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Developmental Biology
The zebrafish down syndrome cell adhesion molecule is involved in
cell movement during embryogenesis
Dean Yimlamai, Liza Konnikova, Larry G. Moss, Daniel G. Jay*
Cellular and Molecular Physiology, Tufts University School of Medicine, 136 Harrison Avenue, M and V 709, Boston, MA 02111, USA
Received for publication 8 June 2004, revised 30 November 2004, accepted 1 December 2004
Available online 20 December 2004
Abstract
The Down syndrome cell adhesion molecule (Dscam) is a protein overexpressed in the brains of Down syndrome patients and
implicated in mental retardation. Dscam is involved in axon guidance and branching in Drosophila, but cellular roles in vertebrates
have yet to be elucidated. To understand its role in vertebrate development, we cloned the zebrafish homolog of Dscam and showed
that it shares high amino acid identity and structure with the mammalian homologs. Zebrafish dscam is highly expressed in developing
neurons, similar to what has been described in Drosophila and mouse. When dscam expression is diminished by morpholino injection,
embryos display few neurons and their axons do not enter stereotyped pathways. Zebrafish dscam is also present at early embryonic
stages including blastulation and gastrulation. Its loss results in early morphogenetic defects. dscam knockdown results in impaired cell
movement during epiboly as well as in subsequent stages. We propose that migrating cells utilize dscam to remodel the developing
embryo.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Danio rerio; Zebrafish; Down syndrome; Dscam; Cell movement; Epiboly; Gastrulation
Introduction
Down syndrome (DS) is the most common form of
genetic birth defect, occurring in approximately one in
every thousand live births. This syndrome is defined by
mental retardation and a set of phenotypic features that is
caused by a trisomy of Chromosome 21 (Lejeune et al.,
1959). Due to this aneuploidy, multiple genes are involved
and it is presumed that overexpression of one or more of
these genes is the cause of the phenotypes seen in DS.
Over three hundred genes have been identified on
Chromosome 21 through genomic sequencing, but it is a
significant challenge to narrow the list of candidate genes
that are involved in DS. One gene that has been linked to
0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2004.12.001
Abbreviations: Dscam, Down syndrome cell adhesion molecule; DS,
Down syndrome; hpf, hours post fertilization; IgCAM, immunoglobulin
cell adhesion molecule; CE, convergent-extension; epi, epiboly.
* Corresponding author. Fax: +1 617 636 0445.
E-mail address: daniel.jay@tufts.edu (D.G. Jay).
DS is the Down syndrome cell adhesion molecule
(Dscam).
Dscam was identified through a positional cloning
strategy for common Down syndrome (DS) phenotypes
(Yamakawa et al., 1998). Dscam is highly expressed
during brain development and is expressed at 20% higher
levels in DS subjects (Saito et al., 2000). Functional
experiments to define the role that Dscam plays in
development have primarily been undertaken in Droso-
phila. Drosophila dscam is also highly expressed in
embryonic and adult CNS. Dscam mutants show disrupted
development of neuronal tracts and perturbed axon
guidance (Hummel et al., 2003; Schmucker et al., 2000;
Wang et al., 2002, 2004; Wojtowicz et al., 2004; Zhan
et al., 2004).
Of particular interest is the finding that Drosophila
Dscam exhibits alternative splicing at multiple sites during
development resulting in over 36,000 potential splice
variants (Schmucker et al., 2000). Expression of these
Dscam variants appears to be regulated in a stage and tissue
279 (2005) 44–57
D. Yimlamai et al. / Developmental Biology 279 (2005) 44–57 45
specific fashion (Celotto and Graveley, 2001; Neves et al.,
2004) with individual cells expressing distinct subsets of
these Dscam isoforms (Neves et al., 2004). Distinct
combinations of Dscam isoforms may act as molecular
labels differentiating cells within a local environment.
Dscams interact with each other through homophilic
adhesion (Agarwala et al., 2000, 2001c; Wojtowicz et al.,
2004), a property that is particularly specific in Drosophila.
Drosophila Dscam isoforms that are identical throughout
their ectodomain, but differ at one exon where identity is
shared up to 90% do not bind in vitro (Wojtowicz et al.,
2004).
In humans and rodents, there are two Dscam paralogs,
Dscam and Dscam-L1 (Agarwala et al., 2001c; Barlow et
al., 2002), neither of which have been described to display
large numbers of alternative splice variants. The two
mammalian Dscams are expressed in complementary
patterns in the CNS (Barlow et al., 2002) and their products
can be found on axons and dendrites (Agarwala et al.,
2001b,c). In the growing embryo, cells in vivo may
segregate or make other preferred cellular contacts depend-
ing on the type of Dscam that is expressed. DS brains show
developmental defects including delayed neuronal lamina-
tion and disorganized arborization (Golden and Hyman,
1994). The nature of these defects suggests that Dscam has
a role in development of cortical malformations, but thus
far cellular mechanisms for vertebrate Dscam are not
known.
The zebrafish (Danio rerio) is an excellent model
system for understanding vertebrate development. The
optical clarity of zebrafish facilitates detailed microscopic
analysis of their embryos. Embryonic development pro-
ceeds rapidly after fertilization with the first cell division
happening within the first hour, and subsequent divisions
proceeding at an increasingly rapid pace. Within 24 h,
zebrafish possess a rudimentary nervous system that is
beginning to form functional connections. In addition,
advances in forward and reverse genetics have made the
zebrafish a powerful system for understanding the biology
of new molecules.
