Dynamic expression and regulation by Fgf8 and Pou2 of the zebrafish
LIM-only gene, lmo4
Mary Ellen Lane*, Alexander P. Runko, Nicole M. Roy, Charles G. Sagerstro ¨m
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street/LRB822, Worcester,
MA 01605, USA
Received 15 May 2002; received in revised form 23 October 2002; accepted 25 October 2002
We report the expression of zebrafish lmo4 during the first 48 h of development. Like its murine ortholog, lmo4 is expressed in somitic
mesoderm, branchial arches, otic vesicles, and limb (pectoral fin) buds. In addition, however, we report zebrafish lmo4 expression in the
developing eye, cardiovascular tissue, and the neural plate and telencephalon. We demonstrate that expression in the rostral hindbrain
requires acerebellar (ace/fgf8) and spiel ohne grenzen (spg/pou2) activity. q 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: lmo4; LIM; Zebrafish; Neural plate; Rostral hindbrain; Telencephalon; Optic primordia; Optic vesicle; Otic vesicle; Olfactory bulb; Retinal
pigment epithelium; Somite; Pharyngeal arch; Endocardium; Zebrafish LG5; acerebellar; spiel ohne grenzen; Fgf8; Pou2
1. Results and discussion
We previously reported cloning of several genes
expressed caudally in the zebrafish gastrula (Sagerstro ¨m et
al., 2001). One of these is 76% identical to human and
mouse LMO4, encoding a 167 amino acid protein with
two LIM domains (Grutz et al., 1998; Kenny et al., 1998;
Racevskis et al., 1999; Tse et al., 1999). LIM domains,
found in functionally diverse proteins, contain tandem
non-DNA binding zinc fingers (Dawid et al., 1998). LMO
proteins are unique in that they lack other functional
domains, and LMO proteins promote formation of multi-
meric transcription regulatory complexes by bridging
factors such as bHLH and GATA proteins (Rabbitts,
1998). LMO proteins also function antagonistically toward
LIM-Homeodomain (Lhx) proteins by competing for bind-
ing to the essential co-factor Ldb/NLI (Rabbitts, 1998). A
single Drosophila LMO gene (dLMO) (Milan and Cohen,
1999; Shoresh et al., 1998; Zeng et al., 1998; Zhu et al.,
1995) and four mammalian genes, LMO 1–4, have been
identified (Rabbitts, 1998). Human LMO2 (Rabbitts et al.,
1999) and LMO4 (Sum et al., 2002; Visvader et al., 2001)
loci are targets of chromosomal translocations associated
with leukemias and breast cancer, suggesting that LMO
genes are also oncogenes.
We mapped zebrafish lmo4 (AY028903) to an interval
between 48.9 and 50.1 cM from the top of linkage group
5 (LG5; data not shown) using a zebrafish radiation hybrid
panel (Geisler et al., 1999). Several zebrafish genes located
on LG5 have orthologs on Human chromosome 1 (Woods et
al., 2000), where human LMO4 resides. Sequence alignment
and phylogenetic analysis (Fig. 1) support the conclusion
that zebrafish lmo4 is orthologous to human LMO4.
sion during gastrula and segmentation stages. Maternal lmo4
blastula stage (Fig. 2B). During gastrulation, zygotic tran-
scripts appear above the germ ring at shield stage (6 hpf,
Fig. 2C) and become distributed broadly in the dorsal ecto-
bud stage (10 hpf, Fig. 2E–G, J) expression is restricted to the
pax2.1 and krox20 (wide and narrowred stripes,respectively,
in Fig. 2F) show that bud stage lmo4 expression is in the
presumptive rostral hindbrain.
Mesendodermal expression is present during gastrula
by the bud and early somite stage (Fig. 2G, H arrowhead).
Presomitic mesoderm and tail bud expression is observed
later (Fig. 2K, S, six-somite stage and T, 14-somite stage).
