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 Pdf file on Expression and function on embryonic development of lissencephaly-1 genes
in zebrafish
Chengfu Sun, Mafei Xu, Zhen
Xing, Zhili Wu, Yiping Li, Tsaiping Li, and Mujun Zhao*
State Key Laboratory of Molecular Biology, *Correspondence address. Tel: +86-21-54921115; Fax: +86-21-54921011; E-mail: [email protected]
Lissencephaly is a severe disease characterized by
brain malformation. The main causative gene of lissencephaly
is LIS1. Mutation or
deletion of LIS1 leads to proliferation and migration deficiency of
neurons in brain development. However, little is known about its biological
function in embryonic development. In this article, we identified the
expression patterns of zebrafish LIS1 gene and investigated
its function in embryonic development. We demonstrated that zebrafish
consisted of two LIS1 genes, LIS
Keywords LIS1; expression;
embryonic development; zebrafish
Received: March 17, 2009 Accepted: April 28, 2009
Introduction
Lissencephaly is a rare brain malformation disease caused by defects in neuronal
migration [1]. Classical lissencephaly has two major
types: Miller–Dieker syndrome (MDS) and
isolated lissencephaly sequence (ILS). All MDS and
some cases of ILS have haploinsufficiency at human
chromosome 17p13.3, from where LIS1 (Lissencephaly-1) gene was isolated [2]. Mutation or deletion of LIS1 leads to proliferation
and migration deficiency of neurons in brain development [3]. Thus, LIS1 is the main causative
gene responsible for classical lissencephaly.
Human LIS1, which
is highly expressed in brain, encodes a protein of 45 kDa
in size. LIS1 protein contains a Lis-H domain at its
N-terminus, followed by a coiled-coil region and seven WD repeats. LIS1
functions as a dimer in vivo. Lis-H domain
and the coiled-coil region are believed to be important for LIS1 dimerization, stability, and localization [4,5], while WD
repeats are required for protein interactions, through which LIS1 interacts
with a variety of proteins, such as tubulin [6], dynein [7], dynactin [7],
CLIP-170 [8], DCX [9], and NudeL [10], conferring its
influences on neuronal migration and mitotic cell division. LIS1 was also
identified as a subunit of platelet-activation factor (PAF) acetylhydrolase
[11], which regulates the physiological activity of PAF.
Although human LIS1 is a brain disease-caused gene, it is much conserved in evolution. The LIS1 gene has also been
identified in other species besides human, such as budding yeast [12], Aspergillus nidulans [13], nematode worm
[14], fruit fly [15], chick [16], and mouse [17]. Zebrafish
(Danio rerio) LIS1 genes have also been cloned, named LIS
Materials and Methods
Zebrafish and cell lines
Zebrafish were provided by the Lab of Germ Cell and Embryonic Developmental Biology
in the Shanghai Institutes for Biological Sciences (
Extraction of RNA and RT–PCR
Total RNA was extracted from zebrafish embryos
and tissues using Trizol reagent (Invitrogen,
Plasmid construction
To generate plasmids pEGFP-C1-LISPlasmid pCS2-lismo-GFP was generated by PCR amplifying of the 5‘-UTR sequences of zebrafish
LIS1b with primers listed in
Table 1 (lismo_BF
and lismo_ER) and inserted into pCS2-GFP (kept in our
Lab). PCR was performed with the same cycling conditions as cloning of LIS
Transient transfection and subcellular localization
Cells were transiently transfected with pEGFP-C1
vector or pEGFP-C1-LIS1 plasmids. Transfections were
performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly,
HEK-293T or HeLa cells were seeded into 24-well
plates, and 0.4 mg of plasmid was mixed with 0.6 ml transfection
agent per well and added to cells. After 24 h transfection,
HEK-293T cells were collected for western blotting analysis; HeLa cells were washed three times with PBS and then fixed
for 10 min in chilled methanol. The nuclei were stained with Hoechst 33258 for
5 min. After several washes, coverslips were sealed
and the cells were examined under a fluorescence microscope (Olympus BX51;
Olympus,
Preparation of antibody
Rabbit polyclonal antibody against the N-terminus of human LIS1 peptide
(residue 1–55 amino acids) fused to N-terminal GST tag was prepared in our
laboratory. The specificity of the antibody was examined by western blotting
analysis.