To better understand Dscam’s role in vertebrate devel-
opment, we cloned the zebrafish homolog. We show that
dscam is present in developing neurons and is expressed
throughout the embryo during early embryogenesis. Loss
of dscam through morpholino knockdown results in a
severe morphologic disruption of the embryo. Treated
embryos show fewer neurons and their axons do not enter
stereotyped pathways, but this phenotype is also associated
with growth defects. These results suggest that dscam
plays an unexpected role earlier in development. We show
that dscam is broadly expressed in the early embryo and
its loss results in impaired morphogenetic movements
during epiboly and gastrulation. At the end of gastrulation,
Dscam knockdown embryos are significantly shorter than
controls and exhibit disorganized mesoderm and prechor-
dal plate. Here we describe a new developmental role for
dscam: dscam facilitates cell migrations that shape the
growing embryo.
Materials and methods
Fish
Wild-type Tqbingen AB zebrafish embryos were col-
lected from our breeding colony and maintained according
to standard procedures. Embryos were maintained at 28.58Cand staged in hours post fertilization (hpf) and by
morphology (Westerfield, 1995).
Cloning and analysis of Dscam orthologs
3V RACE primers were designed against the aligned
human, mouse, and Drosophila Dscam sequences. cDNA
pools were generated from total embryonic RNA from
48 hpf zebrafish using the SMART kit (BD Clontech)
according to the manufacturer’s directions. A 3V 4.7 kb
RACE product was isolated with the following primer, 5V-GGGAACGTGGTCAGCTACCTGAA-3V and the 3VSMART kit primer. Nested 5VRACE to generate the full-
length gene was performed on the same pools with the
following primers used in succession, 5V-CCGCCGAGT-TATTGCATGTGCAG-3V and 5V-CCTTCTGCACGTT-
CAGCAGCTTCAG-3Vwith the SMART kit primers.
DNA and protein sequences were aligned and analyzed
using tools available at the Biologist’s Workbench (http://
workbench.sdsc.edu). ClustalW alignments were prepared
to assess the identity of the cloned sequence against
previously identified homologs. Domain analysis was
performed using HMMPFAM. Rooted phylogenetic trees
were generated using DRAWGRAM. Amino acid sequence
annotation was produced using The Eukaryotic Linear Motif
Resource (http://elm.eu.org).
RT-PCR analysis
RT-PCR was performed on total RNA extracted from
embryos at the desired time point using Trizol Reagent
(Invitrogen, Carlsbad, CA) according to the manufacturer’s
directions. RNA (1 Ag) was subsequently treated with
DNase I (Amplification Grade, Invitrogen) and 1st strand
cDNA synthesized with Superscript III (Invitrogen). A
200 bp dscam fragment was amplified with the following
primers, DSRT1L (5V-AGGAGCTGTTCACGAGCATT-3V)and DSRT1R (5V-TCATCTGCAGCT CGTACCAC-3V).Primers for the transcription factor MAX were used as
control (Schreiber-Agus et al., 1993) on the same samples.
Whole-mount in situ hybridization (WISH)
Embryos were grown to the desired age, fixed over-
night in 4% PFA in PBS, and manually dechorionated.
D. Yimlamai et al. / Developmental Biology 279 (2005) 44–5746
Embryos were stored in 100% methanol until use.
Digoxigenin-labeled sense and anti-sense riboprobes were
synthesized from a 1.2 kb dscam fragment cloned into the
EcoRI/HindIII sites of Bluescript SK+ with the following
primers, DC95L (5V-AGTCAGAATTCCGAGGAGAGCTQACAACGTCC) and DC95R (5V-ATATGAAGCTTCTQ
D. Yimlamai et al. / Developmental Biology 279 (2005) 44–57 47
CTGCCGTTATGCTGTGAA-3V). Hybridization and detec-
tion on whole-mount zebrafish embryos were performed
as described by Broadbent and Read (1999). Wnt11
(cb748), Bmp2b (cb670), Six3b (cb347) were obtained
from the Zebrafish Information Resource Center. A
Dharma probe was generated by RT-PCR with the
following primers, DhaL (5V-AGCAGGCAAACAGCA-GAATC-3V) and DhaR (5V-TCCTCCAGAGCTCGAT-
TAGC-3V). The PCR product was cloned into pCRII for
use. Squint (Feldman et al., 1998), floating head (Talbot et
al., 1995), and axial/foxA2 (Strahle et al., 1993) were
used as previously described. Images were taken on a
Leica MZLIII dissecting microscope (Leica Microsystems,
Bannockburn, IL) equipped with a Hamamatsu CC4742-
95 camera (Hamamatsu Corp., Bridgewater, NJ) with a
Varispec RGB color slider (CRI Inc, Woburn, MA).
Images were recorded on a Macintosh with Openlab
software (Improvision, Lexington, MA).
Dscam antibody production and use
We cloned a 3.5 kb extracellular portion of Dscam into
pFastBAC (Invitrogen) with a 6xHis tag. This vector was
used to synthesize baculovirus that was subsequently used
to infect HiFive cells for antigen production. Secreted
Dscam was collected from the media and purified using Ni-
NTA Agarose (Qiagen, Valencia, CA) according to the
manufacturer’s directions. Mice were immunized with this
antigen using a standard protocol. Eventually one mouse
was sacrificed for hybridoma fusion and screened for
positives by ELISA and immunocytochemistry. The ascites
produced from this is DS#1 (produced by Tufts GRASP).
