Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Development
2001 Apr 01;1288:1335-46. doi: 10.1242/dev.128.8.1335.
Show Gene links
Show Anatomy links
Identification of NKL, a novel Gli-Kruppel zinc-finger protein that promotes neuronal differentiation.
Lamar E
,
Kintner C
,
Goulding M
.
???displayArticle.abstract???
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.
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.