Protein extraction and western blotting analysis
For the preparation of proteins from embryos, zebrafish
embryos of different developmental stages were dechorionated
and cells were scattered by pipette aspiration. After centrifuged at
Whole-mount immunochemistry
Whole-mount immunochemistry was performed according to Hashimoto et al. [22]. Briefly,
embryos of different developmental stages were fixed for 24 h with 4% paraformaldehyde at 4ºC, dechorionated
and washed with PBST (PBS with 1% Tween-20), and blocked with blocking buffer
(PBST containing 10% heat inactivated goat serum). Preimmune
serum or anti-LIS1 antibody (dilution 1:
Capped RNA transcription, morpholino
oligonucleotides, and microinjection
To produce capped lismo-GFP RNA, the
pCS2-lismo-GFP plasmid was linearized by restriction
enzyme NotI, followed by in vitro transcription using the SP6 mMessage mMachine kit (Ambion,
Results
Bioinformatics analysis of zebrafish
LIS1 genes
By GenBank searching, we found that LIS1 homologous genes
existed in 29 species including 18 vertebrates and 11 invertebrates. To infer
evolutionary relationships of zebrafish LIS1 homologues, we
constructed a phylogenetic tree based on the protein
sequences of LIS1 from these 29 species using the neighbor-joining method (Fig. 1). The phylogenetic tree showed that zebrafish
LIS1 (D. rerio-a and D. rerio-b) were evolutionarily
conserved, especially among the vertebrate species. It is interesting that the
similarity of LIS1 proteins between social ameba and human, the positions of
which are very distant in evolution, is still as high as about 64% identical,
suggesting that LIS1 gene appeared very early in evolution and evolved in a much conserved way.
To analyze the relatedness between the zebrafish LIS1
proteins and other animal model species more detailedly,
multiple amino acid sequence alignments of the human, mouse, zebrafish, fruit fly, nematode worm, and yeast were
performed with ClustalX software (ftp:// ftp-igbmc.u-strasbg.fr/pub/ClustalX). Results showed that LIS1
proteins including LIS
Expression patterns of zebrafish
LIS1 transcripts
Alignment of LIS
Expression patterns of zebrafish
LIS1 proteins
To examine the expression of zebrafish LIS1
proteins at different embryonic developmental stages and adult tissues, western
blotting analysis with polyclonal antibody against human LIS1 was used. The
efficiency of the antibody against zebrafish LIS1
protein was examined. It showed that the antibody could recognize exogenous zebrafish LISWhole-mount immunostaining was also used to
investigate the expression patterns of LIS1 proteins during embryogenesis. The
results indicated that zebrafish LIS1 proteins were
distributed all over the embryos from 1-cell stage to 5 dpf,
as well as in the unfertilized eggs (Fig. 4). In the unfertilized eggs [Fig. 4(A–D)], LIS1 proteins were
distributed evenly in the eggs [Fig. 4(B)]; with the advance of cytoplasmic streaming,
cytoplasm was separated from yolk, LIS1 proteins were translocated
to the putative animal pole, and accumulated there gradually [Fig. 4(C,D)]. After
fertilization, LIS1 proteins were accumulated at the animal pole during the
1-cell stage as in unfertilized eggs and then distributed throughout the blastomeres in the cleavage period [Fig. 4(F–H)]. Intensity of LIS1 expressions was much higher in fertilized embryos
than unfertilized eggs. At the 4-cell stage, we found that LIS1 proteins were
not evenly distributed, with lower expression in some areas of blastomeres [Fig. 4(G)]. Generalized distribution of LIS1 proteins persisted from shield stage
to 5-somite stage; it was disturbed after 24 hpf [Fig. 4(I,J,M)]. From 24 hpf to 5 dpf, the LIS1 proteins
were highly expressed in brain than other tissues [Fig. 4(K,L,O)], suggesting that LIS1 proteins are very
important for brain development.
Subcellular localizations of zebrafish
LIS1 proteins
To identify the subcellular localizations of zebrafish LIS1 proteins, we constructed the GFP-fused LIS
Knockdown of zebrafish LIS1
proteins affects embryonic development
Owing to the advantages of zebrafish over other
animal system, especially the external embryonic development, we examined the
effect of LIS1 on zebrafish embryonic development using antisense MO
technology. Three MOs (MO-LIS
Discussion
LIS1 gene has been cloned in a variety of animals, including zebrafish, which was reported by Tsujikawa
et al. [18], while we had
also cloned zebrafish LIS1 genes. Interestingly, zebrafish
contains two LIS1 genes, LISTo investigate the expression of LISStudies in other animal systems suggest that LIS1 plays an important role in reproduction.
For example, depletion of LIS
Funding
This work was supported by grants from National Key S&T Special
Project of China (No. 2008ZX10002-020), and the Shanghai Municipal Commission
for Science and Technology (No. 74319111 and 07DZ22919).
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