The monoclonal antibody used in this study is DS1-11.
Western blots were performed with DS#1 (1:1000) and
DS1-11 (1:100).
Whole-mount immunohistochemistry
Embryos were blocked for 1 h at RT in 20% FBS in PBS.
Antibody incubations were done overnight at 48C in
blocking buffer. Five 1 h washes were performed with
PBS-T before incubation with secondary antibody. Anti-
mouse Alexa 488 was purchased from Molecular Probes
(Invitrogen), anti-mouse HRP was purchased from Dako-
Cytomation (Carpinteria, CA) and diluted according to the
manufacturer’s directions. Peroxidase substrate kit (Vector
Laboratories, Burlingame, CA) was used to develop HRP
Fig. 1. Sequence analysis of Dscams. (A) Amino acid sequence of zebrafish dsc
sequence. Black boxed characters represent hydrophobic sequence. Gray boxe
domains. Intracellular signaling motifs are indicated by underlined sequence and
fibronectin domain; SP, signal peptide; TM, transmembrane domain; WW, WW
binding motif; PDZ, PDZ class I binding motif. (B) Amino acid sequence identit
immunoglobulin domains, shaded rectangles represent fibronectin domains, and
analysis of Dscam family members. Horizontal length is proportional to estimated t
m, mouse; r, rat; zf, zebrafish.
staining according to the manufacturer’s directions. The
following primary antibody concentrations were used: anti-
acetylated tubulin (Sigma, clone 6-11B-1, 1:1000), anti-
DS1-11 (1:100). Confocal images were taken with a Leica
SP2 (Leica Microsystems).
Morpholino injection
Morpholinos were purchased from Gene Tools, LLC
(Philomath, OR). Three morpholinos were designed, DS2M
(5V-CGCTCCTTTCAATCTCCAAACTAAG-3V), against
the 5V UTR of Dscam (�29 to �5), DS2Mmis (5V-CGATCATTTCAAGCTCCAATCTCAG-3V), a correspond-
ing five base mismatch oligo, and DS3M (5V-AAAAGAT-GATGGCCAATATCCACAT-3V), an oligo overlapping with
the ATG start site (+1 to +25). Morpholinos were
resuspended in 1� Danieau Buffer with 0.1% phenol red
and injected into the yolk of one to two cell stage embryos.
10 pl of morpholino was injected using an Eppendorf 5242
Microinjector. Concentrations from 1 to 40 ng/embryos
were tested and assessed for non-specific phenotypes
appearing between DS2M and DS2Mmis oligos.
Labeling cells of early embryos
One or two cell embryos were injected with either a
solution of 0.025% DMNB-FITC dextran (10 kDa, Molec-
ular Probes) or a similar solution also containing DS2M
morpholino in 1� Danieau Buffer. Embryos were allowed
to develop in the dark until the high cell stage. These
embryos were then mounted in 1.2% agarose viewing
chambers and uncaging was performed by exposing a
central area of the embryo to UV light through a DAPI filter
set for 10 s. Embryos were briefly imaged to record the spot
size using a FITC filter set on a Nikon Diaphot 200 and
returned to the incubator to continue development. Dispersal
of the spot was recorded 2 h later for each treated embryo.
Results
Cloning a zebrafish Dscam homolog
To address the role of Dscam in vertebrate development,
we cloned a zebrafish homolog by RT-PCR. We performed
3V and 5V RACE using degenerate primers recognizing
conserved sequences in the human, mouse, and Drosophila
am. Amino acid motifs predicted by ELM are indicated above the shaded
s represent immunoglobulin domains. White boxes represent fibronectin
are conserved in all vertebrate dscams. Ig, immunoglobulin domain; FN
class IV binding motif; T2, Traf2 binding motif; SH2/3, src homology 2/3
y between zebrafish dscam and human Dscam by domain. Ovals represen
the dark rectangle represents a transmembrane domain. (C) Evolutionary
ime from divergence of the gene from the related family member; h, human
,
t
;
D. Yimlamai et al. / Developmental Biology 279 (2005) 44–5748
sequences using the SMART kit. A 6.6 kb cDNA encoding
a 2024 amino acid protein was isolated (Fig. 1A). Several
previously isolated partial ESTs confirm the sequence of this
gene (AL928362, AL919997, CA496783). Zebrafish dscam
contains ten immunoglobulin and six fibronectin domains in
the same order and sequence as other family members. The
amino acid sequence is highly conserved with the human
gene with 59–94% amino acid identity by domain (Fig. 1B).
The cytoplasmic domain of dscam is highly related to
other vertebrate family members. It is predicted to be 401
amino acids in length with several types of signaling motifs
including three WW Class IV binding domains, two Traf2
binding sites, an SH2 and SH3 binding site, and a PDZ
binding domain at the C terminus. Each of these motifs was
highly conserved from human through zebrafish and
suggests likely functionality (Fig. 1A). The more distantly
related gene, Dscam-L1, has conserved several of these
motifs including the WW binding domains, an SH2 binding
site, and the PDZ recognition motif. Common cytoplasmic
molecules probably bind to these sites, although, Dscam and
Dscam-L1 also contains several types of non-overlapping
signal sequences likely conferring specificity.
