Download Induction of cranial and posterior trunk neural crest by exogenous

NOTES
Induction of cranial and
posterior trunk neural crest
by exogenous retinoic acid in
zebrafish
LI Ming, SU Ying & MENG Anming
Department of Biological Sciences and Biotechnology, Protein Science
Laboratory of the MOE, Tsinghua University, Beijing 100084, China
Correspondence should be addressed to Meng Anming (e-mail:
[email protected])
Abstract Retinoic acid (RA) plays an important role in
development of vertebrate embryos. We demonstrate impacts of exogenous RA on the formation of neural crest cells
in zebrafish using specific neural crest markers sox9b and
crestin. Treatment with all-trans RA at 10−7 mmol/L at 50%
epiboly induces sox9b expression in the forebrain and crestin
expression in the forebrain and midbrain, resulting in significant increase of pigment cells in the head derived from
the cranial neural crest. In addition, RA treatment induces
expression of sox9b and crestin in the caudal marginal cells
of the neuroectoderm during early segmentation. Earlier
commitment of these cells to the neural crest fate in the posterior margins leads to abnormal development of the posterior body, probably by preventing mingling of ventral derived and dorsal-derived cells during the formation of the
tailbud.
Keywords: zebrafish, retinoic acid, neural crest, sox9b, crestin.
Retinoic acid (RA), a bioactive form of vitamin A,
has been implicated as an endogenous signaling molecule
in the normal development of vertebrate embryos[1]. Embryos developed in the absence of vitamin A show severe
defects in the cardiovascular system as well as in the central nervous system, including loss or size reduction of
posterior hindbrain, massive death of neural crest cells
and failure of neurite outgrowth[2 5]. The affected embryos can recover after application of exogenous RA or
feeding with vitamin A[5,6]. On the other hand, exposure of
embryos to an excessive amount of RA also results in
abnormal development. The affected tissues or organs
usually are the head and the central nervous system, heart
and limbs[7 11]. The anomalies resulting from either a
depletion or surplus of RA are accompanied by the
misexpression of many genes in the affected regions[3 7].
Zebrafish has become an important model for
studying development of vertebrates because of its high
reproductivity, fertilization and embryonic development
outside the mothers and easy observation and so on. Like
in other vertebrate species, neural crest precursors in zebrafish are located at the junction between the prospective
neural
Chinese Science Bulletin Vol. 47 No. 13 July 2002
plate and the epidermis in the ectoderm during mid- to
late gastrulation. During neurulation, these cells move
toward the dorsal midline until, by the end of neurulation,
they sit on the dorsalmost portion of the formed neural
tube. Thereafter, neural crest cells segregate from the
neuroepithelium, migrate along different pathways, and
ultimately differentiate into a large number of cell types
depending on their initial locations and final destinations[12]. In this study, we demonstrate the impacts of RA
on the formation of neural crest in zebrafish using markers
sox9b and crestin that are expressed in neural crest precursors, migrating neural crest cells and some of their derivatives[13]1).
1 Materials and methods
( ) RA treatment of embryos. AB line of zebrafish was used in this experiment. Collection and culture of
embryos followed the methods described in ref. [14].
When each batch of embryos (about 400) incubated in
Holtfreter solution developed to 50% epiboly, half of them
were transferred to a solution containing 10−7 mmol/L RA
and 0.1% DMSO and the other half to 0.1% DMSO solution as control. The embryos were incubated for 1 h at
28.5
under the dark condition, followed by several
washes with Holtfreter solution and incubation in
0.5×Holtfreter solution. The embryos were fixed in 4%
paraformaldehyde at desired stages.
( ) Whole mount in situ hybridization. Sox9b
cDNA isolated in our lab1) was used. Crestin cDNA[13]
was provided by Dr. Marnie Halpern. Digoxigenin-UTP
labeled antisense RNA probes were generated by in vitro
transcription with SP6 or T7 polymerase. Whole-mount
RNA in situ hybridizations, which were used for examining the amount and spatiotemporal distribution of RNA
expression in embryos, were performed using the protocol
described in ref. [15].
