A Spatial and Temporal Gradient of Fgf Differentially Regulates Distinct Stages of Neural Development in the Zebrafish Inner Ear

Neuroblasts of the statoacoustic ganglion (SAG) initially form in the floor of the otic vesicle during a relatively brief developmental window. They soon delaminate and undergo a protracted phase of proliferation and migration (transit-amplification). Neuroblasts eventually differentiate and extend processes bi-directionally to synapse with hair cells in the inner ear and various targets in the hindbrain. Our studies in zebrafish have shown that Fgf signaling controls multiple phases of this complex developmental process. Moderate levels of Fgf in a gradient emanating from the nascent utricular macula specify SAG neuroblasts in laterally adjacent otic epithelium. At a later stage, differentiating SAG neurons express Fgf5, which serves two functions: First, as SAG neurons accumulate, increasing levels of Fgf exceed an upper threshold that terminates the initial phase of neuroblast specification. Second, elevated Fgf delays differentiation of transit-amplifying cells, balancing the rate of progenitor renewal with neuronal differentiation. Laser-ablation of mature SAG neurons abolishes feedback-inhibition and causes precocious neuronal differentiation. Similar effects are obtained by Fgf5-knockdown or global impairment of Fgf signaling, whereas Fgf misexpression has the opposite effect. Thus Fgf signaling renders SAG development self-regulating, ensuring steady production of an appropriate number of neurons as the larva grows

Crim1 maintains retinal vascular stability during development by regulating endothelial cell Vegfa autocrine signaling

Angiogenesis defines the process in which new vessels grow from existing vessels. Using the mouse retina as a model system, we show that cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is highly expressed in angiogenic endothelial cells. Conditional deletion of the Crim1 gene in vascular endothelial cells (VECs) causes delayed vessel expansion and reduced vessel density. Based on known Vegfa binding by Crim1 and Crim1 expression in retinal vasculature, where angiogenesis is known to be Vegfa dependent, we tested the hypothesis that Crim1 is involved in the regulation of Vegfa signaling. Consistent with this hypothesis, we showed that VEC-specific conditional compound heterozygotes for Crim1 and Vegfa exhibit a phenotype that is more severe than each single heterozygote and indistinguishable from that of the conditional homozygotes. We further showed that human CRIM1 knockdown in cultured VECs results in diminished phosphorylation of VEGFR2, but only when VECs are required to rely on an autocrine source of VEGFA. The effect of CRIM1 knockdown on reducing VEGFR2 phosphorylation was enhanced when VEGFA was also knocked down. Finally, an anti-VEGFA antibody did not enhance the effect of CRIM1 knockdown in reducing VEGFR2 phosphorylation caused by autocrine signaling, but VEGFR2 phosphorylation was completely suppressed by SU5416, a small-molecule VEGFR2 kinase inhibitor. These data are consistent with a model in which Crim1 enhances the autocrine signaling activity of Vegfa in VECs at least in part via Vegfr2

Effects of altering Fgf on SAG maturation.

*<p>Rate of mature neuron production during the indicated interval.</p

Effects of transgene activation on expression of the Fgf reporter <i>etv5b</i>.

<p>All embryos were heat shocked for 30 minutes beginning at 24 hpf. Wild-type and <i>hs:fgf8</i> embryos were heat shocked at 39°C and <i>hs:dnfgfr1</i> embryos were heat shocked at 38°C. (A–C) Cross sections showing <i>etv5b</i> expression in wild-type (A), <i>hs:fgf</i> (B) and <i>hs:dnfgfr1</i> (C) embryos at 26 hpf. (D–I) Dorsal views (anterior up) of wholemounts showing <i>etv5b</i> expression in wild-type (D, G), <i>hs:fgf8</i> (E, H) and <i>hs:dnfgfr1</i> (F, I) embryos at the indicated times. The otic vesicle (ov) is marked. Expression of <i>etv5b</i> remains elevated in <i>hs:fgf8</i> embryos for at least 6 hours after heat shock, whereas <i>etv5b</i> expression is downregulated in <i>hs:dnfgfr1</i> for at least 12 hours after heat shock. Scale bar, 25 µm.</p

Regeneration following SAG ablation.

<p>The icon at the top of the figure indicates that neuronal maturation is the focus of analysis. Manipulations in these experiments (Neuronal maturation group II) are briefly summarized at the top. (A) Accumulation of Isl1⁺ SAG neurons in <i>isl2b:Gfp/+</i> embryos after serial ablation of Gfp-positive neurons (mature) or ablation of Gfp-positive neurons and transit-amplifying cells (t.a. + mature) at 30 hpf and 32 hpf. Neuronal accumulation on the contralateral (non-ablated) side served as a control. (B) Effects of modulating Fgf after serial ablations at 30 hpf and 32 hpf on the total number of Isl1⁺ neurons. Embryos were heat shocked for 30 minutes at 39°C (+/+ and <i>hs:fgf8/+</i> embryos) or 38°C (<i>hs:dnfgfr1/+</i> embryos) beginning at 34 hpf. Data show means and standard deviations of 2–5 specimens per time point.</p

Development of Statoacoustic Ganglion (SAG).

