Zebrafish foggy/spt 5 is required for migration of facial branchiomotor neurons but not for their survival

Abstract

Transcript elongation is a critical step in the production of mature messenger RNAs. Many factors have been identified that are required for transcript elongation, including Spt 5. Studies in yeast determined that spt 5 is required for cell viability, and analyses in Drosophila indicate Spt 5 is localized to sites of active transcription, suggesting it is required generally for transcription. However, the requirement for spt 5 for cell viability in a metazoan organism has not been addressed. We determined that zebrafish foggy/spt 5 is required cell-autonomously for the posterior migration of facial branchiomotor neurons from rhombomere 4 (r4) into r6 and r7 of the hindbrain. These genetic mosaics also give us the unique opportunity to determine whether spt 5 is required for mRNA transcription equivalently at all loci by addressing two processes within the same cell-neuronal migration and cell viability. In a wild-type host, spt 5 null facial branchiomotor neurons survive to at least 5 days postfertilization while failing to migrate posteriorly. This finding indicates that spt 5-dependent transcript elongation is required cell-autonomously for a complex cell migration but not for the survival of these same cells. This work provides evidence that transcript elongation is not a global mechanism equivalently required by all loci and may actually be under more strict developmental regulation.

Fig. 1

fh20^-/- mutant embryos exhibit multiple defects including failure of facial branchiomotor neuron migration. Anterior is to the left in all panels. (B) The morphological phenotype of fh20^-/- embryos includes developmental delay and failure of tail elongation, absence of pigment formation, failure of the ventral retina to close, heart tube hypotrophy accompanied by pericardial edema, and degeneration of somites. The mutation is lethal at about 4 dpf. TUNEL labeling indicates that apoptotic cell death is increased in (D) fh20^-/- compared to (C) wild-type. (F) Facial branchiomotor neurons (asterisks) fail to migrate posteriorly from r4 in isl1-GFP transgenic fh20^-/- embryos compared to (E) wild-type. (G) Full-length wild-type spt5 mRNA injected into fh20^-/-embryos rescues facial branchiomotor neuron migration. (H) PCR genotypes for an absolutely linked microsatellite marker (30020R9) in (1) homozygous wild-type, (2) fh20^+/-, (3) fh20^-/-, and (4) the rescued fh20^-/- embryo shown in (G). In situ hybridization for ngn1 expression indicates a decrease in intensity that correlates with the fh20 mutation; (I) wild-type, (J) fh20^-/-. (K-L) Facial branchiomotor neurons, identified by islet1 in situ hybridization, migrate normally in the m806 point mutant of foggy/spt5 that abolishes the negative effect on transcript elongation. Scalebars = 900 μm (A, B) and 100 μm (C-L).

Fig. 2

fh20 is a hypomorphic cryptic splice allele of foggy/spt5. (A) Genomic sequence analysis indicates the fh20 allele is a C-T transition corresponding to position 1904 of the spt5 mRNA. (B) This base change (red arrow) results in the creation of an AGGT consensus splice donor sequence (underlined), and the eight base region including the change (box in fh20) matches eight bases around the endogenous splice donor (boxes in wt and fh20) for exon 15 (shaded green). Sequence analysis of fh20^-/- cDNA shows a double trace indicating that correctly and incorrectly spliced mRNA transcripts are present in the same fh20 mutant. Thus the fh20 allele is a hypomorph. (C) The misspliced mRNA results in a frameshift, and the protein terminates at amino acid (∼676), prior to the fourth KOW RNA pol II binding motif.

Fig. 3

foggy/spt5 is required cell-autonomously for facial branchiomotor neuron migration. (A) Genetic mosaics were generated by transplanting cells from rhodamine dextran-labeled isl1-GFP transgenic donor cells to unlabeled hosts at gastrula stage (6 hpf), and confocal images were collected at 36 hpf. Donor-derived cells are red, and donor derived motor neurons, which express GFP, are yellow. (B) Wild-type facial motor neurons (arrow) migrate posteriorly in a wild-type host; n=14/14. (C) Wild-type facial motor neurons (arrows) also migrate posteriorly from r4 in a foggy/spt5^fh20 mutant host, though to a lesser extent than in wild-type to wild-type control mosaics; n=14/20. (D) foggy/spt5^fh20 facial motor neurons (arrow) fail to migrate from r4 in a wild-type host; n=11/11. Scalebar = 100μm.

Fig. 4

spt5 is not required for facial branchiomotor neuron survival to at least 5 dpf (A) wild-type transplant hosts containing wild-type donor cells (red) and donor-derived motor neurons (yellow) at 5 dpf. Wild-type facial motor neurons (arrow) in a wild-type host migrate posteriorly, differentiate and extend normal axonal processes from r4. (B) Hypomorphic foggy/spt5^fh20 facial motor neurons (arrow) placed in a wild-type host fail to migrate but are still detectable by GFP expression and have normal morphology with axons extending from r4 at 5 dpf; n=12/13. (C) The isl1-GFP motor neuron phenotype of the foggy/spt5^sk8 null allele at 48 hpf demonstrates that facial motor neurons fail to migrate posteriorly from r4 (asterisk). (D) foggy/spt5^sk8 null facial motor neurons (arrow) placed in a wild-type host fail to migrate normally but have axons extending from r4 at 5 dpf; n=8/8. Scalebars = 40μm. Scalebar in D also corresponds with A and B.