Download Identification and Characterization of Lbh, a Novel

Developmental Biology 233, 291–304 (2001)
doi:10.1006/dbio.2001.0225, available online at http://www.idealibrary.com on
Identification and Characterization of Lbh, a Novel
Conserved Nuclear Protein Expressed during Early
Limb and Heart Development
Karoline J. Briegel and Alexandra L. Joyner 1
Howard Hughes Medical Institute and Developmental Genetics Program, Skirball Institute
of Biomolecular Medicine, and Department of Cell Biology, New York University School
of Medicine, New York, New York 10016
We report the cloning, protein characterization, and expression of a novel vertebrate gene, termed Lbh (Limb-bud-andheart), with a spatiotemporal expression pattern that marks embryologically significant domains in the developing limbs
and heart. Lbh encodes a highly conserved nuclear protein, which in tissue culture cells possesses a transcriptional activator
function. During limb development, expression of Lbh initiates in the ectoderm of the presumptive limb territory in the
lateral body wall. As the limb buds appear, Lbh expression is restricted primarily to the distal ventral limb ectoderm and
the apical ectodermal ridge, and overlaps in these ectodermal compartments with En1 and Fgf8 expression. During heart
formation, Lbh is expressed as early as Nkx2.5 and dHand in the bilateral heart primordia, with the highest levels in the
anterior promyocardium. After heart tube fusion and looping, Lbh expression is confined to the ventricular myocardium,
with the highest intensity in the right ventricle and atrioventricular canal, as well as in the sinus venosus. Based on the
molecular characteristics and the domain-specific expression pattern, it is possible that Lbh functions in synergy with other
genes known to be required for heart and limb development. © 2001 Academic Press
Key Words: mouse embryogenesis; Lbh; Xlcl2; nuclear; transcriptional activation; limb development; cardiogenesis;
pattern formation.
INTRODUCTION
Significant progress has been made toward identifying
genes that pattern body axes and organ rudiments during
vertebrate embryogenesis. It has become evident that morphologically distinct organs, such as the limbs and the
heart, rely on some of the same molecules for patterning
(Wilson, 1998). Patterning of organs is critically dependent
on spatially restricted expression of transcription factors at
specific stages of development. Interestingly, positional
information is often first apparent as defined domains of
gene expression and only later do overt signs of morphological differentiation become visible (Schwabe et al., 1998).
The identification of genes with regionalized expression
patterns in both the forming limbs and heart therefore
1
To whom correspondence should be addressed at New York
University School of Medicine, Developmental Genetics Program,
Skirball Institute, 540 First Avenue, 4th floor, New York, NY
10016. Fax: (212) 263-7760. E-mail: [email protected].
0012-1606/01 $35.00
Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.
should provide additional tools for studying possible
mechanisms of patterning in these two organs.
The mouse forelimb bud becomes visible at approximately embryonic day 9 (E9.0) as a small protrusion from
the lateral body wall, consisting of undifferentiated mesenchymal cells enveloped by an ectodermal sheet. Three
major signaling centers control development of the bud into
a structured limb (Tickle, 1999). A group of mesenchymal
cells in the posterior limb bud, the so-called zone of
polarizing activity (ZPA), controls anteroposterior (A-P)
patterning by secreting the posteriorizing signal Shh. The
limb ectoderm is divided into distinct dorsal and ventral
domains and conveys dorsoventral (D-V) limb polarity as
well as proximodistal (P-D) pattern to the limb mesenchyme (Chen and Johnson, 1999). A specialized epithelium
at the apex of the limb bud, the apical ectodermal ridge
(AER), produces Fgfs that promote outgrowth of the limb
along the P-D axis (Martin, 1998). Furthermore, the dorsal
nonridge ectoderm exerts an instructive dorsalizing property by secreting the signaling molecule Wnt7A, which
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induces dorsal differentiation in the underlying mesenchyme via its target gene, that encodes the LIM homeodomain protein Lmx1B (Parr and McMahon, 1995; Riddle et
al., 1995; Vogel et al., 1995).
Compartmentalization of the limb ectoderm is also evidenced by expression of the homeodomain transcription
factor Engrailed-1 (En1) in the ventral ectoderm and the
ventral AER (Davis and Joyner, 1988). Although no signaling molecules have been attributed to the ventral ectoderm
so far, it appears to provide the requirements for ventral
limb polarity and inhibits dorsal differentiation (MacCabe
et al., 1974; Pautou and Kieny, 1973). These functions have
been mainly attributed to the activity of the En1 protein
(Logan et al., 1997; Loomis et al., 1996, 1998). Loss of En1
function results in development of dorsal structures on the
distal ventral limb, as a result of ectopic Wnt7A expression
in the ventral ectoderm (Loomis et al., 1996). In addition,
En1 is required to correctly position and form a normal AER
at the dorsal–ventral interface (Cygan et al., 1997; Kimmel
et al., 2000; Loomis et al., 1996, 1998). At present the
molecular basis of the interactions between AER and nonridge limb ectoderm during establishment of D-V limb
polarity and AER formation is poorly understood.
The heart arises from two bilateral symmetric regions of
the splanchnic lateral plate mesoderm (LPM) of the mouse
embryo at presomitic stages, which join to form a distinctive cardiac crescent (Fishman and Chien, 1997). Fatemapping studies in the chick have shown that cardiomyocytes display an early A-P pattern, which is manifested by a
difference in contractility between caudal and rostral myocardial cells (Fishman and Chien, 1997; Garcia and Schoenwolf, 1993). Patterning is also evidenced by the different
expression pattern of early cardiac marker genes along the
A-P axis, such as Irx4, dHand, and Cdpg2/Versican in the
anterior, and Tbx5, Msg1, Pop3, and AMHC1 in the posterior myocardium (Andree et al., 2000; Biben and Harvey,
1997; Bruneau et al., 1999, 2000; Dunwoodie et al., 1998;
Mjaatvedt et al., 1998; Yutzey et al., 1994). Subsequently,
the cardiac progenitors converge into a primitive heart tube
at the ventral midline, and the first morphological sign of
left-right (L-R) asymmetry becomes morphologically apparent in the developing murine embryo with a rightward and
ventrocaudal looping of the heart tube (Fishman and Chien,
1997). The looped heart tube early on is regionalized along
its rostrocaudal axis. Domain-specific expression of heartspecific transcription factors defines different segments in
mouse: the outflow tract (conotruncus), right and left ventricles, common atrium, and sinus venosa, which gives rise
to the inflow tract (Fishman and Olson, 1997; Srivastava
and Olson, 2000). Finally, distinct ventricular and atrial
chambers form as a consequence of a balloonlike expansion
of the mitotically active outer compact zone, followed by
septum and valve formation (Christoffels et al., 2000a;
Fishman and Chien, 1997).