It has been reported that Drosophila dscam is a gene
product that displays significant diversity through alter-
native exon splicing (Schmucker et al., 2000), although
neither vertebrate Dscam paralogs have been described to
display a significant number of splice variants. It is of
particular note that Drosophila contains four Dscams, only
one of which displays an extensive number of splice
variants (Dscam1). We closely examined our dscam gene
product to determine if extensive alternative splicing could
exist. The cDNAwas constructed from 4.7 kb, 3Vand 2.1 kb,5V overlapping RACE products. We sequenced several of
these isolated clones in order to determine if there were any
significant splice variants. We did not identify any
alternative splicing in our clones, although PCR or stage-
specific expression may have biased the dscam isoform that
was cloned. Of the two Dscams isolated from other
vertebrate species, the cDNAwe cloned shows significantly
more homology to Dscam than to Dscam-L1 (Fig. 1C).
Zebrafish dscam is expressed in regions of developing
neurons
We examined dscam expression during embryogenesis
by whole-mount in situ hybridization (WISH) analysis using
a dscam probe in wild-type embryos every 2 h following
fertilization. Discrete dscam expression appeared at approx-
imately 22 hpf. We detected strong expression from brain
regions including the midbrain, telencephalon, and dien-
cephalon (Fig. 2E). By 24 hpf, dscam expression spreads
widely throughout the CNS into the hindbrain and spinal
cord (Fig. 2F). At this stage, neuronal precursors in these
regions of the CNS are differentiating and beginning the
process of extending axons and dendrites (Chitnis and
Kuwada, 1990; Wilson et al., 1990). These results agree
with previous work demonstrating that dscam is highly
expressed in regions of developing mouse neurons (Agar-
wala et al., 2001a; Barlow et al., 2002).
dscam expression is maintained in the brain through at
least 5 dpf (Figs. 2I and J), while in the spinal cord
expression is greatly diminished between 2 and 5 dpf (Figs.
2G and I). As the neuropil segregates into more discrete
structures, dscam is localized to several synaptic layers of
the eye (Fig. 2H) as well as brain (Fig. 2J). The dscam
expression suggests that it plays a role in axon tract
development and maintenance.
Dscam knockdown results in growth and axon defects
We investigated the in vivo role of dscam by micro-
injecting morpholino oligos to interfere with expression of
dscam (Nasevicius and Ekker, 2000). Oligos against the 5VUTR (DS2M) and overlapping the ATG start site (DS3M)
were targeted with this strategy. We initially examined
embryos at 28 hpf and found that they exhibited marked
morphological defects generally characterized by overall
shortening of the embryo and disorganization of many
tissues. These embryos were sorted by a phenotypic grading
system that we developed: G0-wild-type appearing; G1-
mild defects such as small brain and/or crooked tail; G2-
moderate defects including moderate anterior–posterior
(AP) axis shortening and/or extremely small eyes and brain;
and G3-severe defect including severe AP axis shortening
and/or complete lack of anterior structures (Figs. 4A–D).
Both morpholinos exhibited similar defects and proportions
(data not shown and Table 1, respectively) that were
indistinguishable, suggesting that they targeted the same
gene. The DS2M oligo was used in the rest of this study as
the 5VUTR is less likely to share sequence conservation
with other genes.
Due to reports in Drosophila that perturbation of dscam
results in disruption of axonal tract development, we
examined early axonal tracts of the brain. It has been
previously described that by 30 hpf, several axon tracts
connecting the two hemispheres as well as various portions
of the brain are well developed and observable with
neuronal markers (Chitnis and Kuwada, 1990; Wilson et
al., 1990). We used an antibody against anti-acetylated
tubulin to label neurons and their processes. DS2M embryos
with a wild-type morphology (G0) displayed normal axon
tracts (Fig. 5A). All of the major tracts were present
including the supraoptic tract (SOT), tract of the posterior
commissure (TPC), and the tract of the post-optic commis-
sure (TPOC). The anterior and post-optic commissures were
also well developed (data not shown). Axon guidance errors
were not observed and these embryos appear equivalent to
non-injected embryos (data not shown). Embryos displaying
mild growth defects (G1) had visibly smaller brains,
displayed few neurons and disorganized axons (Fig. 5B).
Tracts and commissures were not discernable from these
embryos, and the few axons projecting from neuronal
Fig. 2. Expression of zebrafish dscam during early embryogenesis. (A) RT-PCR of dscam at various developmental stages within the first 24 h. Max is used as
loading control. Whole mount in situ hybridization of wild-type embryos at (B) 1 cell, (C) high, (D) 8 somite, (E) 22 hpf, (F) 24 hpf, (G) 2 dpf, (H) high
magnification of 3 dpf eye, (I) 5 dpf, and (J) anterior–dorsal view at 5 dpf. B–D show expression throughout the early embryo. Arrowhead in D indicates the
presumptive eye field. (E) 22 hpf embryo shows initial dscam discrete expression within midbrain, diencephalon, and telencephalon. (F) 24 hpf embryo shows
expanded dscam expression and new expression in hindbrain and spinal cord. (G) 2 dpf embryo shows expanding brain and spinal cord expression. (H)
Multiple layers of the laminated eye show strong dscam expression, particularly IPL and INL. (I) 5 dpf embryo shows expression only in the brain. (J) High
magnification dorsal view of the brain shows dscam expression is expressed discretely throughout. PE, pigmented epithelium; PRL, photoreceptor layer; OPL,
outer plexiform layer; INL, inner nerve layer; IPL, inner plexiform layer; GCL, ganglion cell layer; Ep, epiphysis; DC, diencephalon; HB, hindbrain; MB,
midbrain; TC, telencephalon; OSE, olfactory sensory epithelium.
