Retinoic acid negatively regulates dact3b expression in the hindbrain of zebrafish embryos

Abstract

Wnt signaling plays important roles in normal development as well as pathophysiological conditions. The Dapper antagonist of β-catenin (Dact) proteins are modulators of both canonical and non-canonical Wnt signaling via direct interactions with Dishevelled (Dvl) and Van Gogh like-2 (Vangl2). Here, we report the dynamic expression patterns of two zebrafish dact3 paralogs during early embryonic development. Our whole mount in situ hybridization (WISH) analysis indicates that specific dact3a expression starts by the tailbud stage in adaxial cells. Later, it is expressed in the anterior lateral plate mesoderm, somites, migrating cranial neural crest, and hindbrain neurons. By comparison, dact3b expression initiates on the dorsal side at the dome stage and soon after is expressed in the dorsal forerunner cells (DFCs) during gastrulation. At later stages, dact3b expression becomes restricted to the branchial neurons of the hindbrain and to the second pharyngeal arch. To investigate how zebrafish dact3 gene expression is regulated, we manipulated retinoic acid (RA) signaling during development and found that it negatively regulates dact3b in the hindbrain. Our study is the first to document the expression of the paralogous zebrafish dact3 genes during early development and demonstrate dact3b can be regulated by RA signaling. Therefore, our study opens up new avenues to study Dact3 function in the development of multiple tissues and suggests a previously unappreciated cross regulation of Wnt signaling by RA signaling in the developing vertebrate hindbrain.

Keywords: Dact; Dishevelled; Retinoic acid signaling; Vertebrate development; Wnt signaling.

Figure 1. Sequence alignments of vertebrate Dact3 proteins

A: Comparison of human (Hs), mouse (Mm), and zebrafish (Dr) Dact3 protein sequences revealed conservation of the N-terminal CD1 and LZD domains and the C-terminal PDZ-binding domain. Conserved domains are underlined in red. 6 conserved leucines of the LZD are indicated with red stars. B: Table with the amino acid identity (Y-axis) and similarity scores (X-axis) of Hs, Mm and Dr Dact3 homologs.

Figure 2. Sequence alignments of zebrafish Dact proteins

A: Comparison of zebrafish (Dr) Dact1, Dact2, and Dact3a and Dact3b protein sequences revealed greater conservation of the N-terminal CD1 and C-terminal PDZ-binding domains, with less conservation of the LZD domain. Conserved domains are underlined in red. 5 conserved leucines of the LZD are indicated with red stars. B: Table with the amino acid identity (Y-axis) and similarity scores (X-axis) of Dr Dact proteins.

Figure 3. RT-PCR analysis of dact3a and dact3b gene expression

Expression of dact3a and dact3b analyzed by RT-PCR at the indicated stages. Solo served as a positive control.

Figure 4. Zebrafish dact3a expression from 60% epiboly through 48hpf

A: Specific dact3a expression was not observed at 60% epiboly, even though it is detectable by RT-PCR (Fig. 3). D: Dact3a expression in the adaxial mesoderm at the TB stage (black arrow). B,E: Dact3a expression in the somites (black arrows) and anterior lateral plate mesoderm (white arrowhead) at the 8s stage. C: Expression at the telencephalon (white arrow), spinal cord neurons (black arrowhead), and the forming somites (black arrow) at the 18s stage. F: Mandibular (nc1), hyoid (nc2), and branchial (nc3) neural crest expression and medial eye expression (black arrowhead) at the 18s stage. G–I: Hindbrain expression at 24, 36 and 48hpf (black arrow). At 36 and 48 hpf, dact3a expression is visible in the pectoral fin bud (red arrow). Inset: Cartilage expression (black arrow) at 48hpf. A, C, G, H, I are lateral views. B, D, E, F are dorsal views. Anterior up in all the images.

Figure 5. Dact3a expression in the brain

A: Dact3a expression in the telencephalon (black arrowhead), medial eye (white arrow), neural crest (nc1, 2, 3) and hindbrain neurons (black arrows). B: Expression in the nV (trigeminal) and nVII (facial) cranial branchiomotor neurons (black arrows). C: Expression in the branchiomotor neurons (black arrows). D: Pectoral fin bud expression in two distinct domains (black arrowheads). All the images are dorsal with anterior right.

Figure 6. Zebrafish dact3b expression from dome through 48hpf

A–C, E–G: Dact3b expression in the dorsal blastoderm margin from dome through 60% epiboly stage (black arrow). D: Specific dact3b is not expressed at the 8s stage, even though we could detect it with RT-PCR (Fig. 3). H,I: Dact3b expression in the hindbrain starts at 18s and is maintained through 24 hpf embryos (arrow). J,K: Dact3b expression initiates in the 2^nd pharyngeal arch (white arrowheads) and the dorsal region of the midbrain/tectum (black arrowheads) at 36hpf and is maintained through 48 hpf. Dact3b continues to be expressed in the hindbrain at 36 and 48hpf (black arrows). A–D and H–K are lateral views with dorsal rightward. E–G are dorsal views. In all images, anterior is up.

Figure 7. Dact3b expression in the hindbrain

A: Double WISH for dact3b and krox-20 at 24 hpf. Krox-20 (orange) is expressed in r3 and r5. Dact3b expression is from r2 through r6 (brackets). dact3b is expressed in the adjacent 2^nd pharyngeal arch (black arrowhead). B: Dact3b expression extends more anteriorly past the midbrain-hindbrain boundary (black arrow) and is expressed in the tectum by 36 hpf (white arrowhead). 2^nd pharyngeal arch expression is maintained (black arrowhead). C: By 48 hpf, dact3b expression is increased and is in stripes consistent with anterior branchiomotor neuron expression (black arrow) (Chandrasekhar et al., 1997). Embryos in images were flat mounted. Images are dorsal views with anterior to the left.

Figure 8. RA signaling negatively regulates dact3b expression

A, D: Control embryos B, E: Treatment with exogenous RA extinguishes dact3b expression in the hindbrain (black arrows) and 2^nd pharyngeal arch (white arrow in E). C, F: Inhibition of RA signaling lead to an expansion of dact3b expression domain with the hindbrain (compare the length between arrows in D and F). Images are lateral views with anterior to upward.