Znf703, a novel target of Pax3 and Zic1, regulates hindbrain and neural crest development in Xenopus

Abstract

The transcription factors Pax3 and Zic1 are critical to specify the neural plate border and to promote neural crest formation. In a microarray screen designed to identify genes regulated by Pax3 and Zic1 in Xenopus we isolated Znf703/Nlz1 a transcriptional repressor member of the NET (NocA/Nlz, Elbow, and TLP-1) protein family. At early neurula stage znf703 is expressed in the dorsal ectoderm, spanning the neural plate and neural plate border, with an anterior boundary of expression corresponding to rhombomeres 3 and 4 (r3/r4) in the prospective hindbrain. As a bonafide target of Pax3 and Zic1, znf703 is activated by neural plate border inducing signals, and its expression depends on Pax3 and Zic1 function in the embryo. Znf703 morpholino-mediated knockdown expanded several posterior hindbrain genes, while Znf703 overexpression completely obliterated the expression of these segmental genes, signifying that the transcriptional repressor activity of Znf703 is critical to pattern the hindbrain. Furthermore, snai2 and sox10 expression was severely impaired upon manipulation of Znf703 expression levels in the embryo suggesting that Znf703 participates in neural crest formation downstream of Pax3 and Zic1 in Xenopus.

Keywords: Xenopus; hindbrain; neural crest; pax3; zic1; znf703.

Figure 1. Sequence comparison of Znf703 proteins across species

(A) The predicted amino acid sequences from Xenopus laevis, human, mouse and zebrafish znf703 genes were aligned using ClustalW. Conserved amino acids in all four species or in at least two species are highlighted in black and grey, respectively. Xenopus laevis (Xl), mouse (Mm), human (Hs) and zebrafish (Dr). The zinc finger domain is underlined in red, and the position of the two Cysteines and Histidines are indicated (*). The putative Groucho binding domain is underlined in green.

Figure 2. Developmental expression of znf703

(A–D) By in situ hybridization at the neurula stage (NF stage 13/14) znf703 is detected in the dorso-lateral ectoderm posteriorly, including the neural plate and the neural crest forming region, with a gap of expression at the dorsal midline. (E, F) At stages 16 through 19, znf703 expression persists in the dorsal ectoderm where it appears to be confined to the prospective hindbrain and spinal cord. (G) Transverse section of a stage 16 embryo, dorsal to top, showing znf703 expression in the neural plate (np), neural crest (nc), ventral non-neural ectoderm (nne) and lateral plate mesoderm (lpm). (K–M) Later in development znf703 is diffusely expressed throughout the ectoderm, and enriched in the hatching gland (hg) and the branchial arches (ba). (A, C, E, H, K) dorsal views, anterior to top. (B, D, J, L, M) lateral views, dorsal to top, anterior to right. (F, I) frontal views, dorsal to top. The embryonic stages are indicated in the lower left corner of each panel.

Figure 3. znf703 anterior expression domain corresponds to r3/r4

(A) At stage 15, znf703 is detected dorsally, with a sharp anterior boundary of expression. (B–D) Double in situ hybridization showing the spatial relationship of znf703 with snai2 in the neural crest (B), mafb in r5/r6 (C) and fgf3 in r3/r4 (D). Dorsal views, anterior to top. The yellow lines indicate the position of the most anterior boundary of znf703 expression. The red asterisks indication the position of the most anterior expression domains of snai2, mafb and fgf3. Insets (lower right corner) in panels (B), (E) and (D) depict single in situ hybridization for snai2, mafb and fgf3, respectively.

Figure 4. znf703 is a true target of Pax3 and Zic1

(A–B) Fold induction of snai2 (A) and znf703 (B) by expression of pax3GR and zic1GR in animal cap explants (microarray data; Bae et al., 2014). (C–D) Injection in one blastomere at the 2-cell stage of MOs blocking Pax3 (pax3MO; 40 ng) or Zic1 (zic1MO; 40 ng) function resulted in a strong reduction of snai2 expression(C), while only the most anterior expression domain of znf703 was affected (D) at the neurula stage. The injected side is to the right as indicated by the presence of the lineage tracer (Red-Gal). The percentage of affected embryos is indicated in the upper right corner. Dorsal views, anterior to top.

Figure 5. Regulation of znf703 expression by neural crest inducing signals

(A) mRNAs encoding noggin (1 ng) and wnt1 (100 pg) were injected, alone or in combination with pax3MO, into both blastomeres in the animal pole region at the 2-cell stage. At the blastula stage (NF stage 9), animal cap explants were dissected, cultured for 8 hours at room temperature (equivalent stage 13/14) and analyzed by qRT-PCR. (B) In combination, Noggin and Wnt1 induce snai2 and represse keratin expression. Co-injection of pax3MO blocks snai2 induction by Noggin and Wnt1 and promotes neural fate (sox2 expression). The induction of znf703 by Noggin and Wnt1 is unaffected by injection of pax3MO.

Figure 6. Znf703 is required for posterior hindbrain patterning

(A) The target sequence of Znf703 morpholino antisense oligonucleotide (Znf703MO; grey box) is indicated. The position of the start codon (ATG) is indicated. (B) In vitro coupled transcription/translation reactions with plasmid encoding Znf703. Increasing amounts of Znf703MO, 10 ng (+), 100 ng (++) and 1000 ng (+++), block translation directed by Znf703 mRNA. (C–J) Unilateral injection of znf703MO (45 ng) at the 2-cell stage expanded egr4 (C), hoxb1 (D) and fgf3 (E) expression domains in the hindbrain at the neurula stage (NF stge 14/15). In contrast mafb (F) and the r5 expression domain of egr2 were reduced or lost (G). In morphant embryos the expression domain of sox2 was expanded (H), while the neural crest markers were mildly (snai2; G) or strongly (sox10; H) reduced. The horizontal lines (F) indicate the width of the neural plate on the control (black line) and injected (white line) sides. The injected side is to the right as indicated by the presence of the lineage tracer (Red-Gal). The percentage of affected embryos is indicated in the upper right corner. Dorsal views, anterior to top.

Figure 7. Znf703 overexpression affects hindbrain and neural crest development

(A–H) Unilateral injection of znf703 mRNA (1 ng) at the 2 cell stage significantly reduced the expression domain of egr4 (A), hoxb1 (B), fgf3 (C) mafb (D) and egr2 (E), in the hindbrain at the neurula stage (NF stage 14/15). In these embryos sox2 expression domain was expanded (F), and both neural crest markers, snai2 (G) and sox10 (H), were strongly reduced. The horizontal lines (F) indicate the width of the neural plate on the control (black line) and injected (white line) sides. The injected side is to the right as indicated by the presence of the lineage tracer (Red-Gal). The percentage of affected embryos is indicated in the upper right corner. Dorsal views, anterior to top.