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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 291 292 Briegel and Joyner 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. 293 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). 294 295 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. 296 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 Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 298 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- Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 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. 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