Although formation of the limbs and the heart occurs
quite distinctively, a common theme is that many genes
controlling pattern formation and morphogenesis in both
tissues are regionally expressed. Moreover, the frequent
association of human congenital heart diseases with hand
anomalies (the so-called heart– hand syndromes) suggests
that some of the same molecules are required for both limb
and heart development (Wilson, 1998). Consistent with
this, several factors that are known to regulate limb patterning are also involved in heart formation. For example,
Fgf8 induces patterning of the most distal limb structures,
but also influences the correct looping and patterning of the
heart (Crossley et al., 1996; Meyers and Martin, 1999;
Reifers et al., 2000). In addition, retinoic acid (RA) signaling
can influence certain aspects of limb and heart pattern
formation. Vitamin A-deficient quail embryos develop limb
D-V patterning and AER defects similar to those of En1 null
mutants. Furthermore, these embryos lacking RA signaling
display a frequent reversal of heart looping (Stratford et al.,
1999; Zile et al., 2000). Finally, at the transcriptional level,
Tbx5 and dHand have been shown to play dual roles in limb
and heart development (Basson et al., 1997; Charite et al.,
2000; Fernandez-Teran et al., 2000; Rodriguez-Esteban et
al., 1999; Srivastava et al., 1997; Takeuchi et al., 1999;
Yelon et al., 2000).
We have identified a novel mouse protein, encoded by the
Lbh (Limb-bud-and-heart) gene, which has an expression
pattern that supports the paradigm of common patterning
mechanisms in the limb and the heart. Lbh is a member of
a highly conserved family of small acidic proteins in vertebrates. We show that Lbh localizes to the nucleus and has
transcriptional activation activity in mammalian cells. At
the initial stages of limb outgrowth, Lbh is expressed in the
ventral limb ectoderm and in the AER. In addition, Lbh is
expressed first in the anterior and later predominantly in
the right myocardium, reflecting early A-P patterning and
later L-R asymmetry during cardiogenesis. Taken together,
our biochemical data and expression studies provide evidence that Lbh could act as a transcriptional coactivator
involved in molecular pathways that pattern the limb and
the heart.
MATERIALS AND METHODS
Yeast Two-Hybrid Screen
A NcoI–BamHI fragment of En1 (corresponding to the full-length
open reading frame: amino acids 1– 401) was subcloned into the
yeast Gal4 DNA-binding vector pAS2 (Clontech, Palo Alto, CA)
and used as a bait to screen an embryonic E11 mouse MATCHMAKER cDNA library (Clontech) according to the manufacturer’s
protocol. A total of 1–2 ⫻ 10 5 transformants was screened and 167
His⫹/lacZ⫹ clones were isolated, 36 of which were confirmed in a
secondary yeast interaction screen and subcloned into pBluescript
KS. Clones were tested for coexpression with En1 by whole-mount
RNA in situ hybridization on E9.5–E10.5 mouse embryos with
RNA probes derived from in vitro transcription of selected cDNA
clones in both directions. Yeast clone YP26, which contained 150
nucleotides of a novel cDNA in frame with the Gal4 activator
domain, was selected because of its coexpression with En1 in the
limb bud.
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
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Early Role of Lbh in Formation of Appendages and Heart
FIG. 1. The vertebrate Lbh gene family. (A) Sequence alignment of Lbh proteins from mouse, human, bovine, and Xenopus (Xlcl2;
Z14122). Alignments were done with the CLUSTAL algorithm using MacVector. The dark shading represents identity at a given residue,
whereas light shading represents amino acid residue similarity. Specific protein domains are indicated by patterned boxes (see below) and
␣-helices by brackets. (B) Schematic representation of predicted protein structure of the vertebrate Lbh proteins as exemplified by the
murine Lbh protein. Characteristic for Lbh proteins is an N-terminal hydrophobic stretch (checkered box), a putative nuclear localization
signal (NLS; black box), and a C-terminal glutamate-rich acidic domain (hatched box).
Cloning of the Murine Lbh
Immunofluorescence and Cell Transfection
To isolate a full-length cDNA the partial cDNA from the YP26
yeast clone was used to screen a ␭ZAPII mouse E8.5 cDNA library
(generously provided by S.-J. Lee, Johns Hopkins University, Baltimore, MD) and two ␭gt11 mouse E11 and E15 cDNA libraries
(Clontech). Three different overlapping cDNA clones were isolated
from a total of 5 ⫻ 10 6 recombinants screened. The cDNA clones
were extracted either by in vivo excision or by subcloning into
pBluescript vectors. Clones were sequenced with ABI-Sequencer
(Applied Biosystems/Perkin–Elmer, Foster City, CA) and sequence
analysis was performed with Mac Vector, Sequencer, and NCBI
software programs. Several novel vertebrate ESTs similar to Lbh
were identified in a BLAST search: W82850, AA667294, W62340
(mouse); R58414, T93787, R02324, R02426, T93832 (human);
BE115356 (rat); BE485556 (bovine); AW786872 (pig); and
AW827038 (zebrafish). The accession number of the Xenopus
homolog Xlcl2 is Z14122.
The subcellular localization of Lbh was determined in COS7
cells transiently transfected with a pCDNA3 expression vector
encoding the full-length Lbh with a Flag-epitope tag at the amino
and the carboxy terminal, respectively. As a control, a pCDNA3
expression vector containing the En1 ORF was expressed. At 48 h
after transfection, cells were fixed for 30 min with 4% paraformaldehyde and stained with mouse anti-Flag antibody (Sigma, St.
Louis, MO) or polyclonal EnbHD (Davis and Joyner, 1988) that
recognizes En1, followed by a secondary Cy3 anti-mouse or Cy3
anti-rabbit antibody, respectively (Jackson Laboratories, Bar Harbor, ME). Oligreen (Molecular Probes, Eugene, OR) was included in
the antibody reactions to reveal the cell nuclei. Immunofluorescent
staining was visualized by confocal microscopy.