2 Results and discussion
( ) RA treatment causes loss of some parts of the
head. Previous studies have shown that RA-treated zebrafish embryos often lose the caudal midbrain and rostral
hindbrain, and expression of pax2 in the midbrain is
eliminated[8,16]. In our study, the RA-treated embryos at
the 6-somite stage were first hybridized with pax2 probe
to confirm the effectiveness of our treatment. The hybridization results reveal that, consistent with the results reported by Hill et al.[16], the RA-treated embryos lose the
transverse midbrain band of pax2 expression (data not
shown). This suggests that the RA treatment in our conditions is effective.
( ) RA treatment induces neural crest precursors in
the forebrain. The expression of sox9b in the control
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NOTES
1) Li, M., Zhao, C. T., Wang, Y. et al., Zebrafish sox9b is an early neural crest marker, Development Genes and Evolution, 2002, in press.
Fig. 1. Effects of RA on crestin expression and pigment formation. (a) (i) Expression patterns of crestin detected by in situ hybridization; (j) (m) pictures of living embryos; (a) (i), (l) and (m) dorsal views; (j) and (k) lateral views; (a) (d) flat-mounted dorsal views;
(g) twisting tail in F at a higher magnification. The other labels are the same as in Plate .
embryos starts at 90% epiboly stage (~ 9 h postfertilization), and is restricted to in the anterior edge, lateral mar1106
gins and the midline of the neuroectoderm until the onset
-1). In contrast, all of the RAof segmentation (Plate
Chinese Science Bulletin Vol. 47 No. 13 July 2002
NOTES
treated embryos at the same stage expressed sox9b in the
whole margins surrounding the anterior half of the neuroectoderm and the expression level slightly increases (Plate
-2). After the onset of segmentation, sox9b transcripts
are detected in the cranial neural crest precursors located
in the lateral margins of the prospective midbrain and
hindbrain in the control embryo (Plate
-3, 5, 7). The
diencephalon starts to express sox9b in the lateral domains
at the 10-somite stage (Plate
-9). In the RA-treated
embryos during early segmentation, sox9b- positive cells,
which show a higher expression level than the control,
form a continuous domain around the margins of the remaining head. This indicates that RA treatment induces
sox9b expression in the forebrain, since the forebrain is
not lost by RA treatment. It is likely that RA treatment
promotes neuronal differentiation in the forebrain by
changing the normal fate of cells to a cranial neural crest
fate. The control embryos have sox9b expression in nonneuronal cells of telencephalon and hindbrain at 18-somite
stage through 24 h postfertilization (Plate - 11, 13),
whereas the treated embryos lack or have very weak expression (Plate
-12, 14). This implies that stimulation
of neural crest cells by RA occurs at the expense of cells
with other fates.
Pectoral fins of fish resemble forelimbs of tetrapods
and their bones are derived from cranial neural crest[17].
On day 3, sox9b is expressed in the pectoral fin buds
(Plate -17). In the treated embryos, the stained pectoral
fin buds are elongated along the anterior-posterior axis
(Plate -18), suggesting that RA treatment induces duplication of the pectoral fin buds.
Crestin is a marker expressed in premigratory and
migratory neural crest cells[13]. Its expression is initiated in
the lateral domains of the posterior hindbrain at 6-somite
stage and thereafter extends posterior to the dorsal midline
of the neural keel (fig. 1(c)). RA treatment results in the
expression of crestin in the margins of the whole remaining head that should includes the forebrain and rostral
midbrain, and the increase of expression level in the hindbrain (fig. 1(b), (d), (f), (i)). This further suggests that RA
can induce and promote cranial neural crest fate.
One of cranial neural crest derivatives is pigment.
We note that RA-treated embryos on day 2 have many
more pigment cells in the head skin (fig. 1(j) (m)). This
indicates that at least some of the RA-induced neural crest
cells in the head have differentiated into pigment-synthesizing melanocytes.
( ) RA induces caudal posterior trunk neural crest
cells. Based on the expressions of sox9b and crestin, it
appears that the exposure to RA has little impact on the
formation of rostral trunk neural crest cells (Plate
-3
10, fig. 1(a) (i)). However, the movement of the expression domains of sox9b and crestin in the trunk region
shows delay of closure of the neural keel. For example,
the two lateral domains of sox9b are almost completely
merged at the dorsal midline in 10-somite control embryos
(Plate -9), whereas this happens in the RA-treated emChinese Science Bulletin Vol. 47 No. 13 July 2002
bryos around the 12 14 somite stage, about 30 60 min
later (Plate -10).