<p>(A) Illustration showing the various stages of SAG development. Neuronal precursors (neuroblasts) are specified (1) and delaminate from (2) the floor of the otic vesicle. Neuroblasts undergo a phase of transit-amplification (3) wherein they migrate to a position between the otic vesicle and hindbrain as they continue to proliferate. Neuroblasts finally differentiate into mature neurons (4). (B) Total number of delaminated <i>neurod⁺</i> cells within the SAG counted from serial sections at the indicated times (mean ± standard deviation, n = 2 or greater for each time point). (C) total number of Islet-1-positive SAG neurons at the indicated times (mean of total number ± standard deviation, n = 20 for each time point). (D) <i>neurog1</i> expression at 30 hpf. (E, F) Co-staining for <i>neurod</i> (blue) and BrdU (red) in embryos exposed to BrdU for 6 hours starting at 26 hpf (E) and 96 hpf (F), and then fixed at 32 hpf and 102 hpf, respectively. (G) Co-staining for <i>isl2b:Gfp</i> (green) and phospho-histone H3 (PH3, red) at 32 hpf. Only one mitotic cell (arrow) is seen in the vicinity of the SAG. (H) Co-staining for Islet1 (green) and BrdU (red) at 36 hpf. Only one double-stained cell is visible (arrow). (I–M) Expression of <i>neurod</i> (blue) and Islet-1 (red) at 36 hpf. Mature neurons are labeled with Islet-1 (I) and delaminated progenitor cells express <i>neurod</i> (J). Positions of section-planes in K–M are indicated in (J). (K–L) Transverse sections passing through the anterior (K), middle (L) and posterior (M) regions of the SAG show mostly complementary patterns of <i>neurod</i> and Islet-1. The outer edge of the otic vesicle is outlined in all panels. SAG cells in stages 1–4 of development are indicated in sections, and the position of the utricular macula (u) is indicated. Images of whole-mount specimens (G, I, J) show dorsolateral (G, I) and dorsal (J) views with anterior to the left. Images of transverse sections (C–F, H, K–M) show dorsal to the top and lateral to the left. Scale bar, 25 µm.</p

Model for regulation of SAG development by Fgf.

<p>(A) Neuroblast specification at early stages. A moderate level of Fgf3 and Fgf8 in a gradient generated by the utricular macula specifies neuroblasts in the floor of the otic vesicle (step 1), and nascent neuroblasts quickly delaminate from the otic vesicle (step 2). (B) As development proceeds, neuroblasts establish a pool of transit-amplifying (TA) progenitors (step 3), which eventually differentiate into mature neurons and express Fgf5 (step 4). Rising levels of neuronal Fgf5, combined with Fgf3 and Fgf8 from the growing utricular macula, exceeds an upper threshold that serves to terminate specification of new neuroblasts within the otic vesicle. Neuronal Fgf5 also slows differentiation of progenitors into mature neurons. (C, D) At stages immediately following establishment of the transit-amplifying pool, experimental attenuation of Fgf signaling promotes maturation of neurons at the expense of progenitors (C) whereas elevating Fgf inhibits maturation, expanding the size of the transit-amplifying pool (D).</p

Normal axial patterning in <i>fgf5</i> morphants.

<p>(A–H) Expression of regional patterning markers in control embryos (A–D) and <i>fgf5</i> morphants (E–H). Expression of <i>pax5</i> (A, E) and <i>pou3f3b</i> (B, F) labels anterior and posterior regions, respectively. Expression of <i>otx1a</i> (C, G) and <i>dlx3b</i> (D, H) labels ventromedial and dorsolateral regions, respectively. The otic vesicle is outlined. Images show dorsolateral views with anterior to the left. (I) The total number of hair cells in the utricular (ut) and saccular (sac) maculae of control embryos and <i>fgf5</i> morphants at 32 hpf. Data were obtained by counting GFP-positive hair cells (mean of total number ± standard deviation) in the sensory epithelia of <i>brn3c:Gfp</i> transgenic embryos. Data show means and standard deviations from 20 specimens each. Differences between control and experimental specimens were not statistically significant (p = 0.16 for the utricle, p = 0.67 for the saccule) based on Student's <i>t</i> tests.</p

Effects of altering Fgf on neuroblast specification.

<p>Effects of altering Fgf on neuroblast specification.</p

<i>fgf5</i> from mature neurons terminates the phase of neuroblast specification.

<p>The icon at the top of the figure indicates that analysis focuses on initial formation of neuroblasts. Experimental manipulations in groups IV and V are briefly summarized at the tops of the corresponding data panels. (A–H) Expression of <i>neurog1</i> in control embryos (A–C), a <i>hs:fgf8</i> embryo (D), <i>fgf5</i> morphants (E–G), and a <i>hs:</i>fgf8 embryo injected with <i>fgf5</i>-MO (H) at the indicated stages. Transgenic embryos (D, H) were heat shocked for 30 minutes at 39°C beginning at 24 hpf. Vertical lines in (A, C–E, G, H) indicate the plane of transverse sections in (B, F, and insets in C, D, G and H). (I–L) Expression of <i>isl2b:Gfp</i> at 22 hpf (I, J) and <i>neurog1</i> at 30 hpf (K, L) in a specimen in which mature (<i>fgf5</i>-expressing) neurons were laser-ablated. The same specimen is shown in all panels. Mature SAG neurons expressing <i>isl2b:Gfp</i> were serially ablated on the left side at 22 hpf (J) and 25 hpf (not shown), and the embryo was fixed and sectioned at 30 hpf to examine <i>neurog1</i> expression (L). Images of the unablated right side (I, K) were inverted to facilitate comparison. The surface of the otic vesicle is outlined in all panels. Arrows in sections indicate the edges of <i>neurog1</i> domain in the otic floor. Note that the amount and duration of delamination of <i>neurog1⁺</i> neuroblasts is strongly enhanced by knockdown of <i>fgf5</i> (F, G) or ablation of mature neurons (L). Activation of <i>hs:fgf8</i> reverses the effects of <i>fgf5-</i>MO (H). Scale bar, 25 µm. Transverse sections are shown with lateral to the left and dorsal up. Wholemount images show dorsolateral views with anterior to the left.</p