Maternal and zygotic aldh1a2 activity is required for pancreas development in zebrafish

Abstract

We have isolated and characterized a novel zebrafish pancreas mutant. Mutant embryos lack expression of isl1 and sst in the endocrine pancreas, but retain isl1 expression in the CNS. Non-endocrine endodermal gene expression is less affected in the mutant, with varying degrees of residual expression observed for pdx1, carbA, hhex, prox1, sid4, transferrin and ifabp. In addition, mutant embryos display a swollen pericardium and lack fin buds. Genetic mapping revealed a mutation resulting in a glycine to arginine change in the catalytic domain of the aldh1a2 gene, which is required for the production of retinoic acid from vitamin A. Comparison of our mutant (aldh1a2(um22)) to neckless (aldh1a2(i26)), a previously identified aldh1a2 mutant, revealed similarities in residual endodermal gene expression. In contrast, treatment with DEAB (diethylaminobenzaldehyde), a competitive reversible inhibitor of Aldh enzymes, produces a more severe phenotype with complete loss of endodermal gene expression, indicating that a source of Aldh activity persists in both mutants. We find that mRNA from the aldh1a2(um22) mutant allele is inactive, indicating that it represents a null allele. Instead, the residual Aldh activity is likely due to maternal aldh1a2, since we find that translation-blocking, but not splice-blocking, aldh1a2 morpholinos produce a phenotype similar to DEAB treatment. We conclude that Aldh1a2 is the primary Aldh acting during pancreas development and that maternal Aldh1a2 activity persists in aldh1a2(um22) and aldh1a2(i26) mutant embryos.

Figure 1. 88.21 is a novel aldh1a2 allele.

A, B. Islet1 (isl1) expression was used in a haploid ENU screen to identify mutants in endocrine pancreas development. Dorsal view of 30 hpf wild type embryo with isl1 expression in the CNS and endocrine pancreas (A; black arrow indicates expression in pancreas) and 88.21 mutant embryo with isl1 expression in the CNS, but not in the endoderm (B). C–E. Lateral view of live wild type (C), 88.21 (D), and neckless aldh1a2ⁱ²⁶ (E) embryos at day 5. F. Linkage analysis using CA repeat markers on pooled genomic DNA from 88.21 mutants and pooled genomic wild type DNA. Marker z10441 amplifies a 450 bp band and a faint 500 bp band in the mutant pool compared to a faint 450 bp band and a 500 bp band in the wild type pool. Marker z8693 amplifies two bands at 250 bp and 300 bp in the mutant pool compared to 250 bp, 300 bp as well as a 400 bp band in the wild type pool. White arrow points to lack of 400 bp band in mutant. G. Schematic drawing of part of linkage group 7 (LG7), showing the location of z10441 and z8693 and aldh1a2 (in red) in reference to these markers. H–J. Sequence analysis of pooled 88.21 mutant (MT) genomic DNA and pooled wild type (WT) genomic DNA (H, J), as well as of individual mutant (MT) and wild type (WT) embryos (I). 88.21 fish carry a mutation that converts Gly⁴⁸⁴ to Arg (in red, and outlined in brackets in J) located in the catalytic domain. K. Schematic of Aldh1a2 protein and the location of the aldh1a2 mutant alleles aldh1a2ⁱ²⁶, aldh1a2^u11 and 88.21/aldh1a2^um22.

Figure 2. Wild type aldh1a2 mRNA rescues 88.21 fin bud development.

Dorsal views of 48 hpf embryos with sonic hedgehog (shh) expression in purple. A. Uninjected wild type embryo with shh expression in the CNS and fin buds (black arrows). B. aldh1a2^um22 mutant embryos lack shh expression in the fin buds. C. aldh1a2^um22 mutant embryo injected with aldh1a2 wild type mRNA shows rescued fin bud expression (black arrows). D. aldh1a2^um22 mutant embryo injected with aldh1a2^um22 mutant mRNA is not rescued.

Figure 3. aldh1a2^um22 and aldh1a2ⁱ²⁶ mutant embryos retain some endoderm gene expression at 24 and 30 hpf.

DMSO treated wild type embryos (A, E, I, M), DEAB-treated wild type embryos (B, F, J, N), embryos from an incross of aldh1a2^um22 heterozygotes (C, G, K, O) and embryos from an incross of aldh1a2ⁱ²⁶ heterozygotes (D, H, L, P) were assayed for expression of ins at 24 hpf (A–D; black arrows indicate residual expression), pdx1 at 24 hpf (E–H; black arrows indicate residual expression), hhex at 30 hpf (I–L; residual expression is indicated in pancreas (arrow) and liver (arrowhead)) and prox1 at 30 hpf (M–P; residual expression is indicated in liver (arrowhead)). Embryos are in dorsal view with anterior to the left. See Table 2 for quantification.

Figure 4. aldh1a2^um22 and aldh1a2ⁱ²⁶ mutant embryos retain some endoderm gene expression at 48 and 72 hpf.

DMSO treated wild type embryos (A, E, I, M, Q), DEAB-treated wild type embryos (B, F, J, N, R), embryos from an incross of aldh1a2^um22 heterozygotes (C, G, K, O, S) and embryos from an incross of aldh1a2ⁱ²⁶ heterozygotes (D, H, L, P, T) were assayed for expression of pdx1 at 48 hpf (A–D), sid4 at 48 hpf (E–H), carbA at 72 hpf (I–L), ifabp at 72 hpf (M–P) and transf at 72 hpf (Q–T). Gene expression is observed in the intestine (open arrows), liver (black arrowheads) and pancreas (black arrows). Embryos are in dorsal view with anterior to the left. See Table 2 for quantification.

Figure 5. aldh1a2 is maternally expressed and aldh1a2 translational morpholino knocks down endoderm expression.

A. PCR of 3 and 6 hpf wild type embryos using primers targeting exon1-2 and exon10-11 of aldh1a2 reveals aldh1a2 expression already at 3 hpf. A no DNA sample and amplification of tubulin is used as negative and positive controls. B–G. Wild type embryos were injected with either 950 uM aldh1a2 mismatch (mm) morpholino (MO; B, D, F) or 950 uM of aldh1a2 translational (tMO; C, E, G) and assayed for expression of hhex (B, C), prox1 (D, E) or pdx1 (F, G). Embryos are in dorsal view with anterior to the left.