RA treatment affects the development of neural crest
in the posterior trunk. At the onset of segmentation, sox9b
is not expressed in the marginal cells of the caudal neuroectoderm (Plate
-3, 5). The RA treatment induces
sox9b expression in these cells, resulting in a U-shaped
band surrounding the caudal neuroectoderm (Plate
-6).
The tailbud of the RA-treated embryos detaches from yolk
and undergoes eversion prematurely (Plate
-4), and
subsequently develops a shorter tail, which may be related
to change in fate of the marginal cells in the caudal neuroectoderm. Kanki and Ho [18] demonstrate that the tailbud
during normal development forms by mingling the dorsaland ventral-derived cells and the ventral-derived cells will
give rise to paraxial mesoderm derivatives during the tail
extension. We propose that the early specification of the
dorsal marginal cells to the neural crest fate in the RAtreated embryos prevents the mingling of ventral-derived
and dorsal derived cells and hence affects the tail development.
During the late segmentation period, the tail of all
RA-treated embryos starts to twist, probably at the position for the normal joining of the ventral-derived and
dorsal derived cells. In that particular region the expression of both sox9b and crestin occurs ventrolaterally and
-11 16). The cells in the twisting
is enhanced (Plate
region die during pharyngula period and many caudal finlike structures appear at the tail tip (picture not shown).
Since caudal fin is derived from trunk neural crest[19], the
neural crest cells in the marigin of the caudal neuroectoderm, induced by RA during early segmentation, should
largely differentiate into fin ectomesenchymal precursors
but not melanocytes.
In conclusion, the exposure of the gastrula embryos
to excessive amount of RA induces cranial and caudal
trunk neural crest cells. This induction may be partly mediated by sox9b and crestin.
Acknowledgements We thank Marnie Halpern for crestin cDNA. This
work was supported by the National Natural Science Foundation of
China (Grant Nos. 30025020 and 39970360) and TRAPOYT of the
MOE.
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cis-acting element located in
the bovine foamy virus
internal promoter possesses
the properties of a transcriptional enhancer
QIAO Wentao, GUO Chunguang, WANG Shuhui,
WANG Jinzhong, CHEN Qimin & GENG Yunqi
College of Life Sciences, Nankai University, Tianjin 300071, China
Correspondence should be addressed to Qiao Wentao (e-mail:
[email protected])
Abstract Bovine foamy virus encodes a transcriptional
transactivitor, Tas or Borf-1, which governs the level of viral
transcripts initiated by both the promoter in the long terminal repeat (LTR) and the internal promoter (IP) located in
the env gene through their cis-acting targets. We have identified and characterized a 72 bp Tas (Borf-1) responsive element located in BFV3026 internal promoter (TRE IP) by deletion mutant and transient expression assay. This cis-acting
target element in the internal promoter has the properties of
a transcriptional enhancer which functions independently of
its orientation, position and also in heterologous promoters
(BFV LTR and bovine immunodeficiency virus, BIV LTR).
Alignments reveal that there are positional similarity and
sequence homology among BFV TRE IP, SFV-1 TRE IP proximal element and SFV-3 TRE IPII, which suggests that this
kind of cis-acting elements possesses some common functional character.
Keywords: bovine foamy virus (BFV), internal promoter (IP), Tas
responsive element (TRE), enhancer.
Foamy viruses (FVs), a member of the Spumavirnae
of Retroviridae, possess a complex genome organization
as well as complex means of gene expression regulation.
The transcription of its genes is dependent of two distinct
promoter elements, the long terminal repeat (LTR) pr omoter and the newly discovered internal promoter (IP)
located towards the 3 end of the env genes. LTR
regulates the expression of the viral structural genes,
gag, pol and env for virion proteins, while IP directs the
expression of the viral auxiliary proteins [1 3]. One of these auxiliary proteins is a potent transcriptional transacti
vator, termed Tas (Borf-1 in bovine foamy virus), which is
critical of foamy virus replication. The existence of two
kinds of promoters induces that foamy viruses utilize a
gene regu- lation mechanism seen in complex DNA viruses but not other retroviruses: multiple promoters[4].
Recent work indicates that Tas is a DNA binding protein,
which transactivates both the IP and the LTR promoter,
(Received March 11, 2002)
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Chinese Science Bulletin Vol. 47 No. 13 July 2002