To test transcriptional activity of Lbh, a BamHI–KpnI fragment
comprising the full-length ORF of Lbh was cloned in frame with a
Gal4 DNA-binding domain in the pSG424 expression vector (Sadowski and Ptashne, 1989) and cotransfected with a UAS–
luciferase reporter plasmid (Zinszner et al., 1994). As an activator
control a Gal4 DBD-Tls fusion construct (Zinszner et al., 1994) was
used. Transfection efficiency was monitored by coexpression of a
Renilla luciferase reporter gene (Promega). Briefly, 0.5 ␮g of UAS–
luciferase reporter, 0.1 ␮g of Renilla luciferase construct, and 1 ␮g
of each activator plasmid were mixed with 6 ␮l of FuGENE6 (Roche
Molecular Diagnostic Systems, Alameda, CA) and added to a 10-cm
dish of 3T3 NIH fibroblasts. Cells were harvested 48 h following
transfection for luciferase assays. A minimum of three transfection
reactions were performed per activator construct. Transactivation
In Vitro Translation
The Lbh open reading frame (ORF; amino acids 1–105) was
expressed in pCDNA3 (Invitrogen, San Diego, CA). Recombinant
Lbh protein was produced by in vitro transcription and translation
using the TnT-reticulocyte lysate (Promega, Madison, WI) and
protein was labeled by [ 35S]-methionine incorporation during
translation. An aliquot of the translation product was separated
on a denaturing SDS gel. Protein bands were detected by autoradiography.
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
FIG. 2. Biochemical characterization and cellular localization of the Lbh protein. (A) In vitro translation of a full-length Lbh cDNA clone subcloned
into pBSIIKS(⫺) in the presence of [35S]-methionine gives rise to a protein with a molecular weight (MW) of ⬃16 kDa, indicated by an arrow (lane 2). Lane
1 is control translation reaction with pBSIIKS(⫺) vector alone. (B) Confocal immunofluorescence analysis of Lbh cellular localization. Flag-epitopetagged Lbh contructs were transfected into COS7 cells and recombinant proteins were detected with a Flag-specific antibody (red, a, b). As a nuclear
control, En1 protein was expressed and detected with an ␣-En antibody (blue; c). Nuclei were counterstained with oligreen (green), and overlays of
protein and nuclear counterstainings are shown in d and e. Note the predominantly nuclear localization of both an N-terminally (NFlagLbh) and
C-terminally epitope-tagged Lbh protein (NFlagLbh). (C) Transcriptional activity of Lbh. Scheme of various Gal4 DNA-binding (GDB) fusion proteins,
which were transiently coexpressed with a luciferase reporter construct driven by Gal4-binding sites (UAS) in NIH 3T3 cells. The entire ORF (amino
acids 1–105) of Lbh fused in frame with the GDB (GDB-Lbh) activated reporter gene expression ⬎ 10-fold. As negative controls, mock-transfected or cells
transfected with the Gal4 DNA-binding domain (GDB) alone were used. A construct containing a strong transcriptional activation domain of Tls
(GDB-Tls) served as positive control. Luciferase activity was measured by luminescence and the fold transactivation was determined as described under
Materials and Methods. DBD, DNA-binding domain (gray box). (D) Northern blot analysis of Lbh expression in mouse embryonic and adult tissues.
CLONTECH multiple-tissue Northern blot membranes with normalized amounts of 2 ␮g poly(A)⫹ RNA were hybridized with a 32P-labeled 0.9-kb PCR
fragment comprising the Lbh 5⬘UTR and coding region. A single ⬃3.2-kb Lbh-specific transcript (indicated by an arrow) was detected at embryonic day
7 (E7.0; lane 1). Lbh mRNA levels increased and persisted between E11 and E17 (lanes 2–4). In adult tissues, Lbh mRNA is most abundantly detected
in the heart and with varying levels in the brain, spleen, lung, liver, and kidney, but not in muscle or testis (lanes 5–12).
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Early Role of Lbh in Formation of Appendages and Heart
FIG. 3. Expression of Lbh during early mouse gestation. (A–E) Whole-mount RNA in situ hybridization analysis of Lbh expression during
mouse embryonic stages E7.5 to E10.5. (A) Frontal view of a presomitic headfold stage embryo (E7.5) showing the onset of Lbh expression in the
anterior promyocardium (pmc), as indicated by a red arrow, and in the foregut (fg). (B–E) Lbh expression in the heart and gut (gt) persists through
stages E8.0 –E10.5 and appears in the branchial arches (ba) (C, D), the ventral ectodermal ridge (ver) of the tail bud (C), and from E9.0 onward in
the ventral ectoderm of the forelimbs (fl) and hindlimbs (hl) (D, E). (F–J) Whole-mount embryos were sectioned transversely to demonstrate
expression in foregut epithelium (F) and the ventral ectodermal ridge (G) at E8.75. (H) At E9.5 Lbh expression becomes is detected in the otic
vesicles (ov) and oral epithelium (oe) of the first branchial arch. From E10.5 onward, Lbh is also expressed in neural crest-derived sensory neurons
of the dorsal root (I) and trigeminal ganglia (J), respectively. at, atrium; drg, dorsal root ganglia; lv, left ventricle; ma, mandible; mx, maxillary; np,
neuropore; nt, neural tube; rv, right ventricle; sv, sinus venosus; trg, trigeminal ganglia; ve, ventricle.
activity was determined from the average of measured luciferase
activities, normalized against cell numbers and transfection efficiencies (Briegel et al., 1993).
Nucleotide Sequence Accession Number
In Situ Hybridization
RESULTS
Embryos from either CD1 or Swiss Webster mice (purchased
from Taconic Farms, Germantown, NY) were dissected in PBS and
fixed overnight in 4% PFA. A 1.1-kb fragment of the Lbh cDNA
contained in cDNA clone 14.1, which comprises the last 14 AA of
the ORF plus the entire 3⬘UTR, was used as a riboprobe. The other
probes used were to En1 (Wurst et al., 1994), Fgf8 (Crossley and
Martin, 1995), Wnt7A (Parr et al., 1993), Nkx2.5 (Lints et al., 1993),
dHand (Srivastava et al., 1995), and Carp (Zou et al., 1997).
Antisense RNA was in vitro transcribed using the DIG detection
system (Roche Molecular Diagnostic Systems). Whole-mount in
situ hybridization was performed essentially as described in
Wilkinson (1992). For ectoderm analysis, proteinase K (ProtK)
treatment was performed with 0.5 ␮g ProtK/ml for 5 min at 37°C.
