Lamar E et al. (2001), Identification of NKL, a novel Gli-Kruppel zinc...

XB-ART-9359

Development 2001 Apr 01;1288:1335-46. doi: 10.1242/dev.128.8.1335.

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Identification of NKL, a novel Gli-Kruppel zinc-finger protein that promotes neuronal differentiation.

Lamar E , Kintner C , Goulding M .

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The proneural basic helix-loop-helix proteins play a crucial role in promoting the differentiation of postmitotic neurons from neural precursors. However, recent evidence from flies and frogs indicates that additional factors act together with the proneural bHLH proteins to promote neurogenesis. We have identified a novel zinc finger protein, neuronal Kruppel-like protein (NKL), that positively regulates neurogenesis in vertebrates. NKL is expressed in Xenopus primary neurons and in differentiating neuronal precursors in the intermediate zone of the mouse and chick neural tube. In frog embryos, NKL is induced by overexpression of Neurogenin (Ngn), arguing that NKL is downstream of the proneural determination genes. Our results show that NKL and a NKL/VP16 fusion protein promote differentiation of neuronal precursors in the embryonic chick spinal cord. Following in ovo misexpression of NKL, neuroepithelial cells exit the cell cycle and differentiate into neurons. Similarly, NKL/VP16 induces extra primary neurons in frogs and upregulates expression of the neural differentiation factors, Xath3 and MyT1, as well as the neuronal markers, N-tubulin and elrC. Our findings establish NKL as a novel positive regulator of neuronal differentiation and provide further evidence that non-bHLH transcription factors function in the neuronal differentiation pathway activated by the vertebrate neuronal determination genes.

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Species referenced: Xenopus laevis
Genes referenced: drg1 elavl3 gli1 glis2 isl1 lhx1 myc myt1 neurod4 neurog1 pax2 tcf3 tra tubb2b

