Limits...
Targeted germ line disruptions reveal general and species-specific roles for paralog group 1 hox genes in zebrafish.

Weicksel SE, Gupta A, Zannino DA, Wolfe SA, Sagerström CG - BMC Dev. Biol. (2014)

Bottom Line: We also demonstrate that Hoxb1b regulates nucleosome organization at the hoxb1a promoter and that retinoic acid acts independently of hoxb1b to activate hoxb1a expression.We generated several novel germ line mutants for zebrafish hoxb1a and hoxb1b.Lastly, our data reveal independent regulation of hoxb1a expression by retinoic acid and Hoxb1b in zebrafish.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St,/LRB815, Worcester, MA 01605-2324, USA. charles.sagerstrom@umassmed.edu.

ABSTRACT

Background: The developing vertebrate hindbrain is transiently segmented into rhombomeres by a process requiring Hox activity. Hox genes control specification of rhombomere fates, as well as the stereotypic differentiation of rhombomere-specific neuronal populations. Accordingly, germ line disruption of the paralog group 1 (PG1) Hox genes Hoxa1 and Hoxb1 causes defects in hindbrain segmentation and neuron formation in mice. However, antisense-mediated interference with zebrafish hoxb1a and hoxb1b (analogous to murine Hoxb1 and Hoxa1, respectively) produces phenotypes that are qualitatively and quantitatively distinct from those observed in the mouse. This suggests that PG1 Hox genes may have species-specific functions, or that anti-sense mediated interference may not completely inactivate Hox function in zebrafish.

Results: Using zinc finger and TALEN technologies, we disrupted hoxb1a and hoxb1b in the zebrafish germ line to establish mutant lines for each gene. We find that zebrafish hoxb1a germ line mutants have a more severe phenotype than reported for Hoxb1a antisense treatment. This phenotype is similar to that observed in Hoxb1 knock out mice, suggesting that Hoxb1/hoxb1a have the same function in both species. Zebrafish hoxb1b germ line mutants also have a more severe phenotype than reported for hoxb1b antisense treatment (e.g. in the effect on Mauthner neuron differentiation), but this phenotype differs from that observed in Hoxa1 knock out mice (e.g. in the specification of rhombomere 5 (r5) and r6), suggesting that Hoxa1/hoxb1b have species-specific activities. We also demonstrate that Hoxb1b regulates nucleosome organization at the hoxb1a promoter and that retinoic acid acts independently of hoxb1b to activate hoxb1a expression.

Conclusions: We generated several novel germ line mutants for zebrafish hoxb1a and hoxb1b. Our analyses indicate that Hoxb1 and hoxb1a have comparable functions in zebrafish and mouse, suggesting a conserved function for these genes. In contrast, while Hoxa1 and hoxb1b share functions in the formation of r3 and r4, they differ with regards to r5 and r6, where Hoxa1 appears to control formation of r5, but not r6, in the mouse, whereas hoxb1b regulates formation of r6, but not r5, in zebrafish. Lastly, our data reveal independent regulation of hoxb1a expression by retinoic acid and Hoxb1b in zebrafish.

Show MeSH
Zebrafish hoxb1b is required for hindbrain segmentation. A-O. Wild type (A, E, I, M), hoxb1a−/−(B, F, J, N), hoxb1b−/−(C, G, K, O) or doubly hoxb1a−/−;hoxb1b−/− embryos (D, H, L) were assayed by in situ hybridization for expression of hoxb1a in r4 (blue stain in A-D), krox20 in r3/r5 (red stain in A-D, I-O and blue stain in panels E-H), pax2 at the midbrain-hindbrain boundary (blue stain in E-H), fgf3 in r4 (blue stain in I-L), hoxd4a in r7 (blue stain in E-H) and hoxb3a in r5/r6 (blue stain in M-O). P. Quantification of segmentation defects in hoxb1b−/− embryos. Rhombomere lengths were measured as indicated in the inset. MHB-r6 measures the full distance from the anterior limit of the MHB to the posterior limit of r6. p-values were computed using Students’ t-test and error bars represent standard error. N = 10 embryos. All embryos are flat mounted in dorsal view with anterior to the top. A-H and M-O are at 22hpf, while I-L are at 14hpf. r = rhombomere; MHB = midbrain/hindbrain boundary.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4061917&req=5

Figure 2: Zebrafish hoxb1b is required for hindbrain segmentation. A-O. Wild type (A, E, I, M), hoxb1a−/−(B, F, J, N), hoxb1b−/−(C, G, K, O) or doubly hoxb1a−/−;hoxb1b−/− embryos (D, H, L) were assayed by in situ hybridization for expression of hoxb1a in r4 (blue stain in A-D), krox20 in r3/r5 (red stain in A-D, I-O and blue stain in panels E-H), pax2 at the midbrain-hindbrain boundary (blue stain in E-H), fgf3 in r4 (blue stain in I-L), hoxd4a in r7 (blue stain in E-H) and hoxb3a in r5/r6 (blue stain in M-O). P. Quantification of segmentation defects in hoxb1b−/− embryos. Rhombomere lengths were measured as indicated in the inset. MHB-r6 measures the full distance from the anterior limit of the MHB to the posterior limit of r6. p-values were computed using Students’ t-test and error bars represent standard error. N = 10 embryos. All embryos are flat mounted in dorsal view with anterior to the top. A-H and M-O are at 22hpf, while I-L are at 14hpf. r = rhombomere; MHB = midbrain/hindbrain boundary.