D. Yimlamai et al. / Developmental Biology 279 (2005) 44–57 49
regions did not appear to project in the direction of known
tracts or commissures. The axons that were present appeared
to take tortuous courses through the brain. Moderate to
severe phenotypes showed no neurons by anti-acetylated
tubulin staining (data not shown).
The spectrum of dscam phenotypes is likely due to
differential uptake of the morpholino resulting in various
levels of dscam expression. We developed polyclonal and
monoclonal antibodies against dscam and confirmed their
specificity by Western blotting and immunohistochemistry
on dscam-transfected Cos-7 cells (Fig. 3A and data not
shown). Western blots of microinjected zebrafish embryos
show variable amounts of knockdown as compared to
controls (Fig. 4A). Although the axonal tract defects
observed in Grade 1 embryos may reflect a role that dscam
plays in axon guidance, it is also possible that generalized
Table 1
Zebrafish dscam knockdown results in growth defects
dscam ng/embryo Phenotypic grade Total
0 1 2 3
DS2Mmis 10 68
(100%)
68
DS2Mmis 20 65
(58%)
48
(42%)
113
DS2M 10 112
(49%)
77
(34%)
28
(12%)
11
(5%)
228
DS2M 20 33
(22%)
26
(17%)
62
(42%)
28
(19%)
149
DS3M 10 61
(41%)
48
(32%)
32
(21%)
8
(5%)
149
DS3M 20 39
(26%)
43
(29%)
28
(19%)
40
(27%)
150
Equivalent amounts of DS2M, DS3M, or DS2Mmis morpholinos were
injected into 1 to 2 cell embryos as described in Materials and methods and
assessed for growth defects at 24 hpf (see Fig. 4).
D. Yimlamai et al. / Developmental Biology 279 (2005) 44–5750
growth defects cause axons to take different paths to their
targets because the local environment is significantly
different from the wild-type scenario. Also, because Grade
2 and 3 embryos show no observable neurons and they
displayed severe growth defects, these results suggested that
dscam may play an earlier developmental role to pattern the
embryo.
Dscam is a gene expressed throughout early development
We performed RT-PCR for dscam from embryo extracts
at various stages of embryonic development to determine if
dscam is present at embryonic stages that were not initially
detected by WISH. We were able to detect dscam at all
Fig. 3. dscam protein is expressed early in development. (A) Immunoblot of an
expressing dscam. Arrowhead indicates dscam at 220 kDa. (B) Secondary antibody
antibody alone, 90% epiboly. (E) a-dscam monoclonal, 90% epiboly.
embryonic stages examined (Fig. 2A). dscam is a mater-
nally expressed transcript that diminishes until near the
mid-blastula transition when zygotic transcription begins.
dscam is present throughout several developmental stages
including blastulation, gastrulation, neurulation, somito-
genesis, and organogenesis. Its presence suggests that
dscam could participate in development at any of these
stages. We repeated dscam WISH at stages earlier than 22
hpf and found staining could be detected throughout the
embryo at 1 cell (Fig. 2B), high (Fig. 2C), and 8 somite
(Fig. 2D) stages.
We used dscam monoclonal antibodies to determine if
the dscam protein is localized to particular cell populations
in the early embryo. Immunostaining of early embryos
showed strong staining at the 32 cell stage as well as at 90%
epiboly (Figs. 3B–E). These results suggest that dscam
could be utilized at the earliest stages of cell division. dscam
expression is broadly expressed superficially at 90% epiboly
(Fig. 3E) with diffuse staining appearing throughout
sectioned embryos (data not shown). These data suggest it
may play a role in multiple cell types. Since dscam is
present during epiboly, we asked if its knockdown inhibits
developmental mechanisms at this time.
dscam knockdown results in impaired cell movement during
epiboly
During epiboly and gastrulation, developmental pro-
grams regulating cell fate and movement cooperate in
proper embryo formation (Heisenberg and Tada, 2002;
Keller et al., 2003; Schier, 2001; Wallingford et al., 2002). If
dscam plays a role at this time, embryos should display
ti-dscam polyclonal and monoclonal antibodies against Cos-7 cell lysates
alone, 32 cell stage. (C) a-dscam monoclonal, 32 cell stage. (D) Secondary
Fig. 4. Zebrafish dscam knockdown results in growth defects. (A) Grade 0 (G0), wild-type appearance. (B) Grade 1 (G1), mild growth defects. (C) Grade 2
(G2), moderate defects. (D) Grade 3 (G3), severe defects. (E) Western blot analysis of individual zebrafish injected with DS2Mmis (10 ng) or DS2M (10 ng).
Arrowhead indicates dscam expression at 220 kDa.
D. Yimlamai et al. / Developmental Biology 279 (2005) 44–57 51
observable differences. We examined control and dscam
morphants at the completion of gastrulation and noted
significant shortening of the anterior–posterior axis (Figs.