For section analyses whole-mount embryos were refixed in 4% PFA
for 30 min after staining, immersed in 30% sucrose, and embedded
in OCT. Cryosections of 8 –30 ␮m were produced.
Isolation of the Lbh Gene
The GenBank accession number for the mouse Lbh cDNA
sequence is AF317517.
In an effort to identify transcriptional cofactors of the
homeodomain protein En1 that function in limb patterning,
we isolated a novel mouse cDNA in a yeast two-hybrid
screen, in which the full-length En1 protein was used as
bait (see Materials and Methods). Although further biochemical characterization indicated that En1 and this putative cofactor do not interact directly with each other, we
further characterized this gene and its protein product
because of its unique expression pattern in the ventral limb
and in the anterior heart primordia. Based on the gene’s
expression pattern it was termed Limb-bud-and-heart (Lbh).
The complete coding sequence of Lbh was obtained from
three overlapping cDNA clones isolated from different
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
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Briegel and Joyner
mouse embryonic cDNA libraries (E8.5 and E15). Lbh
encodes a single open reading frame of 105 amino acids
with an isoelectric point of 4.04 and a predicted molecular
weight of 12 kDa (Fig. 1). When translated in vitro and
separated on a denaturing SDS gel, Lbh has a molecular
weight of approximately 16 kDa (Fig. 2A). Sequence analysis revealed that Lbh is a member of a novel gene family in
vertebrates. Alignment of the predicted mouse Lbh protein
sequence with other mouse (W82850, AA667294,
AA500226, W62340), human (R02324, T93787, R58414,
R02426, T93832), and bovine ESTs (BE485556) contained in
the NCBI database, and a previously identified homolog in
Xenopus, Xlcl2 (Paris et al., 1988; Paris and Philippe, 1990),
showed a high degree of conservation (77–90%) throughout
the primary protein structure (Fig. 1A). The mouse, human,
and bovine Lbh proteins share 90% amino acid identity,
while mouse Lbh shares 77% and human and bovine Lbh
80% amino acid identity with the frog Xlcl2 protein (Fig.
1A). ESTs encoding partial Lbh-coding regions in other
genomes, such as rat (BE115356), pig (AW786872), and
zebrafish (AW827038), were also found. Moreover, Zooblot
analysis predicts an as-yet unidentified ortholog in chick
(data not shown).
A search of different protein databases failed to identify
any previously known protein motifs. However, comparison of cross-species amino acid residue identities among
Lbh proteins revealed several interesting features. All Lbh
proteins contain an amino terminal hydrophobic region of
nine amino acids immediately downstream of the initiation
methionine. A putative nuclear localization signal (NLS) at
amino acid positions 56 – 64 (Jans et al., 2000) and an acidic
glutamate-rich domain at the carboxy terminus spanning
amino acids 64 –105 are also apparent in Lbh. Stretches of
acidic amino acid residues are a common motif in otherwise often unstructured transcriptional activation domains
(Ptashne and Gann, 1997). In addition, the Lbh open reading
frame predicts two ␣ helices, one located in the N-terminal
domain at amino acid positions 15–23 and another one right
upstream of the acidic domain (amino acids 84 –93), which
could act as protein–protein interaction interfaces. Taken
together, these results suggest that Lbh family members are
likely to be nuclear proteins consisting of a carboxy terminal acidic domain, which could act as a transcriptional
activation domain.
Biochemical Characterization of the Lbh Protein
As a first step in characterizing the murine Lbh protein in
more detail, we sought to determine its subcellular localization. Various Flag-epitope-tagged versions of the Lbh
protein were transiently expressed in COS7 cells. Both an
N-terminally and a C-terminally tagged Flag-Lbh protein
(N FlagLbh, C FlagLbh) were predominantly detected in the
nucleus by a Flag-specific antibody (Fig. 2B). As expected, an
En-specific antibody detected nuclear localization of En1
protein, which was used as a control. Some minor fraction
of C FlagLbh protein was also detected in the cytoplasm and
in some organelles (Fig. 2B). Thus, Lbh is a predominantly
nuclear protein.
Because the presence of an acidic domain in the Lbh
protein raised the question of whether Lbh proteins might
be involved in transcriptional gene regulation, we studied
Lbh transcriptional activity in a Gal4 reporter assay (Sadowski and Ptashne, 1989). The whole open reading frame
of Lbh was fused at its C-terminus to the Gal4 DNAbinding domain (DBD) and transiently coexpressed in
NIH3T3 cells with a luciferase reporter construct containing UAS promoter elements (Fig. 2C). The Gal4DBD (GDB)
alone was used as a negative control, and a Gal4DBD–Tls
fusion protein (GDB-Tls) served as a positive control for
transcriptional activation. The adipocyte-specific factor Tls
was previously shown to be a strong transactivator in
mammalian cells (Zinszner et al., 1994). While the
Gal4DBD alone did not activate reporter gene transcription,
Gal4DBD–Lbh (GDB-Lbh) induced a 10- to 20-fold increase
of luciferase activity and Gal4DBD–Tls a 200-fold increase
over the basal level (Fig. 2D). These data show that Lbh has
the potential to act as a transcriptional activator in mammalian cells.
Expression of Lbh during Early Mouse
Embryogenesis (E7.5–E11)
Lbh expression during mouse embryogenesis and in adult
tissues was first studied by Northern blot hybridization
analysis. A single approximately 3.2-kb Lbh transcript was
found to be expressed at low level in Day 7.0 postcoitum
(E7.0) embryos, and at relatively high levels at E11 and later
(Fig. 2D). In adults, Lbh is most abundantly expressed in the
heart, as well as at lower levels in the brain, spleen, lung,
liver, and kidney. Interestingly, Lbh mRNA was not detected in skeletal muscle, indicating that Lbh is specific to
cardiac myocytes in adults. This was confirmed by RNA in
situ analysis of postnatal mouse hearts at P5 (data not
shown).
To examine the spatial and temporal expression pattern
of Lbh during early mouse embryogenesis, whole-mount
RNA in situ analysis was performed between E7.5 and E11.