Mentions: We first examined the expression of several rhombomere-restricted genes – pax2 (expressed at the midbrain-hindbrain boundary; MHB), krox20 (expressed in r3 and r5), hoxb1a (expressed in r4), hoxb3a (expressed in r5 and r6) and hoxd4a (expressed in r7 and r8). For this purpose, heterozygous hoxb1b+/um197 F1 fish were in-crossed and the resulting embryos were assayed at 22hpf by in situ hybridization followed by genotyping. We find that homozygous hoxb1bum197/um197 mutant embryos express krox20 in r3 and r5, as well as hoxb1a in r4 (Figure 2C). However, the size of r3 is increased and the size of r4 is decreased in hoxb1b mutants relative to wild type (or heterozygous) embryos (Figure 2A; Additional file 3: Table S2). To address the possibility that the Zb1b-3 ZFN might have introduced off-target mutations in the hoxb1bum197 line that could contribute to this phenotype, we also examined in-crosses of the hoxb1bum196 and hoxb1bum195 lines, as well as pair-wise inter-crosses among all three lines. We find that mutant embryos derived from all such crosses exhibit the same phenotype and that this phenotype segregates with the hoxb1b mutation (Additional file 3: Table S2 and Additional file 4: Figure S2), suggesting that it is due to disruption of the hoxb1b gene. Furthermore, the PROGNOS on-line tool [37] revealed five exonic sites in the top fifty potential off-target sites for Zb1b-3, but neither of these sites resides on the same chromosome as the hoxb1b gene, and they would therefore segregate independently of the hoxb1b mutation in our crosses. Further analysis of hoxb1bum197/um197 mutant embryos revealed expression of pax2, hoxb3a and hoxd4a in the expected domains (Figure 2G, O). In addition to the enlargement of r3 and the reduction of r4 noted above, this analysis also revealed an apparent reduction of r6 – as evidenced by a smaller gap between r5 krox20 staining and r7 hoxd4a staining (brackets in Figure 2E, G), as well as by a reduction in the size of the hoxb3a expression domain (brackets in Figure 2M, O) in mutant embryos relative to wild type embryos.


Targeted germ line disruptions reveal general and species-specific roles for paralog group 1 hox genes in zebrafish.

Weicksel SE, Gupta A, Zannino DA, Wolfe SA, Sagerström CG - BMC Dev. Biol. (2014)

Zebrafish hoxb1b is required for hindbrain segmentation. A-O. Wild type (A, E, I, M), hoxb1a−/−(B, F, J, N), hoxb1b−/−(C, G, K, O) or doubly hoxb1a−/−;hoxb1b−/− embryos (D, H, L) were assayed by in situ hybridization for expression of hoxb1a in r4 (blue stain in A-D), krox20 in r3/r5 (red stain in A-D, I-O and blue stain in panels E-H), pax2 at the midbrain-hindbrain boundary (blue stain in E-H), fgf3 in r4 (blue stain in I-L), hoxd4a in r7 (blue stain in E-H) and hoxb3a in r5/r6 (blue stain in M-O). P. Quantification of segmentation defects in hoxb1b−/− embryos. Rhombomere lengths were measured as indicated in the inset. MHB-r6 measures the full distance from the anterior limit of the MHB to the posterior limit of r6. p-values were computed using Students’ t-test and error bars represent standard error. N = 10 embryos. All embryos are flat mounted in dorsal view with anterior to the top. A-H and M-O are at 22hpf, while I-L are at 14hpf. r = rhombomere; MHB = midbrain/hindbrain boundary.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4061917&req=5

