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First published online 16 September 2003
doi: 10.1242/dev.00722

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Hedgehog signaling is directly required for the development of zebrafish dorsal root ganglia neurons

¹ Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195-7420 USA
² Department of Biological Structure, University of Washington, Seattle, WA 98195-7420 USA
³ Department of Biology, University of Massachusetts, Amherst, MA 01003, USA

View larger version (63K): [in a new window]   Fig. 1. Loss of Hh signaling disrupts DRG development. Confocal stacks of lateral views of embryos processed for anti-Hu at 60 hpf. (A) Normal pattern of DRG neurons (arrowheads) in wild-type (WT) embryos. (B) syu mutants lack DRG in many segments (asterisks), but abnormal clusters of neuronal cells (arrows) also appear. (C) WT embryos treated with 6.0 µg/ml of cyclopamine during gastrulation show similar phenotypes.

View larger version (69K): [in a new window]   Fig. 3. Large neuronal cluster formation in the absence of Hh signaling does not require Ngn1 function. Wild-type embryos were injected with ngn1 morpholino (ngn1 MO) at the 1-cell stage. Injected embryos were treated with 6 µg/ml cyclopamine beginning at dome stage. Embryos were fixed at 72 hpf and processed for anti-Hu immunoreactivity. Images are confocal stacks of lateral views of whole-mount embryos. (A) Control embryo showing normal DRG (arrow) and Rohon-Beard (RB) neurons (arrowhead). (B) ngn1 MO-injected embryo showing absence of DRG and RB neurons. The remaining Hu+ cells are spinal cord neurons. (C) ngn1 MO-injected embryo that was also treated with cyclopamine (cyc) showing large neuronal cluster (arrow).

View larger version (125K): [in a new window]   Fig. 2. Loss of Hh signaling appears to disrupt integrity of the spinal cord. (A-C) Isl-1/2 is shown in red; Lim1/2 is shown in green. (A) Wild-type (WT) expression of Isl-1/2 in DRG neurons (arrowheads) just lateral to ventral motor neuron expression and ventral to Lim-1/2 neuron expression. (B) Large clusters (asterisks) predominantly express Lim in smu mutant embryos. (C) Some neurons in large clusters (asterisks) in dtr mutants express Islet. (D-F) Hu staining is shown in red; zn-5 is shown in green. (D) WT embryos show expression of both Hu (cell bodies) and zn-5 (axons) in motor neurons as well as DRG neurons (arrowheads). (E) In smu mutant embryos, large clusters (asterisks) express Hu but not zn-5. (F) Some neurons in large clusters (asterisks) in dtr mutant embryos express both Hu and zn-5. (G,H) Acetylated tubulin is shown as red. (G) Acetylated tubulin is normally expressed in motor neuron axons as well as DRG axons (arrowheads). (H,I) Large clusters (asterisks) in smu and dtr mutant embryos display acetylated tubulin-positive projections. Both Hu and acetylated tubulin are red in I. (J-L) Confocal images of transverse sections showing pHuC::GFP in green and Isl-1/2 in red. (J) WT expression of HuC and Isl-1/2 in DRG neurons (arrowheads) located lateral to the spinal cord. (K,L) Clusters of Isl-1/2+ and HuC+ cells at the ventrolateral edge of the spinal cord (asterisks) in embryos treated with cyclopamine at 24 hpf.

View larger version (72K): [in a new window]   Fig. 5. Hh signaling is required for expression of ngn1 and neurod in DRG precursors. yot, smu and wild-type embryos were processed for in situ hybridization at 31-36 hpf to reveal ngn1 or neurod mRNA expression. (A) ngn1 shows broad expression in the spinal cord in addition to its DRG precursor expression (arrowheads), whereas (B) neurod expression in the trunk is restricted to DRG precursors (arrowheads). (C,D) In yot and smu embryos, spinal cord staining is maintained, but ngn1 and neurod expression in DRG precursors is eliminated.

View larger version (24K): [in a new window]   Fig. 4. The severity of the DRG defects corresponds to the level of Hh signaling. Graphs show the percentage of normal DRG neurons, missing DRG neurons (zero) and abnormal neuronal clusters (large) for yot, con, dtr, smu and syu mutant embryos. The bottom graphs show DRG phenotypes from embryos that were treated starting at dome stage with the concentration of cyclopamine indicated. Embryos were fixed and processed for anti-Hu immunoreactivity at 60 hpf.

View larger version (69K): [in a new window]   Fig. 6. Loss of Hh signaling does not result in a general disruption of neural crest development. smu embryos (B,D) and wild-type (WT) siblings (A,C) were probed with crestin to examine migratory neural crest. In smu embryos (B,D), levels of crestin expression are comparable to WT and neural crest do migrate ventrally. However, neural crest cells in smu embryos do not appear to be restricted to the mid-point of the somite (D). In contrast, WT embryos treated with 6 µg/ml cyclopamine beginning at 18 hpf and fixed at 36 hpf show no obvious defects in migration (F) compared with untreated siblings (E). Despite normal patterning and migration, DRG phenotypes in embryos cyclopamine-treated at 18 hpf (G) are similar to the severe disruption of Hh signaling in mutants (Fig. 4).

View larger version (91K): [in a new window]   Fig. 7. ptc1 transcripts are upregulated in presumptive DRG precursors. Wild-type embryos (36 hpf) were processed for whole-mount in situ hybridization, embedded in araldite and transverse sections were cut at trunk levels. (A) crestin is expressed in migrating neural crest cells (arrowhead). (B) ngn1 is expressed in neural crest in the nascent DRG (arrowhead). (C,D) Cells expressing high levels of ptc1 (arrowheads) are found in positions corresponding to expression of ngn1 and crestin. (E,F) Adjacent serial sections were probed for ngn1 (E) or ptc (F).

View larger version (52K): [in a new window]   Fig. 8. Transplanted wild-type (WT) DRG precursor cells rescue DRG development in smu and yot backgrounds. (A) Lateral view of a smu host labeled with anti-Hu antibody viewed by confocal microscopy. (B) WT cells are shown in red in the same optical section. (C) Photoshop merge of A and B showing co-localization of labeled WT cells and Hu+ DRG neurons. (D) Lateral views of a yot host embryo labeled with anti-Hu and anti-zn5 (both in green). Hu expression can be seen in the spinal cord and in two separate ganglia (arrows) lateral to the spinal cord. zn-5 expression can be seen in secondary motor neuron projections (arrowheads). Notice the motor neuron projections are truncated, consistent with previously reported outgrowth defects in yot mutants (Brand et al., 1996). (E) The same yot mutant host embryo shown in D showing WT donor-derived cells (red). (F) Photoshop merge of D and E showing co-localization of labeled WT cells and Hu+ DRG neurons.

View larger version (24K): [in a new window]   Fig. 9. Temporal sensitivity to cyclopamine. Embryos were treated with 6.0 µg/ml cyclopamine beginning at the indicated times and allowed to develop in the presence of cyclopamine until 60 hpf when they were scored for DRG phenotypes. The X-axis indicates somite level; 1 is the most rostral somite, 33 is the most caudal somite, and the Y-axis shows the percentage of embryos with `normal DRG'. DRG were scored in at least 20 embryos for each treatment.