Znf703 is a novel RA target in the neural plate border

Abstract

Znf703 is an RAR- and Wnt-inducible transcription factor that exhibits a complex expression pattern in the developing embryo: Znf703 mRNA is found in the early circumblastoporal ring, then later throughout the neural plate and its border, and subsequently in the mid/hindbrain and somites. We show that Znf703 has a different and separable function in early mesoderm versus neural crest and placode development. Independent of its early knockdown phenotype on Gdf3 and Wnt8, Znf703 disrupts patterning of distinct neural crest migratory streams normally delineated by Sox10, Twist, and Foxd3 and inhibits otocyst formation and otic expression of Sox10 and Eya1. Furthermore, Znf703 promotes massive overgrowth of SOX2+ cells, disrupting the SoxB1 balance at the neural plate border. Despite prominent expression in other neural plate border-derived cranial and sensory domains, Znf703 is selectively absent from the otocyst, suggesting that Znf703 must be specifically cleared or down-regulated for proper otic development. We show that mutation of the putative Groucho-repression domain does not ameliorate Znf703 effects on mesoderm, neural crest, and placodes. We instead provide evidence that Znf703 requires the Buttonhead domain for transcriptional repression.

Figure 1

Znf703 is a conserved transcriptional repressor. (A) Phylogenetic tree of Znf703 sequences aligned and constructed with MAFFT (see materials and methods). The scale bar represents the divergence distance of 0.2 amino acid substitutions per site of the Znf703 sequence. The tree segregates organisms appropriately with the exception of Amphioxus. We attempted to add more sequences to improve this unexpected result, but Amphioxus persistently segregated with mollusk sequences. (B) Cos7 cells were transfected with 5:5:1 DNA ratio of reporter (Gal4-Luc): β-gal: effector (Znf703). The y-axes represent relative light units measured by the luminometer normalized to β-gal activity. Basal reporter activity (Gal4 alone) is repressed by Znf703. Mutating the FKPY domain or deletion of the N-terminus up until the Buttonhead (Btd) domain still represses transcriptional activity. Deletion beyond the Btd domain relieves repression. Statistical significance was determined using one-way ANOVA, and Bonferroni post-hoc test in GraphPad Prism v5.0 (***P ≤ 0.001). (C) Conservation of the Btd domain across the animal kingdom, visualized with WebLogo.

Figure 2

Expression of Znf703 and Znf503 across developmental time. (A) Whole mount in situ hybridization of Znf703 expression at Nieuwkoop and Faber developmental stages 11 (vegetal view, dorsal at the top), 14 (dorsal view, anterior on the left), 19 (dorsal and anterior views), and 25 (lateral and dorsal views, anterior on the right). WISH of Znf503 expression can be found in Fig. S1. (B) Double WISH at stage 19 reveals the spatial relationship between Znf703 and midbrain (Engrailed 2) and hindbrain (Krox20) markers. (C) Znf703 expression in a chick embryo at Hamburger Hamilton (HH) Stage 16 (lateral view; anterior on the right). MB = midbrain; HB = hindbrain. (D) QPCR showing Znf703 and Znf503 gene expression averaging two biological replicates over developmental time. Error bars = S.E.M. The y-axis represents 2^−ΔCt values (adjusted for primer efficiency), normalized to reference gene, Histone H4.

Figure 3

Znf703 expression is modulated by RAR-selective agonist TTNPB. WISH from embryos treated at stage 6/7 with 1 µM TTNPB or control vehicle (0.1% EtOH). TTNPB greatly expands expression of Znf703 into the dorsal and anterior domains, relative to control vehicle. Stage 11 embryos are shown in vegetal view with the dorsal lip at the top. Stage 12 and 16 are shown in dorsal view with anterior to the right. Stage 20 embryos are shown in anterior view. Stage 30 embryos are shown in lateral view with anterior to the right.