Rostral somite expression is restricted to anterior epithelium
Mechanisms of Development 119S (2002) S185–S189
0925-4773/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved.
* Corresponding author. Present address: Department of Biochemistry
and Cell Biology, Rice University, 6100 Main Street, Houston, TX
E-mail address: firstname.lastname@example.org (M.E. Lane).
M.E. Lane et al. / Mechanisms of Development 119S (2002) S185–S189
Fig. 1. (A) Alignmentofthe amino acid sequenceof zebrafish(Dr.) LMO4with severalmembersof the mouse (Mm.),human(Hs.),and zebrafishLMO family.
Alignment was made using Clustal X version 1.8. Hs. DAT-1 is the protein in the database that gives the closest match to Mm. LMO3 as published by Grutz et
al. (1998). Majority residues are shaded black. (B) Phylogenetic analysis of LMO family members from zebrafish, mouse, and human. The sequences were
aligned as in (A). The phylogenetic tree was constructed with Phylip 4.0, using a Jones–Taylor–Thornton Matrix and neighbor joining tree with Bootstrap
values of 500. The accession numbers are: Mm. LMO1: NM057173.1, Mm. LMO2: NM008505.1, Mm. LMO4: NM010723.1, Hs. LMO1: AJ277662.1, Hs.
LMO2: NM005574.2, Hs. DAT: NP061110, Hs. LMO4: NM006769.2, Dr. LMO1: AF398514.1, Dr. LMO2: NM131111.1, Dr. LMO4: AY028903.
and the mesenchymal core (Fig. 2U, 14-somite stage).
Expression in deep cells underlying the hindbrain is visible
at the six-somite stage (arrow in Fig. 2K).
Decreased expression in the rostral hindbrain by the
three-somite stage is visible in lateral views of whole-
mount embryos (Fig. 2H) though mesodermal expression
in this region of the embryo is still visible. Dynamic expres-
sion inthe anteriorneuroepithelium begins aroundthe three-
somite stage (Fig. 2H, I) at the anterior edge of the neural
plate in the presumptive telencephalon, and persists in the
dorsoanterior neural keel at the six-somite stage (Fig. 2K,
L). By the ten-somite stage (Fig. 2M), expression is visible
through much of the optic vesicles and telencephalon. By
the 14- and 18-somite stages (Fig. 2N, O), optic vesicle
expression is strongest in the medial areas that will form
the optic stalk and retinal pigment epithelium. Expression at
the 21-somite stage is restricted to the optic stalk (Fig. 2P,
Q) and the pigmented epithelium (Fig. 2R).
Expression is seen in the otic vesicle (Fig. 2V) and
presumptive branchial arch region (Fig. 2W) at the 21-
somite stage, as well as in the anterior sensory ganglia
We next examined expression between the prim-5 (24
hpf) and high pec stage (48 hpf). Cardiovascular expression
is observed at the prim-5 stage in the endocardium along the
anteroposterior extent of the tube (Fig. 3A) and in the
midline vasculature (Fig. 3B). Expression in the pharyngeal
arch mesenchyme, heart and fin buds is observed at 36 hpf
(Fig. 3C, D).
Telencephalic expression is a persistent aspect of lmo4
M.E. Lane et al. / Mechanisms of Development 119S (2002) S185–S189
Fig. 2. Expression of lmo4 through the 24-somite stage. Whole-mount in situ hybridization with an lmo4 probe was performed as reported (Sagerstro ¨m et al.,
1996). (A, B) Cleavage stages. Maternal lmo4 expression is visible at the two-cell stage (A), but is not detectable by late blastula stage (B). (C–G) Gastrula
stages. (C) Lateral view of a shield stage (6 hpf) close-upshowing lmo4 expressionis excluded from the margin of the shield (arrow) above the forerunnercells
(arrowhead). Dorsal views of (D) 75% epiboly (8 hpf), (E) bud stage (10 hpf), (F) double in situ hybridization showing lmo4 expression in blue and pax2.1
(upperstripe)andthe rostralstripe(presumptive R3)ofkrox20in red,in a budstage embryo.(G)Lateral viewofbudstage (10hpf)embryosdemonstratingthat
zygotic lmo4 is restricted to the rostralmost portion of this domain by the end of gastrulation. The arrow in (G) points to mesendodermal staining. (H, I) Three-
somite stage. (H) Lateral view showing decreasing expression in the ectoderm at the three-somite stage (11 hpf), expression in the somitic mesoderm
(arrowhead) and low expression in the presomitic mesoderm. (I) Animal pole view of the embryo in (H), showing expression in the anterior neural plate.