5C and D). Gastrulation begins at approximately 50%
epiboly, when the dorsal shield appears displaying the first
morphologic sign of anterior–posterior polarity. We fol-
lowed control and dscam morphants from 50% epiboly
through the tailbud stage to determine if there were
observable morphologic differences.
Control and dscam morphants demonstrated several
differences at 50% epiboly: dscam morphants do not form
a prominent dorsal shield, their blastoderm is significantly
thickened, and the blastoderm of dscam morphants appears
to be bilayered (Fig. 6). This phenotype continues to worsen
through gastrulation. At 60% epiboly, DS2M embryos have
an excess number of cells at the animal pole with respect to
controls. Also, appearance of the polster is not obvious until
much later (see arrow, Fig. 6). The polster although visible
in DS2M embryos at 85% epiboly is extremely thickened,
possibly due to a lack of cell migration from the animal pole
where the polster is visible. By the completion of
gastrulation, DS2M embryos are shorter, thinner, and have
an excess number of cells near the anterior margin of the
embryo.
Fig. 5. dscam morphants show axon tract defects. (A) G0 embryo shows normal axon tracts. (B) G1 embryo shows few neurons and disorganized axons. Ep,
epiphysis; DC, diencephalon; SOT, supraoptic tract; TC, telencephalon; TPOC, tract of the post-optic commissure; TPC, tract of the posterior commissure. (C)
Control and (D) dscam knockdown embryos at tailbud stage. Arrowhead represents tailbud. Arrow represents leading edge of the polster.
D. Yimlamai et al. / Developmental Biology 279 (2005) 44–5752
Previous work suggested that dscam is a molecule
involved in cell adhesion and/or guidance, but the morpho-
logic deficits observed could be due to dscam playing a role
in cellular differentiation. dscam knockdown at early stages
may lead to loss or expansion of tissues resulting in
morphologic disruption. We used marker genes in situ to
determine if dscam knockdown disrupts the presence or
organization of early cell populations.
Patterning of the zebrafish embryo is governed by
expression of maternally derived genes that convey posi-
tional information. Squint (sqt) is initially expressed along
the yolk syncytial layer and induces ventral cells into
endoderm and mesoderm (Feldman et al., 1998). This gene
Fig. 6. Time lapse images of control and dscam morphants during gastrulation. Ima
represents the tailbud. Arrow represents leading edge of the polster.
was present in control and knockdown embryos, but in
knockdown embryos it appeared more diffusely distributed
along the border of the yolk syncytial layer at 30% epiboly
(Fig. 7C). Cells expressing the signaling molecule Wnt11
(Heisenberg et al., 2000) demonstrated a similar diffuse
pattern of expression. These results appear to suggest that
cellular induction in Dscam morphants occurs over longer
distances or cells are unable to reorganize themselves
properly. We observed significant differences in the
expression of other signaling molecules as well. At 50%
epiboly, Bmp2b appeared to be significantly diminished in
the area of the shield (Fig. 7D), while the Bmp antagonist
Dharma (Fig. 7B) (Fekany et al., 1999) was absent in
ges are of a single embryo photographed at the indicated stages. Arrowhead
Fig. 7. Marker gene in situ of dscam knockdown embryos. (A and C) Animal pole view. (B and D) Lateral view, dorsal is right, animal pole is up. (E and F)
Dorsal view, tailbud is at the bottom. (G, H, and J) Lateral view, tailbud is at the bottom. (I) Rostral view. (A and C) Wnt11 and Squint/Nodal at 30% epiboly,
respectively. (B and D) Dharma and Bmp2b at 50% epiboly, respectively. (E and F) Axial and floating head at 90% epiboly, respectively. (E and F) Axial and
floating head at 1 somite, respectively. (I and J) Six3b at 8 somites. All controls show representative samples. DS2M injections show severe phenotype in at
least five embryos in samples of twenty.
D. Yimlamai et al. / Developmental Biology 279 (2005) 44–57 53
Dscam morphants implying a requirement for Dscam in
normal induction of these molecules.
As gastrulation begins (50% epiboly), cells of the
developing zebrafish embryo participate in a coordinated
set of movements termed convergent-extension (CE) (Hei-
senberg and Tada, 2002; Wallingford et al., 2002). We used
the pattern of in situ markers for particular cell populations
to assess the overall development of the embryo and if CE
movements are occurring normally. The mesoderm markers
axial/foxA2 (axl, Strahle et al., 1993) and floating head (flh,
Talbot et al., 1995) demonstrate that dscam knockdown
results in less mesoderm migration to the midline at 90%
epiboly, producing staining patterns that are wider than
controls (Figs. 7E and F, respectively). These embryos are
also significantly shorter than controls and the phenotype
progressively worsens through somitogenesis (Figs. 7G and
H). The effects of dscam knockdown are not limited to
mesoderm. A marker of the prechordal plate, six3b (Seo et
al., 1998), shows that dscam knockdown does not inhibit its
specification. However, similar to the effects seen in
mesoderm, neural plate formation is more disorganized
than controls and is much thinner (Figs. 7I and J).
In order to directly address if Dscam may be an important
component of cell movement during epiboly, we labeled
small groups of cells centrally at the high cell stage in
control and Dscam morphant embryos (Figs. 8A and B).