Figure 2: Zebrafish hoxb1b is required for hindbrain segmentation. A-O. Wild type (A, E, I, M), hoxb1a−/−(B, F, J, N), hoxb1b−/−(C, G, K, O) or doubly hoxb1a−/−;hoxb1b−/− embryos (D, H, L) were assayed by in situ hybridization for expression of hoxb1a in r4 (blue stain in A-D), krox20 in r3/r5 (red stain in A-D, I-O and blue stain in panels E-H), pax2 at the midbrain-hindbrain boundary (blue stain in E-H), fgf3 in r4 (blue stain in I-L), hoxd4a in r7 (blue stain in E-H) and hoxb3a in r5/r6 (blue stain in M-O). P. Quantification of segmentation defects in hoxb1b−/− embryos. Rhombomere lengths were measured as indicated in the inset. MHB-r6 measures the full distance from the anterior limit of the MHB to the posterior limit of r6. p-values were computed using Students’ t-test and error bars represent standard error. N = 10 embryos. All embryos are flat mounted in dorsal view with anterior to the top. A-H and M-O are at 22hpf, while I-L are at 14hpf. r = rhombomere; MHB = midbrain/hindbrain boundary.
Mentions: We first examined the expression of several rhombomere-restricted genes – pax2 (expressed at the midbrain-hindbrain boundary; MHB), krox20 (expressed in r3 and r5), hoxb1a (expressed in r4), hoxb3a (expressed in r5 and r6) and hoxd4a (expressed in r7 and r8). For this purpose, heterozygous hoxb1b+/um197 F1 fish were in-crossed and the resulting embryos were assayed at 22hpf by in situ hybridization followed by genotyping. We find that homozygous hoxb1bum197/um197 mutant embryos express krox20 in r3 and r5, as well as hoxb1a in r4 (Figure 2C). However, the size of r3 is increased and the size of r4 is decreased in hoxb1b mutants relative to wild type (or heterozygous) embryos (Figure 2A; Additional file 3: Table S2). To address the possibility that the Zb1b-3 ZFN might have introduced off-target mutations in the hoxb1bum197 line that could contribute to this phenotype, we also examined in-crosses of the hoxb1bum196 and hoxb1bum195 lines, as well as pair-wise inter-crosses among all three lines. We find that mutant embryos derived from all such crosses exhibit the same phenotype and that this phenotype segregates with the hoxb1b mutation (Additional file 3: Table S2 and Additional file 4: Figure S2), suggesting that it is due to disruption of the hoxb1b gene. Furthermore, the PROGNOS on-line tool [37] revealed five exonic sites in the top fifty potential off-target sites for Zb1b-3, but neither of these sites resides on the same chromosome as the hoxb1b gene, and they would therefore segregate independently of the hoxb1b mutation in our crosses. Further analysis of hoxb1bum197/um197 mutant embryos revealed expression of pax2, hoxb3a and hoxd4a in the expected domains (Figure 2G, O). In addition to the enlargement of r3 and the reduction of r4 noted above, this analysis also revealed an apparent reduction of r6 – as evidenced by a smaller gap between r5 krox20 staining and r7 hoxd4a staining (brackets in Figure 2E, G), as well as by a reduction in the size of the hoxb3a expression domain (brackets in Figure 2M, O) in mutant embryos relative to wild type embryos.

Bottom Line: We also demonstrate that Hoxb1b regulates nucleosome organization at the hoxb1a promoter and that retinoic acid acts independently of hoxb1b to activate hoxb1a expression.We generated several novel germ line mutants for zebrafish hoxb1a and hoxb1b.Lastly, our data reveal independent regulation of hoxb1a expression by retinoic acid and Hoxb1b in zebrafish.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St,/LRB815, Worcester, MA 01605-2324, USA. charles.sagerstrom@umassmed.edu.

ABSTRACT

Background: The developing vertebrate hindbrain is transiently segmented into rhombomeres by a process requiring Hox activity. Hox genes control specification of rhombomere fates, as well as the stereotypic differentiation of rhombomere-specific neuronal populations. Accordingly, germ line disruption of the paralog group 1 (PG1) Hox genes Hoxa1 and Hoxb1 causes defects in hindbrain segmentation and neuron formation in mice. However, antisense-mediated interference with zebrafish hoxb1a and hoxb1b (analogous to murine Hoxb1 and Hoxa1, respectively) produces phenotypes that are qualitatively and quantitatively distinct from those observed in the mouse. This suggests that PG1 Hox genes may have species-specific functions, or that anti-sense mediated interference may not completely inactivate Hox function in zebrafish.

Results: Using zinc finger and TALEN technologies, we disrupted hoxb1a and hoxb1b in the zebrafish germ line to establish mutant lines for each gene. We find that zebrafish hoxb1a germ line mutants have a more severe phenotype than reported for Hoxb1a antisense treatment. This phenotype is similar to that observed in Hoxb1 knock out mice, suggesting that Hoxb1/hoxb1a have the same function in both species. Zebrafish hoxb1b germ line mutants also have a more severe phenotype than reported for hoxb1b antisense treatment (e.g. in the effect on Mauthner neuron differentiation), but this phenotype differs from that observed in Hoxa1 knock out mice (e.g. in the specification of rhombomere 5 (r5) and r6), suggesting that Hoxa1/hoxb1b have species-specific activities. We also demonstrate that Hoxb1b regulates nucleosome organization at the hoxb1a promoter and that retinoic acid acts independently of hoxb1b to activate hoxb1a expression.

Conclusions: We generated several novel germ line mutants for zebrafish hoxb1a and hoxb1b. Our analyses indicate that Hoxb1 and hoxb1a have comparable functions in zebrafish and mouse, suggesting a conserved function for these genes. In contrast, while Hoxa1 and hoxb1b share functions in the formation of r3 and r4, they differ with regards to r5 and r6, where Hoxa1 appears to control formation of r5, but not r6, in the mouse, whereas hoxb1b regulates formation of r6, but not r5, in zebrafish. Lastly, our data reveal independent regulation of hoxb1a expression by retinoic acid and Hoxb1b in zebrafish.

Show MeSH