(J, J0) Optical sectionthrough the rostralhindbrainarea ofa bud stage embryoand a six-somite stage embryo,showingexpressionin the neural plate(np) at bud
but not six-somite stage. (K, L) Six-somite stage. (K) Neural plate expression is restricted to the anterior. Mesodermal expression persists in the somites and
head mesoderm (arrow) and expression in presomitic mesoderm becomes detectable. (L) Animal pole view of embryo in (K). (M–R) Expression in the optic
primordia from the ten-somite to the 21-somite stage. (M) Ten-somite stage: expression in the telencephalon (t) and much of the optic primordia. (N)
Expression at the 14-somite stage is resolved to the presumptive stalk and pigmented epithelium (rpe, arrowhead). (O) Expression at the 18-somite stage
in the optic stalk and rpe (arrowhead). (P) Dorsal, (Q) ventral and (R) lateral view of the optic primordia of 21-somite stage embryos. The arrowhead in (R)
points to a retinal pigment cell. (S–U) Expression in somitic, presomitic and tail mesoderm. (S) Dorsal view of the tail bud presomitic mesoderm at the six-
somite stage (see also (J)). (T) Lateral view of tail mesoderm at the 14-somite stage. (U) Expression in rostral somites at the 14-somite stage. (V–X) Twenty-
one-somite stage. (V) Dorsal view showing expression in the otic vesicle. (W) Same embryo as in (V) in a ventral focal plane, showing expression in the
pharyngeal arch mesenchyme ventral to the otic vesicle. (X) Lateral view showing expression in the anterior sensory ganglia.
expression. This becomes restricted to the olfactory bulb by
the 21-somite stage (Fig. 2P) and remains at least through 48
hpf (Fig. 3E). Expression in the presumptive hindbrain
decreases significantly by early somitogenesis, however
expression in a subset of rostral hindbrain cells is observed
at 48 hpf (Fig. 3F).
While similarities in the expression of zebrafish lmo4 and
that of its murine ortholog are observed (Hermanson et al.,
1999; Kenny et al., 1998; Sugihara et al., 1998), we note
additional aspects. In particular, expression in the rostral
hindbrain during gastrula stages, which has not been
described in mouse, suggests regulation by genes involved
in midbrain–hindbrain boundary (MHB) formation. We
examined expression in embryos mutant at the acerebellar
(ace), spiel ohne grenzen (spg) and no ishtmus (noi) loci,
which encode fgf8 (Reifers et al., 1998), pou2 (Burgess et
al., 2002; Hauptmann et al., 2002; Reim and Brand, 2002)
and pax2.1 (Brand et al., 1996), respectively. We observed
normal expression of lmo4 in embryos from noi/pax2.1
heterozygotes at all stages and no differences in lmo4
expression were detected prior to bud stage in embryos
homozygous for ace/fgf8 or spg/pou2. spg embryos, distin-
guishable by reduced noi/pax2.1 expression, show reduced
lmo4 expression (Fig. 4A–D, asterisks indicate noi/pax2.1
expression, brackets in Fig. 4A, B indicate metencephalic
lmo4 expression). ace mutant embryos showed similarly
reduced lmo4 expression (Fig. 4E–H). Histological sections
(Fig. 4G, H) and a higher magnification view (Fig. 4C, D;
focal plane in the ectoderm) did not allow assignment of
residual staining to a germ layer, but showed greatly
reduced expression throughout.
