Ectopically expressed Slc34a2a sense-antisense transcripts cause a cerebellar phenotype in zebrafish embryos depending on RNA complementarity and Dicer

Abstract

Natural antisense transcripts (NATs) are complementary to protein coding genes and potentially regulate their expression. Despite widespread occurrence of NATs in the genomes of higher eukaryotes, their biological role and mechanism of action is poorly understood. Zebrafish embryos offer a unique model system to study sense-antisense transcript interplay at whole organism level. Here, we investigate putative antisense transcript-mediated mechanisms by ectopically co-expressing the complementary transcripts during early zebrafish development. In zebrafish the gene Slc34a2a (Na-phosphate transporter) is bi-directionally transcribed, the NAT predominantly during early development up to 48 hours after fertilization. Declining levels of the NAT, Slc34a2a(as), coincide with an increase of the sense transcript. At that time, sense and antisense transcripts co-localize in the endoderm at near equal amounts. Ectopic expression of the sense transcript during embryogenesis leads to specific failure to develop a cerebellum. The defect is RNA-mediated and dependent on sense-antisense complementarity. Overexpression of a Slc34a2a paralogue (Slc34a2b) or the NAT itself had no phenotypic consequences. Knockdown of Dicer rescued the brain defect suggesting that RNA interference is required to mediate the phenotype. Our results corroborate previous reports of Slc34a2a-related endo-siRNAs in two days old zebrafish embryos and emphasize the importance of coordinated expression of sense-antisense transcripts. Our findings suggest that RNAi is involved in gene regulation by certain natural antisense RNAs.

Fig 1. Expression of Slc34a2a and related transcripts during zebrafish embryogenesis.

(A) Schematic representation of the Slc34a2a, Slc34a2a(as) and Rbpja loci. The antisense transcript Slc34a2a(as) is depicted in red. (B) RT-qPCR analysis of Slc34a2a, Slc34a2a(as) and Rbpja transcripts including the paralog Slc34a2b. Based on negative controls using RNA as an input, the detection limit was set at a ΔCt of 12 which is in agreement with ISH results. (C) Demonstration of Slc34a2a, Slc34a2a(as), Rbpja, Slc34a2b and Shh (Sonic Hedgehog) transcripts at progressing stages of development by whole mount ISH.

Fig 2. Injection of Slc34a2a RNA into fertilized zebrafish eggs.

A) Visual classification of malformations depending on the severity of the defect: Level 1, wild type; level 2, one organ affected (size, shape or function, e.g. heart rate); level 3, 2–3 organs affected, level 4, multiple malformations; level 5, developmental arrest. B) Phenotypic characterization of zebrafish embryos injected with various RNAs, Slc34a2a (554 embryos in total), antisense (94 embryos) and Slc34a2b (538 embryos). C) (i) Anatomy of a 48 hpf zebrafish embryo: Y, yolk sac; E, eye; O, otic vesicle (ear); R1/R7, rhombomeres; M, mesencephalon; C, cerebellum, in red. (ii) non injected wild type embryo; (iii) Slc34a2a injected embryo; (iv) wild type embryo, Eng2 stained; (v) Slc34a2a injected embryo, Eng2 stained; (vi, vii) Slc34a2a(as) and Slc34a2b Eng2 stained.

Fig 3. Injection of Slc34a2a RNA and related constructs into fertilized zebrafish eggs.

A) ISH of wild type and injected embryos at 24 hpf. Horizontal labels at the top indicate the injected material, vertical labels, left, represent the probes used for ISH. B) RT-qPCR of injected zebrafish embryos; Slc34a2a, Slc34a2a(as) and Slc34a2b RNA was injected as indicated with the different colour from brown to blue and assayed after 10 and 24 hpf. The left group represents RT-qPCR reactions with Slc34a2a-specific primers; the middle group with Slc34a2a(as)-specific primers and the right group with Slc34a2b-specific primers. The values for non-injected controls are indicated with grey, transparent boxed.

Fig 4. Injection of non- protein coding Slc34a2a RNA and Slc34a2a fragments interfere with zebrafish development.

A) Schematic representation of a zebrafish head at 48 hpf; forebrain, blue; eyes, yellow; otic vesicles, green and cerebellum, red. Middle and left, wild type and Slc34a2a-FS injected embryo, respectively. Red arrows indicate the position of the cerebellum. B) Phenotypic quantification of Slc34a2a and Slc34a2a-FS injected embryos (364 Slc34a2a-FS injected embryos were assessed). C) Schematic representation of the fragments generated, even numbers represent sense orientation; uneven numbers, antisense orientation. The large black boxes represent exons comprised in the relevant fragments, the open boxes are exons that are not represented in the injected fragments. The small boxes in red indicate potential sites of hybridization of the injected fragments with an endogenous transcript on the opposite strand. D) Top view of 48 hpf embryos with the fragments (Frag) injected as indicated. E) Phenotypic assessment of injected embryos (90 or more per RNA). F) Eng2 stained embryos injected with the indicated fragments and the relevant controls. All the embryos were tested in parallel with the same solutions and under identical conditions to allow for a comparison of the relative intensities.

Fig 5. Morpholino knockdown of Slc34a2a(as) and Dicer.

A) RT-qPCR quantification of Slc34a2a, Slc34a2a(as) and Slc34a2b after splice site morpholino injection at 24 hpf. Wild type non injected controls, light blue bars; 5 ng splice-site MO injected embryos are in dark blue; 5 ng scrambled MO injected embryos are in blue. B) Phenotypic characterization of MO injected embryos at 24 and 48 hpf. The injected oligonucleotides and quantities are indicated below the bars. Phenotypic scaling was performed as described in Fig 2. C) Rescue of cerebellum development by Dicer knockdown. Phenotypic assessment of embryos injected with combinations of Dicer MO, p53 MO and Slc34a2a. D) 48 hpf zebrafish embryos injected with Dicer MO, p53 MO and Slc34a2a as indicated in the pictures. In the upper panel, heads with red arrows indicating the cerebellum are shown; the lower panel shows ISH of embryos with an Eng2 probe.