Embryos were then sectioned to determine the specific cell
types expressing Lbh in the various tissues. By this method,
weak Lbh expression was detected as early as the headfold
stage (E7.5) in the anterior mesoendoderm, specifically in
cells of the presumptive cardiac mesoderm and the primitive gut endoderm (Fig. 3A). A similar domain of expression
was also seen at the 1–10 somitic stage, with intensified
expression in the bilaterally symmetrical crescent of the
heart primordia and the caudally extending gut tube (Fig.
3B). Lbh expression remains in the myocardium and gut
endothelium throughout development (Figs. 3C–3F; and
data not shown). By late E8.75 new domains of expression
were visible, including staining in the branchial arches and
the ventral ectodermal ridge (VER), a morphologically distinct epithelium on the ventral surface at the tip of the tail
(Figs. 3C and 3G). At E9.5 Lbh expression was seen in the
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
Early Role of Lbh in Formation of Appendages and Heart
297
otic vesicles, the oral epithelium, the ventral limb ectoderm, and in neural crest-derived sensory neurons of the
dorsal root ganglia, trigeminal ganglia, and acoustic ganglia
(Figs. 3D and 3H–3J; and data not shown). From E10.5
onward, expression of Lbh became more dynamic in the
limbs, heart, and branchial arches and appeared in the
forming urogenital ridge (data not shown).
abuts the atria in this region (Figs. 5D and 5H). Although it
is not clear at present whether this border constitutes a
compartment boundary between ventricular and atrial
myocardium, the AVC myocardium appears to generate the
cues for endocardial cushion formation (Fishman and
Chien, 1997; Srivastava and Olson, 2000). At E10.5 the
right-sidedness of Lbh expression in the ventricular myocardium became even more apparent, with a decrease in
expression levels in the outflow tract (Fig. 5E) and an
increase of expression in the forming ventricular septum,
reflecting progressive segmentation between the RV and LV
(Figs. 5E, 5H, and 5I). By E12, when the different heart
chambers have been established, the L-R asymmetry of Lbh
expression in the ventricular myocardium was lost and
expression was found to be distributed more uniformly (Fig.
5I). Little or no expression of Lbh was detected in the
endocardium, in the cardiac jelly, or in endocardial- and
cardiac neural crest-derived structures (AV- and conal cushions, valves, and atrial septum) (Figs. 5I–5M). Taken together, these observations suggest that Lbh could play a role
at the very early stages of cardiogenesis, as well as in
regionalization of the heart tube.
Lbh Is an Early Marker for Cardiogenesis and
Becomes Regionally Restricted after Heart Looping
Because Lbh was found to be expressed in cardiac progenitor cells, we undertook a more detailed analysis of Lbh
expression in the heart. Shortly after gastrulation, cardiomyocytes originate in the anterior lateral mesoderm. One of
the earliest molecular markers for the cardiac lineage is
Nkx2.5, whereas dHand represents an early marker for
cardiac regionalization (Biben and Harvey, 1997; Lints et
al., 1993). To determine the onset of cardiac Lbh expression, we compared expression of Lbh in E7.5–E8.5 embryos
with that in Nkx2.5, dHand, and Carp, a Nkx2.5 target
gene, respectively (Zou et al., 1997). As shown in Fig. 4, Lbh
mRNA was already detected, albeit at low levels, in the
presumptive heart primordium of early headfold stage embryos (Fig. 4A; see also Fig. 3A) and overlapped with Nkx2.5
and dHand (Figs. 4B and 4C). At this stage, the Carp gene is
not yet expressed, but comes on after the 2 somitic stage
(Figs. 4D, 4H, 4L, 4P). Interestingly, while Nkx2.5 and
dHand were distributed uniformly throughout the myocardial plate at the 0 –5 somitic stages (Figs. 4B, 4C, 4F, 4G, 4J,
4K, 4N, and 4O), Lbh expression initially was stronger in
the anterior myocardium until approximately the 5 somitic
stage (Figs. 3A, 4A, 4E, and 4J). At the same time Lbh
expression levels gradually increased. When the myocardial
plate fuses to form a linear heart tube (6 –7 somitic stages)
and during the morphogenetic process of heart looping
(8 –10 somitic stages), Lbh, much like the other cardiac
marker genes, appeared to be more evenly expressed in the
linear heart tube (Figs. 4M– 4P and 5A). A strong expression
of Lbh was also visible in the sinus venosus (SV), which was
retained in the arising inflow tract later in development
(Figs. 3C, 3D, and 5A–5D).
After heart looping (E9.0), Lbh expression reflected the
beginning regionalization in the heart tube. Lbh became
restricted to the anterior myocardium of the outflow tract
and right ventricle (RV), with reduced levels of expression
in the left ventricle (LV) (Figs. 5C, 5G, and 5H). No Lbh
expression was detected in the posterior atria, which differentiate later than the ventricular myocardium (Figs. 5D and
5H–5K). In addition, Lbh transcripts localized to the outer
proliferative compact layer of the ventricular myocardium
and were detected to a much lesser extent in the trabeculae
(Figs. 5C, 5H, and 5I). High levels of Lbh transcripts were
also detected in the atrioventricular canal (AVC), from
where the tricuspid and mitral valves originate (Figs. 5D,
5E, and 5H). Most notably, a sharp boundary was observed
at the limit of the ventricular Lbh expression domain that
Lbh Expression Marks Ventral Limb Ectoderm and
the AER during Early Limb Bud Outgrowth
It is striking that expression of Lbh in the forming limb
buds is remarkably similar to that of the ventral expression
domain of En1 (Figs. 3D, 6, and 7). To date En1 has been the
only limb marker gene that is selectively expressed in the
ventral nonridge ectoderm and the ventral part of the AER
(Davis and Joyner, 1988). Thus, the specific expression of
Lbh during limb development was examined more closely
by whole-mount and section RNA in situ analyses and
compared with expression of other limb ectodermal markers, such as En1, Fgf8, and Wnt7A (Figs. 6 and 7). Lbh was
first detected at E9.0 in the ectoderm of the presumptive
forelimb field on the lateral body wall (Figs. 6A and 6K).
Expression of Lbh in the hindlimb buds was visible approximately 1 day later and showed a similar pattern (Figs.
6E– 6H). During the earliest stages of limb bud outgrowth
(stages 0.2–1.5), when D-V limb patterning is controlled by
the nonridge ectoderm (Chen and Johnson, 1999), expression of Lbh was found to be confined to the ventral limb
ectoderm, including the pre-AER, which at these stages of
development is localized ventrally (Figs. 6B, 6C, and 6I– 6K).