Fig. 8. Dscam is required for cell dispersal during epiboly. (A) Wild-type embryo at high cell stage showing bright spot of FITC-dextran labeled cells after
uncaging. (B) DS2M injected embryo at high cell stage showing a similar size spot of FITC-dextran labeled cells. (C) Awild-type embryo imaged after 2 h of
development illustrating how cells within the original spot widely disperse across the embryo. This embryo is an example of Grade IV dispersal. (D) A DS2M
embryo imaged 2 h after uncaging shows most cells contained within the original spot. Limited migration from the spot has occurred as the border of the
original spot is irregular. This is an example of Grade I dispersal. (E) Microinjection of DS2M (n = 36) shifts population of embryos exhibiting less dispersal of
cells within the uncaged spot toward Grade I as compared to wild-type embryos (n = 28). This shift is statistically significant by v2, P = 0.01. Grade I, uncaged
spot is condensed with slight cell migration. Grade II, spot is slightly dispersed. Grade III, spot is moderately dispersed. Grade IV, spot is widely dispersed.
D. Yimlamai et al. / Developmental Biology 279 (2005) 44–5754
This method labeled both superficial and deep cells of the
early embryo. Two hours later, we examined the placement
of these cells out of the initial spot to determine if significant
cell movement had occurred. As epiboly begins following
the high cell stage, cells begin to migrate to the periphery
through radial cell intercalation (Warga and Kimmel, 1990).
The result of this behavior is that cells in close proximity at
the beginning of epiboly become uniformly distributed
throughout the embryo. An example of normal cellular
behavior can be seen in Fig. 8C where cells of the original
D. Yimlamai et al. / Developmental Biology 279 (2005) 44–57 55
spot have disseminated across the embryo. Labeled cells in
Dscam morphants after 2 h showed much less dispersal
than controls (Fig. 8D) with a significant shift in the
population of embryos displaying less migration (Fig. 8E,
v2, P = 0.01).
Our results suggest that most dscam knockdown defects
are the results of aberrant or delayed cell movement. Normal
organization of the embryo is disturbed as observed in time
lapse images and marker gene WISH. Cells in dscam
morphants exhibit less movement from their initial position
at the start of epiboly than controls. An additional defect we
observed is that the induction of some genes is influenced
by dscam expression. This may be primarily due to their
activation through dscam or a secondary defect due to
improper formation of an inductive tissue.
Discussion
Understanding the role that Dscam plays in development
of the vertebrate embryo may lead to valuable insights into
its associated disease, Down syndrome (DS). Previous
studies have primarily focused upon potential roles of
Dscam in the nervous system due to its strong expression in
the mouse and human CNS (Agarwala et al., 2001a;
Yamakawa et al., 1998). Examinations of brains from
normal and DS patients have shown that Dscam is
overexpressed and is associated with certain types of
CNS pathology (Saito et al., 2000). Drosophila mutants
have been useful in providing insight into cellular roles for
Dscam in neural development (Hummel et al., 2003;
Schmucker et al., 2000; Wang et al., 2002, 2004;
Wojtowicz et al., 2004; Zhan et al., 2004), but descriptions
for the molecular role of vertebrate Dscams have been
limited.
Here we report the sequence of the zebrafish homolog
that is highly related to the gene found on human
Chromosome 21. It shares high structural and sequence
homology to all other Dscams in their extracellular
domains. Two orthologs of Dscam exist in humans and
rodents and it is a distinct possibility that four dscam genes
may exist in the zebrafish due to genomic duplication in the
ray fin fish lineage. Few splice variants for vertebrates have
been reported in the literature in contrast to reports in
Drosophila of several thousand possible splice variants
(Celotto and Graveley, 2001; Schmucker et al., 2000). We
have not observed extensive splice variants for zebrafish
dscam.
The vertebrate Dscams differ greatly from their Droso-
phila counterparts with respect to their intracellular
domains. The intracellular portion of zebrafish dscam shares
83% amino acid identity to the human homolog. The
intracellular portions of Drosophila and vertebrate Dscams
do not share significant homology, but there are some
signaling motifs that exist in both groups and may be
functionally conserved between Drosophila and zebrafish.
SH2/SH3 binding motifs can be found in both species. In
Drosophila, the SH3 adaptor protein Dock binds to dscam
in a cooperative fashion through its SH2/SH3 binding
motifs (Schmucker et al., 2000). Vertebrate dscams may
bind nck (vertebrate homolog of Dock) through SH2/SH3
motifs, although the structure appears to have changed over
time. Vertebrate Dscams may have evolved new modifica-
tions to their signaling cascades as human Dscam directly
binds and activates one of nck’s downstream targets, pak1
(Li and Guan, 2004). All vertebrate Dscams contain a C-
terminal consensus PDZ binding motif (YTLV) that is
similar to the C-terminus of Drosophila dscam (TMAV).
Dscams likely utilize PDZ binding proteins to localize itself
to particular microdomains. It has been previously described
that Dscam can be found on the axons and dendrites of
Purkinje cells (Agarwala et al., 2001b). PDZ domains may
localize Dscam to synapses, allowing it to stabilize synaptic
connections in the CNS.
Zebrafish dscam is highly expressed in the developing
brain. Discrete expression begins in the forebrain and
midbrain, expanding in the following hours into the hind-
brain and spinal cord (Figs. 2E–J). These results are
strikingly similar to previous work identifying areas of
neurogenesis in the zebrafish brain (Chitnis and Kuwada,
1990; Wilson et al., 1990). The expression patterns are
similar to those in other systems (Agarwala et al., 2001a,c;
Barlow et al., 2002; Hummel et al., 2003; Schmucker et al.,
2000; Wang et al., 2002; Yamakawa et al., 1998),
suggesting dscam is used in shaping the nervous system.