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	Fig. 1. Sequence alignment of mouse, Xenopus and chick NKL. (A) The fulllength predicted amino acid sequence of mouse (mNKL) and Xenopus (XNKL) cDNA clones and the deduced chick (cNKL) sequence derived from a partial cDNA clone (see Materials and Methods). The zinc-finger domain is in red and underlining marks the five tandem fingers separated by consensus (GEKPY) linker regions. The initiation codon of the Xenopus clone was determined by the position of an in frame stop codon at –18 and the optimal context of the start codon (GenBank Accession Number AF249341). The initiation codon of mNKL was determined based on the homology between the N termini of the Xenopus and mouse proteins. (B) Alignment of the zinc-finger domain of NKL and related Gli and Zic proteins; identical residues are shaded, and the five tandem zinc fingers are underlined. Note NKL lacks the 10 amino acid insert found in the first finger of all Zic proteins and exhibits C2H2 spacing different from either Glis or Zics in fingers 3, 4 and 5. hNKL refers to the probable human NKL homolog; DmEST refers to a Drosophila EST encoding a zinc finger approximately 62% identical to NKL. Ci (Cubitus interruptus) and Opa (Odd-paired) are Drosophila homologs of Gli and Zic proteins, respectively. Tra-1 is a C. elegans protein homologous to Gli. (C) EMSA of a tagged mouse NKL zinc-finger domain on a 32P-labeled Gli-1 binding site (B1; Kinzler and Vogelstein, 1990). Sequence of B1 template is shown; underlining indicates bases contacted by Gli-1 protein based on the crystal structure (Pavletich and Pabo, 1993). Brackets indicate shifted band of tagged NKL alone (lower), which is supershifted in the presence of the anti-Myc 9E10 antibody (upper). Lanes: 1, template only; 2, reticulocyte lysate only; 3, Myc-tagged NKL; 4, Myctagged NKL plus anti-Myc 9E10 antibody; 5, Myc-tagged NKL plus 10´ B1 competitor; 6, Myc-tagged NKL plus 100´ B1 competitor; 7, Myc-tagged NKL plus 10´ GC-rich competitor; 8, Myc-tagged NKL plus 100´ GC-rich competitor.
	Fig. 2. Expression pattern of NKL in mouse, chick and Xenopus embryos. (A-C) In mouse, mNKL is expressed in the intermediate zone (arrows) of E10.5 hindbrain (A) and spinal cord (B), as well as in the DRG (arrowhead in B). At E12.5 mNKL is expressed in a narrow band of cells in the ventricular zone (vz) (C). In chick stage 25 embryos (D) cNKL is expressed in the intermediate zone (arrow) of the spinal cord as it is in the E10.5 mouse. (E) In Xenopus stage 13.5 embryos staining is apparent in the neural plate in lateral (L) and medial (M) stripes of primary neurons. (F) An anterior view of a stage 19 embryo indicates NKL hybridization in the trigeminal ganglion (arrow). (G) A dorsal view of columns of NKL-positive cells in the neuraxis (arrow). Note also the onset of NKL staining in the branchial arches (arrowhead). In tailbud stage (32) embryos NKL staining is seen in the neuraxis, the head, the eye and the branchial arches (H). (I,J) Transverse vibratome sections taken immediately posterior to the hindbrain through the neural tube of stage 24 embryos stained for NKL (I) and N-tubulin (J).
	Fig. 3. NKL expression is induced by overexpression of XNGN- 1. Xenopus embryos were injected in one blastomere of two-cell embryos with mRNAs encoding XNGN- 1 and a b-galactosidase tracer, and allowed to develop to the neural tube stage (stage 23). In all panels, anterior is to the left. (A) A dorsal view of a XNGN-1-injected embryo showing ectopic patches of N-tubulin expression on the injected (upper) side (65% (19/29) of embryos injected). (B) The injected side of a XNGN-1-injected embryo probed with antisense RNA for NKL. Note ectopic NKL staining in the ectoderm outside the neural tube (arrow), as well as a mild increase in the level of endogenous NKL staining in the neural tube (arrowhead). (C) The uninjected side, where hybridization signals indicate endogenous NKL expression (arrowhead). Ectopic NKL was seen in 60% (18/30) of embryos injected.
	Fig. 4. Overexpression of NKL promotes premature differentiation of neuronal precursors in the chick neural tube. (A-F) Double-label immunofluorescence of Stage 18 (E3.5) chick embryos electroporated with MT-NKL (A-C), a construct expressing the nuclear localized Myc tag (MT) only (D,E), and a tagged form of XNGN-1 (MT-XNGN-1) (F). Electroporated embryos were stained with anti-Myc and BrdU: BrdU+ cells are green, Myc+ cells are red, and cells co-expressing both are yellow. All embryos were given a 1 hour pulse of BrdU in the neural tube before sacrificing. 10 mm cryostat sections through the forelimb level of the spinal cord were reacted with anti-Myc and anti-BrdU antibodies and the appropriate secondaries (see Materials and Methods). Note that MT-XNGN-1 and MT to a lesser extent are not restricted to the nucleus.
	MTFig. 5. NKL-overexpressing cells in the chick spinal cord are neurons. (A,B) Stage 18 embryos electroporated at stage 12 with the Myc tag alone (A) or MT-NKL (B) and stained with antibodies to both Myc (red) and TuJ1 (green). Only the electroporated sides are shown. (B) Note cluster of double-labeled MT-NKL/TuJ1-postive cells in the intermediate zone (arrowheads). (C,D) Embryos electroporated with MTNKL are stained with anti-Myc (red nuclei) and Lim1/2 antibodies (green nuclei). Merged image is shown in (C) and double-labeled cells are indicated by arrowheads. Myc-only cells are shown in (D). Electroporated side is to the right, as in (E-I). MT-NKL constructs electroporated into the intermediate and ventral regions of the spinal cord are also Pax2 (E) and Isl1 (G) positive, respectively (arrowheads in E and G). Note that MT-NKL-positive cells ventral to the Pax2 domain in are Pax2 negative (arrow in E). In (E-H) Pax2- or Isl1-positive nuclei are seen as green; Mycpositive cells are red; and nuclei that co-express both are yellow. (I) Stage 18 embryos electroporated at stage 12 with MT-Ngn1 are reacted with antibodies to Pax2 (green) and Myc (red). As in (E), Myc-positive cells ventral to the Pax2 domain (compare to the untransfected side) are labeled with Myc only.
	Fig. 6. Overexpression of NKL/VP16 phenocopies overexpression of wild-type MT-NKL in the chick neural tube. (A) Chick embryos were electroporated with MT-NKL/VP16, given a 1 hour BrdU pulse, and analyzed for Myc (red) and BrdU (green) expression as described in Fig. 4. Note that the majority of Myc-positive cells are BrdU-. (B) Embryos electroporated with the Myc-tagged NKL zincfinger domain (MT-NKLzf) and analyzed by the protocol described for (A). (C) Embryos electroporated with a tagged deletion mutant of NKL containing an intact zinc-finger domain but lacking the Nterminal 144 amino acids of the protein (MT-NKLDN). (D) Embryos electroporated with a deletion mutant lacking the C terminal 128 amino acids but retaining the intact zinc-finger domain (MTNKL DC). (E) Chick embryos electroporated with MT-NKL/EnR at Stage 10, sacrificed at Stage 14, and reacted with antibodies to Myc only. Note what appear to be clones of Myc-positive cells, indicative of proliferating cells. (F) Quantification of cell counts from data presented here and in Fig. 4. The y-axis shows the total number of Myc+/BrdU+ (yellow) cells divided by the total number of Mycpositive (red) cells. Cells lining the lumen of the neural tube and migrating crest cells were excluded from the analysis. Injected constructs include: WT (full-length NKL), myc (Myc only), zf (tagged NKL zinc-finger only), DN (tagged NKL N-terminal deletion) and DC (tagged NKL C-terminal deletion). Three or more embryos were analyzed for each construct. Numbers of cells counted were 593 (WT), 539 (myc), 390 (zf), 245 (DN), and 196 (DC).
	Fig. 7. Overexpression of NKL/EnR and NKL/VP16 fusion proteins promotes changes in neuronal markers in Xenopus embryos. Xenopus embryos were injected with mRNA encoding a b- galactosidase tracer and mRNAs encoding NKL/EnR (A-C), or NKL/VP16 (D-I) fusion proteins. Embryos were injected with 40 pg of test RNA. RNA was injected into one blastomere at the two-cell stage and probed with antisense RNAs for the indicated neuronal markers. (A,B,D,E,G-I) Expression of N-tubulin, Xath3, elrC and MyT1 expression in representative embryos at neural plate stages. (C,F) Show neurofilament expression in cross sections through stage 26 embryos. In all embryos the injected side is to the right. Changes in gene expression are indicated by arrows.