Significantly, the most distal limit of the ventral Lbh
expression domain at all stages respected the dorsal AER
border, which abuts the dorsal ectoderm that expresses
Wnt7A (Figs. 6C, 6I– 6L, 7G, and 7H). By stages 2–3, when
the AER is thickening and becoming constricted to the tip
of the limb bud, Lbh RNA was detected in both the
maturing AER and the distal ventral limb ectoderm (Figs.
6H and 6L). The proximal limit of ventral ectodermal
expression of Lbh was found to be the more distal than
proximal extreme of the En1 expression domain (Figs. 7B
and 7D; and data not shown), indicating that Lbh was not
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Briegel and Joyner
7A, and 7G). At all stages of limb development examined,
Lbh was expressed evenly along the A-P axis of the AER
(Fig. 6G). AER expression of Lbh persisted until the ridge
disappeared as a distinct morphological structure at about
E12.5 (Fig. 6M; and data not shown). In summary, our
results show that during limb outgrowth, when the distal
limb ectoderm confers D-V polarity to the underlying
mesenchyme, Lbh is expressed with a clear D-V asymmetry
in the distal limb ectoderm. Furthermore, Lbh is expressed throughout the AER, suggesting that Lbh could be
involved in mechanisms controlling both D-V and P-D limb
patterning.
To test the possibility that the ventral expression domain
of Lbh in the limb ectoderm is dependent on En1, a marker
gene expression analysis was performed in En1 lki/lki mutants
(Loomis et al., 1998). As shown in Fig. 7, Lbh expression
appeared normal and was expressed strongly in the ventrally expanded AER and ectopic AER on the ventral side of
an En1 lki/lki mutant limb (Figs. 7I and 7K) (Loomis et al.,
1998; Cygan et al., 1998). Fgf8 expression confirmed the
AER phenotype in the mutant (Fig. 7K). In these mutants Wnt7A is expressed ectopically in the ventral limb
ectoderm and contributes to the aberrant AER formation
(Loomis et al., 1998), but seems to have no effect on ventral
Lbh expression (Figs. 7I and 7L). Lbh was also expressed in
Wnt7A and Lmx1B mutants (Chen and Johnson, 1999).
Thus, Lbh expression appears to be largely independent of
the activity of known D-V-patterning molecules.
FIG. 4. Lbh is an early marker for cardiogenesis. Onset of Lbh
expression in cardiomyocytes (A, E, I, M) was compared with
different early cardiac genes Nkx2.5 (B, F, J, N), dHand (C, G, K, O),
and Carp (D, H, L, P). (A–D) Presomitic headfold stage embryos
viewed from the right side. (E–P) Frontal view of embryos at the 1–2
somitic (E–H), 5 somitic (I–L), and 6 somitic (M–P) stages, respectively. Lbh mRNA is weakly detected in the forming myocardial
plate from the presomitic headfold stage (E7.5) onward (A, B, I) and
appears to overlap with expression of Nkx2.5 (B, F, J) and dHand (C,
G, K). Carp expression is not yet detected at these early stages (D,
H), but comes on between somitic stages 2–5 (H, L). Note that Lbh
is more strongly expressed in anterior cardiomyocytes of the
cardiac crescent (red arrows). At the 6 somitic stage, when cardiomyocytes converge to form a linear heart tube, Lbh and the other
cardiac markers are expressed uniformly throughout the myocardium (M, N, O, P). A, anterior; P, posterior.
expressed in the proximal nonridge ventral ectoderm. Furthermore, whereas En1 is expressed only in the ventral
AER, Lbh expression extended throughout the entire AER,
similar to the expression of Fgf8 (Figs. 7B, 7D, and 7F).
In contrast, no Lbh expression was detected in the most
distal dorsal ectoderm, where Wnt7A is expressed (Figs. 7B
and 7H). Only after approximately stage 1 of limb outgrowth did a minor Lbh expression domain in the proximal
posterior-dorsal nonridge ectoderm become apparent and
there overlapped with Wnt7A expression (Figs. 6D, 6H, 6M,
DISCUSSION
Our study describes the identification and first functional
characterization of Lbh, a member of a new conserved gene
family in vertebrates. We have demonstrated that Lbh
encodes a nuclear protein with transcriptional activation
activity in mammalian cells. Most remarkably, the spatiotemporal expression of Lbh reflects the onset of formation
and early regionalization of both the limbs and the heart. In
the limb bud, Lbh expression displays a dorsoventral asymmetry distally, being initially restricted to the ventral
ectodermal compartment and the AER. In the heart, Lbh
expression initiates in the myocardial plate at the presomitic stage, with the highest levels in the anterior myocardium. During heart chamber formation, Lbh is expressed
transiently in the right ventricle, as well as in both the
atrioventricular canal and the inflow tract. Thus, Lbh could
represent a new cell-intrinsic player in the molecularpatterning mechanisms that control limb development and
cardiogenesis.
Are Lbh Proteins Transcriptional Cofactors?
Murine Lbh is a member of a new conserved family of
small, acidic proteins in vertebrates. A homolog of Lbh in
Xenopus, Xlcl2, was identified as a maternal RNA that
becomes polyadenylated and possibly activated in fertilized
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
299
Early Role of Lbh in Formation of Appendages and Heart
FIG. 5. Lbh expression becomes regionalized soon after cardiac looping. (A–E) Whole-mount in situ hybridization analysis showing
regionalized and dynamic expression of Lbh during heart formation between E8.5 and E10.5. During cardiac jogging (A) and looping (B)
stages, Lbh appears to be expressed throughout the heart tube (ht) and in the sinus venosa (sv), with exception of the atrium (at), which
forms from posterior myocardium (A). (C, D) At E9.5, Lbh is restricted to the outer curvature (yellow arrow) of the outflow tract (ot) and
right ventricle (rv), as well as the atrioventricular canal (AVC), sinus venosus (sv), and at lower levels in the left ventricle (lv). The red arrow
indicates the sharp border of the Lbh expression domain at the atrioventricular (AV) boundary. (E) At E10.5, Lbh expression becomes further
restricted to the right ventricle, AVC, and the forming ventricular septum (indicated by black arrow). The weak staining in the right atrium
(ra) represents shining through of the Lbh-expressing vena cava, which is derived from the sinus venosus. La, left atrium; v, ventricle. (F–I)
Transverse sections of whole-mount embryos from E8.0 to E12.5. (F) At the myocardial plate stage (E8.0), Lbh is expressed with bilateral
symmetry in the promyocardium (pmc). (G, H) Soon after cardiac looping, Lbh expression in the ventricular myocardium acquires
temporary right-sidedness, which is lost by E12.5 (I). Red arrows delineate the AV-border, yellow arrow the outer compact zone of the right
ventricle, and yellow stars the trabeculae, which express Lbh to a lower extent compared to that of the compact zone. Lbh is not expressed
in the atria or the atrioventricular (AV) cushions (marked by black stars). (J–M) Sagittal sections of E9.0 to E12.5 embryos hybridized with
35
S-labeled Lbh antisense probe, showing a strong staining in the ventricular myocardium and the sinus venosus (sv). No staining is
observed in the endocardium (J, K; white stars), the atrioventricular endocardial cushion (avec; K), or the endocardial cushion of
aorticopulmonary septum (ecap; L, M). Sections have been deliberately overexposed to delineate the tissue anatomy. As, aortic sac.