Figure 4

Overexpression of Znf703 mRNA inhibits neural crest migration. Embryos were injected unilaterally at 2- or 4-cell stage with either 0.5 ng Znf703 (A–F’) or control mCherry. (A–C) Znf703 mRNA reduces the lateral and anterior expression of Sox10, Twist, and Foxd3 in stage 19 embryos (shown in anterior view). Injected side is to the right of the dotted line, and is indicated by the magenta β-gal lineage tracer. (D’–F’) Znf703 mRNA inhibits Sox10 patterning, Twist, and Tfap2a in stage 27 embryos (shown in lateral view). (D–F) Uninjected side of the same embryo. Fractions represent the portion of embryos displaying the phenotype.

Figure 5

Overexpression of Znf703 mRNA reduces expression of Gdf3 and Wnt8, but not T or Fgf8. Embryos were injected unilaterally at 2- or 4-cell stage with either 0.5 ng Znf703 or control mCherry mRNA. Injected side is to the right of the dotted line, and is indicated by the magenta ß-gal lineage tracer. (A–C,F) Znf703 mRNA causes loss of Gdf3, knockdown of Wnt8, and weak knockdown or no change of T and Fgf8. Embryos are shown at stage 10.5/11 in vegetal view. (D,E) Control mRNA did not have any effect on Gdf3 or Wnt8. Fractions represent the portion of embryos displaying the phenotype. NC = No change.

Figure 6

Znf703 impairs Sox10 expression at tailbud stage, when induced after stage 12 up until stage 19. (A) Schematic of ligand-inducible transcriptional repression by hGR-Znf703. In the absence of dexamethasone (DEX), hGR-ZNF703 is tethered in the cytoplasm by HSP90. In the presence of DEX, hGR-ZNF703 is free to enter the nucleus. Question marks indicate that Znf703 is not thought to bind DNA directly. (B–E’) Embryos were injected unilaterally with 0.2 ng hGR-Znf703 mRNA at 2- or 4-cell stage, then treated with 5 µM DEX or 0.05% DMSO vehicle at the stages indicated. Injected side is indicated by the magenta β-gal mRNA lineage tracer. Znf703 blurs the migratory streams of Sox10 expression when Znf703 is induced after gastrulation and prior to stage 19. Little effect on Sox10 is observed when embryos are treated with DEX at stage 19. DMSO treated embryos are pictured in Fig. S4. All embryos are shown in lateral view with anterior on the right, at stage 27. Fractions represent the portion of embryos displaying the phenotype in one time-course experiment from the same clutch of embryos. This experiment was repeated an additional time, and very similar results were obtained.

Figure 7

Znf703 is normally absent from the otocyst. Znf703 overexpression causes aberrant SOX2 expression and disappearance of otocyst. (A–D) Znf703 is absent from the otocyst as marked by Eya1 and Sox10, and lateral line placode as marked by Eya1, shown in lateral view at stage 27. Ot = otocyst; ll = lateral line; eb = epibranchial placodes. Embryos were injected unilaterally at the 2- or 4-cell stage with 0.5 ng Znf703 mRNA (E,G–I) or 0.2 ng hGR-Znf703 mRNA and treated with DEX at stage 12 (F). (E,F) Maximum intensity projections of Dapi nuclear stain and/or SOX2 from confocal images of transverse sections (expansion of SOX2 observed in 8/8 embryos sectioned). Otocysts were not found on the injected side in 60% of embryos. In the remaining 40% of embryos, the otocyst was reduced in size (red arrow). (G) Sox10 expression marks otocysts that are significantly reduced in size and positioned more rostral/ventral in the head compared to control (Stage 27 embryo in lateral view). (H) Maximum intensity projection of Dapi (false-colored in white) from confocal images through a laterally-mounted stage 27 embryo. (I) Eya1 expression is reduced, particularly in the otic vesicle on the injected side (Stage 27 embryo in lateral view) (15/15 embryos). Red boxes in H and I highlight the otocyst on the uninjected side and its absence on the injected side.