hedgehog signaling, but their numbers were increased in embryos overexpressing <i>shhb</i>. We manipulated the amounts of kinase that antagonizes the Hedgehog signaling pathway, protein kinase A (PKA), by treating embryos with PKA activator forskolin or by injecting mRNAs encoding its constitutively active catalytic subunit (<i>PKA*</i>) and dominant negative regulatory subunit (<i>PKI</i>) into zebrafish embryos. <i>PKA*</i> misexpression or forskolin treatment decreased GFP cell numbers, while <i>PKI</i> misexpression led to ectopic production of GFP cells. Our data indicate that the Hedgehog-PKA pathway participates in the development of gnrh3-expressing neurons during embryogenesis.</p></div

Co-localization <i>gnrh3</i> mRNA and GFP in transgenic fish expressing <i>GFP-LacZ</i> under the control of the <i>gnrh3</i> promoter.

<p>A–D, 3 dpf, E–H, 4 dpf. A and E, bright field (BF) view of the embryos. B and F, green color indicated <i>gnrh3</i> mRNA detection by <i>in situ</i> hybridization. C and G, GFP immunostaining is shown as red signal. D and H, the merged pictures show co-localization of <i>gnrh3</i> and GFP signals in the olfactory bulb (OB) at 3 dpf and 4 dpf. The anterior is to the left in all panels, and arrows point to the olfactory bulb.</p

Depletion of <i>gnrh3</i> expression by morpholinos causes reduction of gnrh3 cells.

<p>A–D, <i>in situ</i> hybridization with the <i>gnrh3</i> probe. E–F, fluorescence detection of GFP cells. G–H, detection of LacZ-expressing cells. After injection of <i>gnrh3-</i>MO1, <i>gnrh3</i> and GFP expression was reduced at 48 hpf (A, C and E) and LacZ cells in the forebrain was reduced at 7 dpf (G). B, D, F and H, Gnrh3 cells neurons after the injection of control sense MO1 (Ctrl-MO1).</p

Aberrant gnrh3 neuron numbers in embryos with perturbed Hh signaling pathways.

<p>Embryos from heterozygote mating were examined for <i>gnrh3</i> expression and the numbers of embryos were scored. Alternatively embryos from wildtype parents were injected with morpholinos or mRNAs before scoring ectopic GFP and <i>gnrh3</i> expression at 2 dpf.</p

Gnrh3 controls the number of gnrh3 neurons.

<p>Gnrh3 neurons are detected as GFP expressing cells. A. The number of GFP cells at different time points after the injection of control MO, <i>gnrh3-</i>MO1, <i>gnrh3</i> RNA, <i>or β-gal</i> RNA. Depletion of <i>gnrh3</i> expression by <i>gnrh3-</i>MO1 (MO1) reduces the number of GFP cells, and over-expression of <i>gnrh3</i> increases GFP cell numbers. B, <i>gnrh2</i> mRNA rescues the <i>gnrh3</i>-MO1 morphants. Uninjected embryos and embryos injected with control (ctrl-MO1) and <i>β-gal</i> RNA are controls. *<i>P</i><0.05. **<i>P</i><0.01. Twenty embryos were counted for each data point, and the same injection experiments were repeated two to four times for each time point.</p