oocytes (Paris et al., 1988; Paris and Philippe, 1990). Although the function of the Lbh/Xlcl2 class of proteins has
not previously been addressed, the available expression data
in Xenopus and our expression studies in mouse show that
both genes are expressed in the embryonic and adult heart
and gut, as well as in adult spleen (Gawantka et al., 1998;
Paris and Philippe, 1990). In addition, we identified homologous ESTs in many other vertebrate species, indicating that
Lbh and Xlcl2 are the founding members of a novel gene
family. The Lbh/Xlcl2 proteins share 77–90% sequence
identity over the entire length of the protein of 103–105
amino acids, suggesting a significant functional conservation.
By transient expression of epitope-tagged Lbh proteins in
COS cells, we have shown that Lbh predominantly localizes to the nucleus. Consistent with this finding, Lbh/Xlcl2
proteins contain a conserved putative nuclear localization
signal characterized by eight basic amino acid residues (Jans
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
300
Briegel and Joyner
FIG. 6. Lbh expression during different stages of limb bud initiation and outgrowth. (A–H) Whole-mount in situ hybridization analysis,
showing different stages of limb outgrowth of forelimb (A–C) and hindlimb (E–H) buds, respectively. (A) Lbh mRNA is detected prior to
limb outgrowth in the ectoderm of the presumptive limb field (indicated by blue arrow). (B, C, E, F) During the earliest stages of limb
outgrowth (stages 0.5–1.5) Lbh is restricted to the ventral limb ectoderm. (D, H) At stages 2–5 of limb outgrowth, expression of Lbh in the
maturing apical ectodermal ridge (AER) becomes distinguishable from expression in the distal ventral limb ectoderm. A minor expression
domain is also apparent in the proximal posterior dorsal ectoderm (indicated by black stars). (I–M) Transverse sections through hind- and
forelimb buds of whole-mount embryos shown above at different stages of limb outgrowth. Red arrows mark the most distal border of the
ventral Lbh expression domain with the dorsal ectoderm, which coincides with the dorsal AER border. D, dorsal; v, ventral; A, anterior;
P, posterior.
et al., 2000). The finding that Lbh/Xlcl2 proteins share an
acidic domain rich in negatively charged glutamate residues
at their C-terminus raises the question of whether these
proteins are transcriptional regulators (Ptashne and Gann,
1997). There are several lines of evidence supporting the
idea that Lbh/Xlcl2 proteins act as transcriptional coactivators. First, when fused to the Gal4 DNA-binding domain,
Lbh can activate gene expression in a transcriptional reporter assay (Sadowski and Ptashne, 1989). Second, the
primary protein structure does not predict a DNA-binding
domain; thus, it seems unlikely that Lbh binds to DNA by
itself. Instead, Lbh could form a protein complex with other
DNA-binding proteins and activate transcription in a synergistic fashion. Third, Lbh proteins contain two ␣ helices,
which could act as protein–protein interfaces. Taken together our data provide the first evidence that Lbh/Xlcl2
proteins could play a role in tissue-specific gene regulation.
Characteristics of Embryonic Lbh Expression
During mouse embryogenesis, Lbh is first detected in the
mesoendoderm that forms the anterior gut and the heart. At
midgestation, Lbh expression is also evident in the limb
bud ectoderm and other specialized epithelia (ventral ectodermal ridge, oral epithelium, and otic vesicles), as well as
in the branchial arches and some neural crest derivatives.
The spatiotemporal expression pattern of Lbh suggests
several recurring themes. First, Lbh expression initiates
early during tissue formation. Second, Lbh expression reflects patterning and regionalization of the tissues where it
is expressed. Third, once early patterning has occurred, the
tissue-specific expression of Lbh becomes more dynamic.
Finally, Lbh is expressed in a variety of signaling centers
that regulate growth and patterning of the embryo.
During cardiogenesis. Lbh transcripts are expressed in
cardiomyocytes as soon as the cardiac crescent forms from
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
301
Early Role of Lbh in Formation of Appendages and Heart
FIG. 7. (A–H) Comparison of Lbh expression (A, B) with the
limb-ectodermal markers En1 (C, D), Fgf8 (E, F), and Wnt7A (G, H)
in wildtype (A–H) or En1 mutant (I–L) limbs at E10.5 (A–H).
Whole-mount in situ hybridization analysis (A, C, E, G) and cross
sections through the forelimbs (B, D, F, H, I–L). (J) X-gal staining
reveals expression of lacZ from the En1 promoter as a marker for
ventral limb ectoderm and ventral AER. Red arrows indicate
formation of ectopic AERs ventrally in En1 lki/lki mutant mice. An
ectopic AER in the En1 mutant is indicated by a star. The dorsal
and ventral borders of the AER are delineated by red and black
arrowheads, respectively. The blue arrow shows a transient middle
border within the AER reflected by En1 expression. D, dorsal; V,
ventral.
the anterior lateral mesoderm shortly after gastrulation.
Although regionalization in the heart does not become
morphologically distinguishable until after cardiac looping,
precursors of different cardiac chambers seem to be specified much earlier (reviewed in Fishman and Chien, 1997).