Unfortunately, we were unable to produce in vitro tran-
scribed mRNA and were therefore unable to directly address
the role overexpressed Dscam has on embryonic growth as
a model for DS. Until this point, the primary interest in
Dscam has focused on its roles in neural and heart develop-
ment due to its strong and specific expression in these
organs. In this study, we reveal that Dscam is broadly
expressed during blastulation. Future work should consider
the impact excess Dscam expression has upon overall growth
and development beginning shortly after fertilization.
We have used morpholino oligos in this study to
knockdown dscam levels to discern its in vivo role. Mild
growth defects are associated with zebrafish embryos
displaying fewer neurons and misguided axons. Coupled
with strong dscam expression from neuronal regions
involved in axonogenesis, these data suggest zebrafish
dscam plays a role in nervous system development.
However, our experiments do not eliminate the possibility
that general growth defects affect axon guidance.
Our work demonstrates that dscam plays a role in early
morphogenesis of the embryo. We show that dscam is a
maternally expressed gene that is upregulated before and
throughout gastrulation. Zebrafish dscam is broadly
expressed through at least somitogenesis. dscam morphants
begin to display morphologic defects shortly after the start
of epiboly. By the beginning of gastrulation, many dscam
morphants appear thickened and bilayered as seen in Fig. 6.
D. Yimlamai et al. / Developmental Biology 279 (2005) 44–5756
Consistent with this phenotype, we show that dscam
morphants exhibit cell movement defects after the onset of
epiboly (Fig. 8), suggesting that cells are unable to move to
their proper spatial position. Since Dscams interact homo-
philically in vitro (Agarwala et al., 2000, 2001c) and appear
to mediate specific cell–cell contact in vivo (Wojtowicz et
al., 2004), Dscam may be utilized in a manner that provides
adhesion and traction against other cells. Therefore, a
bilayered Dscam morphant may be a sign of inappropriate
adhesion to neighboring cells. Since our data show that
many cells in early embryo express dscam, any cell could
move with respect to another cell by regulating adhesion to
its neighbors. A number of immunoglobulin cell adhesion
molecules such as Dscam have been demonstrated to
mediate migration in vivo through homophilic adhesion
(Ishida et al., 2003; Izumoto et al., 1996; Nakada et al.,
2000; Wright et al., 1999).
It appears that a primary consequence of dscam knock-
down is a lack of convergent-extension in mesodermal cells
and disorganization in the prechordal plate. This indicates
that dscam is involved with different types of cell move-
ment through various developmental stages. Marker gene in
situ of knockdown embryos demonstrates that dscam does
not appear to interfere with cell fate, since knockdown
embryos have the correct cell layers in the approximately
correct position. In contrast, expression of Bmp2b and
Dharma in the shield was downregulated in Dscam
morphants. Dharma antagonizes Bmp2b expression (Leung
et al., 2003) and would be expected to upregulate Bmp2b
expression in this study as Dharma appeared to be absent.
This may indicate that morphologic deficits caused by
Dscam knockdown have subsequent consequences upon
normal gene expression.
Here, we show Dscam knockdown is required in zebra-
fish during epiboly when cells begin migrating to envelope
the yolk and take their eventual positions in the vertebrate
embryo. This situation is likely to be analogous to the fetal
development of rare cases of monosomy Chromosome 21.
Case reports of full, molecularly confirmed monosomy 21
describe severe intrauterine growth retardation, microce-
phaly, brain abnormalities, and congenital heart defects as
common traits (Mori et al., 2004; Pellissier et al., 1987;
Wisniewski et al., 1983). Specifically, partial deletion of
21q22.2, the chromosomal region containing Dscam, is
associated with microcephaly and intrauterine growth
retardation (Matsumoto et al., 1997). The work here
supports the notion that Dscam may be an important
component of embryonic and cephalic growth.
Recently, it has been suggested that Dscam is involved
in Drosophila dorsal–ventral patterning (Stathopoulos et
al., 2002). Perturbing the maternal Dorsal gradient results in
significant changes in dscam expression raising the
possibility that it functions in early Drosophila develop-
ment. In wild-type Drosophila, it is primarily expressed in
neuroectoderm although its expression can be found else-
where to a lesser degree. Our results show that dscam plays
a role during early embryonic vertebrate development. In
Grade 3/4 zebrafish dscam knockdown embryos, the
neuroectodermal-derived tissues are severely affected (Figs.
4C and D), and although mesoderm derived tissues are
present, they often appear disorganized. The use of Dscams
in cell movement during early development thus appears to
be an evolutionarily conserved mechanism for embryonic
morphogenesis.
Acknowledgments
The authors would like to thank members of the Jay
laboratory and Dr. Jennifer Moss (Tufts University) for
helpful discussions; Punita Koustabhan for excellent fish
care and technical assistance; the Tufts GRASP center for
antibody production and cell culture, NIH program grant
(DK34928); the Zebrafish International Resource Center for
providing cDNA clones, NIH Grant (RR15402); and the
useful comments of the anonymous reviewers of the
manuscript. This work was supported by NIH grants
(EY11992, NS34699) to D.G.J.
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