Knockdown of a galectin-1-like protein in zebrafish (Danio rerio) causes defects in skeletal muscle development

Abstract

We previously identified and characterized four galectin-1-like proteins in zebrafish, Drgal1-L1, Drgal1-L2, Drgal1-L3, and one splice variant of Drgal1-L2, of distinct ontogenic expression. Drgal1-L1 is maternal; Drgal1-L2 is zygotic and strongly expressed in the notochord, while Drgal1-L3 is both maternal and zygotic. Knockdown experiments in zebrafish embryos using a morpholino-modified antisense oligo targeted to the 5'-UTR sequence of Drgal1-L2 resulted in a phenotype with a bent tail and disorganized muscle fibers. This effect was dose-dependent as follows: 62-66% at 17 ng, 29-35% at 5.7 ng, 21-28% at 1.9 ng, and 14-17% at 0.6 ng. However, no (or a negligible number of) Drgal1-L1 knockdown embryos showed similar morphological defects, indicating that the observed effects are sequence-specific, and not due to the toxicity of the morpholino-modified oligos. Further, ectopic expression of native Drgal1-L2 specifically rescued the phenotype, as co-injection of the full-length sense Drgal1-L2 mRNA with Drgal1-L2-MO yielded 60-62% normal embryos. As the notochord serves as the primary source of signaling molecules required for proper patterning of adjacent tissues, such as neural tube, somites, and heart, these results suggest that galectins produced by the notochord play a key role in somitic cell differentiation and development.

Fig. 1

Amino acid sequence comparison of the Drgal1-L2 with the mammalian galectin-1 or galectin-1-like proteins from lower vertebrates. Amino acid sequence of the galectins are aligned by ClustalW program (clustalw. genome.ad.jp/). The amino acids shown in shaded boxes interact with N-acetyllactosamine as determined from 3-D structure of the bovine galectin-1 [22]

Fig. 2

Gene organization of Drgal1-L2. The vertical boxes represent exons, which are numbered at the top. The size of each exon (in bp) is indicated at the bottom. The horizontal boxes represent introns, whose sizes are indicated in kb

Fig. 3

Blocking of Drgal1-L2 protein expression by Drgal1-L2-MO. A In vitro blocking of Drgal1-L2 protein in a rabbit reticulocyte system. SDS-PAGE/autoradiography analysis of the in vitro labeled Drgal1-L2 translation products. Lane 1: translation reaction in the absence of Drgal1-L2-MO (positive control); lanes 2 and 3: reactions in the presence of 170 ng and 340 ng of Drgal1-L2-MO, respectively. B Whole mount immunostaining of an uninjected embryo probed with anti-Drgal1-L2 antibodies (positive control). Lateral and dorsal view of a 27 hpf embryo. C In vivo blocking of Drgal1-L2 protein as shown by whole mount immunostaining of an embryo injected with Drgal1-L2-MO, and probed with anti-Drgal1-L2 antibodies. Lateral and dorsal view of a 27 hpf embryo. D Dorsal and lateral view of the embryo showing the short/bent tail macroscopic phenotype. E, F Dorsal and lateral view of the trunk showing disorganized muscle fibers in the Drgal1-L2-MO-injected embryo (F), as compared to the wild type control embryo (E)

Fig. 4

Dorsal and lateral view of embryos showing the effect(s) of Drgal1-L2 gene expression knockdown. A, B Whole mount immunostaining of (A) wild type (uninjected) control and (B) Drgal1-L2-MO injected embryos (24 hpf) with F59 antibody. C, D Whole mount in situ hybridization of (C) uninjected and (D) Drgal1-L2-MO injected embryos (19 hpf) probed with myod antisense oligos. Compared to the wild type control embryos (A, C), the myofibers in Drgal1-L2-MO injected embryos (B, D) appeared less organized