Fgf8 signaling, detected by activation of the ERK-MAPK
(Curran and Grainger, 2000; Umbhauer et al., 1995), is
disrupted in the rostral hindbrain in ace embryos (Fig. 4I,
J), and lmo4 expression may be induced by Fgf. Indeed,
implantation of an bFgf-soaked Affigel bead led to ectopic
lmo4 expression (Fig. 4K, L).
2. Materials and methods
See figure legends.
Mutant strains were kindly provided by Alex Schier,
M.E. Lane et al. / Mechanisms of Development 119S (2002) S185–S189
Fig. 3. Expression during pharyngula stages (24–48 hpf). (A, B) Prim-5
stage (24hpf). (A)Ventral viewof the heartprimordiumwithrostraltoward
the top, showing expression in the endocardium (ec). (B) Lateral view of
the trunk region (rostral to the left) showing expression in midline vascu-
lature (arrowhead). Expression is also detectable in the ventral mesoderm
(vm). (C,D) Prim-25stage (36hpf).(C)Expression in thetelencephalon(t),
pharyngeal arch mesenchyme (arrowheads in C, D), and proximal fin buds
(arrow in C). (D) Higher magnification ventral view of the larva in (C),
showing expression in the pharyngeal arches (p1 and p2 and arrowhead).
(E, F) Expression in the neuroepithelium at 48 hpf. (E) Dorsal view with
rostral toward the top, showing expression in the olfactory bulb adjacent to
the nasal pits (np). (F) Lateral view showing expression in the rostral
hindbrain, adjacent to the cerebellum (c). Abbreviations: floor plate (fp),
heart (h), myocardium (mc), otic vesicle (ov), notochord (n), spinal cord
Fig. 4. Lmo4 expression in spg/pou2 and ace/fgf8 mutant embryos and in
response to soluble Fgf. Embryos of various genotypes (as shown in the top
right corner of each panel) were analyzed with various probes (as indicated
in the lower right corner of each panel). In situ hybridization and immu-
nostaining were done as reported (Sagerstro ¨m et al., 2001; Schulte-Merker
et al., 1992). (A, C, E, G, I, K) Wild type embryos; (B, D) spg mutant
embryos; (F, H, J) ace mutant embryos. (K, L) Wild type embryos into
which an Affi-Gel bead soaked in MBS (K) or 0.5 mg/ml Fgf (L) was
implanted at shield stage (6 hpf). (A–D) were hybridized to noi/pax2.1
(indicated by asterisks) and lmo4 probes (brackets in A, B). (C, D) are
focused in the plane of the ectoderm. (E–H) and (K, L) were hybridized
to a lmo4 probe and (I, J) were stained with the dP-ERK antibody (Sigma
Cat. # M8159) at a dilution of 1:1000. All whole-mount embryos are dorsal
views with anterior to the top. Transverse 10 mm sections in (G, H) are cut
through the presumptive rostral hindbrain area that shows maximal lmo4
expressionin wildtype bud stage embryos.Dorsal is at the top. All embryos
are at bud stage (10 hpf) except (C, D), which are at the two-somite stage.
Didier Stainer, and the Tu ¨bingen Stock Center. We wish to Download full-text
thank Tatjana Piotrowski and Emily Walsh for helpful
comments on the expression pattern, and Damian Dalle
Nogare for assistance with sequence alignment. We thank
the reviewers for helpful comments on the manuscript.
Laboratory support form Hazel Sive during the initial
phase of this work is acknowledged. This work was
supported by NIH grant HD39156 to C.G.S., and M.E.L.
was supported by a Burroughs-Wellcome Postdoctoral
Fellowship administered by the Life Science Research
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