We found that the initial expression of Lbh in the cardiac
crescent is highest in anterior cardiomyocytes and almost
not detected in more posterior cardiomyocytes that join the
lateral plate mesoderm. Consistently, expression of Lbh is
highest in structures derived from anterior promyocardium,
such as the outflow tract and right ventricle, once looping
has occurred. A remarkably similar, but not entirely identical, expression pattern has been described for the homeobox gene Irx4, which is downstream of Nkx2.5 and
dHand and required for the specification of ventricular
myocardium (Bao et al., 1999; Bruneau et al., 2000). After
cardiac looping, Lbh expression also resembles the right
ventricular-specific expression domain of dHand, which is
required for specification and survival of this chamber
(Srivastava et al., 1995, 1997; Yelon et al., 2000). Thus, early
Lbh expression reflects not only early A-P patterning of the
myocardium before a linear heart tube is formed but also
segmentation of the heart tube after cardiac looping.
Furthermore, Lbh is expressed in other segments of the
heart, the AVC, and the SV, but not in the atria. Intriguingly, cardiac chamber formation is thought to be regulated
by a combinatorial code of widely expressed cardiac transcription factors, such as Nkx2.5 and Mef2C and more
chamber-specific cofactors, such as the basic helix–loop–
helix (bHLH) transcription factors dHand and eHand,
which are predominantly expressed in the primitive right
and left ventricular segments, respectively (Fishman and
Chien, 1997; Srivastava and Olson, 2000). For example,
deletion of both Nkx2.5 and dHand in mice results in
hypoplasia of the right ventricle (Lyons et al., 1995; Srivastava et al., 1997). Our finding that Lbh can act as a
transcriptional activator, therefore, makes Lbh a good candidate for a chamber-specific cofactor of the cardiac transcription factors known to be required for chamber specification.
Another interesting feature of cardiac Lbh expression is
that the most posterior limit of its ventricular expression
domain forms a sharp boundary and coincides with the
atrioventricular (AV) border that separates ventricular from
atrial myocardium, where Lbh is not expressed. It is currently not understood how this border is established. However, retinoic acid signaling could play a role in regulating
AV-border formation, in that it is required for atrial specification (Xavier-Neto et al., 1999 and references therein).
The AV border secretes signaling molecules of the TGF␤
family that induce the endothelial–mesenchymal transition
of endocardial cells during AV cushion and valve formation
(Fishman and Chien, 1997).
Taken together, Lbh expression coincides with functionally and morphologically distinct domains of the forming
heart. Because of the transcriptional activation property of
Lbh, it is tempting to speculate that Lbh could have a
cell-intrinsic role in heart patterning and AV-valve formation. We have performed transgenic misexpression studies
during myocardial development, which indeed indicate that
Lbh can interfere with the molecular pathways that regulate valve formation and septation (Briegel, K. and Joyner,
A. L., manuscript in preparation).
During limb formation. Lbh transcripts are present in
the ectoderm of limb bud territories prior to overt morphological signs of limb bud outgrowth. At this stage the
presumptive limb ectoderm appears to become specified
into distinct dorsal and ventral domains by as-yet unknown
signals emanating from the somites and the lateral somatopleuric mesoderm (Michaud et al., 1997). As the limb bud
extends distally, it is obvious that Lbh expression is con-
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302
Briegel and Joyner
fined to both the distal ventral ectoderm and the developing
AER.
Given the crucial role of En1 in limb patterning (Loomis
et al., 1996, 1998) and our discovery that Lbh is another
molecule expressed in the distal ventral limb ectoderm and
AER, it is therefore tempting to speculate that Lbh might
also be involved in aspects of D-V limb patterning and AER
formation. Interestingly, Lbh is expressed in the limbs of
En1 null mutants, indicating that Lbh is not downstream of
En1, but could act in a separate pathway. Another feature of
Lbh expression in the limb is that the most distal border of
the ventral ectodermal Lbh expression domain coincides
with the permanent boundary between the AER and the
dorsal ectoderm (Kimmel et al., 2000). In contrast, En1
expression defines a middle transient border within the
AER (Loomis et al., 1996; Kimmel et al., 2000). Thus, Lbh
expression might be involved in conveying cell-specific
properties common to AER and ventral ectodermal cells.
After the dorsal D-V boundary is formed, a minor Lbh
expression domain becomes apparent in the posteriordorsal ectoderm. A-P pattern within the dorsal ectoderm is
also suggested by expression of Connexin 43 and a formin
promoter-lacZ reporter construct in transgenic mice (Chan
et al., 1995; Meyer et al., 1997).
Finally, as in the AER, Lbh is coexpressed with members
of the Fgf, Bmp, and Wnt families of secreted molecules in
other epithelial signaling centers. The ventral ectodermal
ridge (VER) at the ventral tip of the tail expresses Fgf17 and
Bmp2 along with Lbh and has been shown to induce the
ventral tail mesenchyme (Goldman et al., 2000). Likewise,
Lbh is coexpressed with Fgf8 in the ectoderm of the
branchial arches and the otic vesicles (Crossley and Martin,
1995; Mahmood et al., 1995).
In conclusion, Lbh represents a member of a new class of
nuclear coactivators in vertebrates. The finding that Lbh
expression during embryogenesis marks specific developmentally significant domains and compartment boundaries
in the developing limb and heart raises the intriguing
possibility that Lbh proteins could be involved in specifying
positional information in a cell-autonomous manner during
vertebrate pattern formation. A possible scenario is that
Lbh proteins act in synergy with other transcription factors
to integrate information from multiple extracellular signals
and, in turn, to induce the necessary cellular changes
required for tissue specification and morphogenesis. Further functional studies will elucidate the roles of Lbh
proteins in development and identify not only their protein
partners but also the signals to which they respond.
ACKNOWLEDGMENTS
We thank Se-Jin Lee, David Ron, Gail Martin, Andy McMahon,
Kenneth Chien, and Eric Olson for reagents. We are grateful to C.
Loomis and D. Yelon for comments on the manuscript. We also
thank N. Dahmane, M. Horowitz, C. Jain, and the members of the
Joyner lab for discussion and encouragement. This work was
supported by a grant from the NIH. A.L.J. is an Investigator of the
Howard Hughes Medical Institute. K.J.B. was a recipient of a
long-term postdoctoral fellowship from the Human Frontiers of
Science Organization (HFSPO) and is currently a Howard Hughes
Research Associate.
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Received for publication November 21, 2000,
Revised February 5, 2001
Accepted February 5, 2001
Published online April 6, 2001
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.