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(Circulation Research. 1997;80:452-462.)
© 1997 American Heart Association, Inc.

Articles

Embryonic Expression of the Gax Homeodomain Protein in Cardiac, Smooth, and Skeletal Muscle

Hal A. Skopicki, Gary E. Lyons, Gina Schatteman, Roy C. Smith, Vicente Andrés, Sabine Schirm, Jeffrey Isner, , Kenneth Walsh

From the Division of Cardiovascular Research (H.A.S., G.S., R.C.S., V.A., J.I., K.W.), St. Elizabeth's Medical Center and Tufts University School of Medicine, Boston, Mass; the Cardiac Unit (H.A.S.), Massachusetts General Hospital, Boston; the Department of Anatomy (G.E.L.), University of Wisconsin Medical School, Madison; and the Department of Cardiovascular Research (S.S.), Berlex Biosciences, Richmond, Calif.

Correspondence to Kenneth Walsh, PhD, Division of Cardiovascular Research, St. Elizabeth's Medical Center, 736 Cambridge St, Boston, MA 02135. E-mail kwalsh{at}OPAL.TUFTS.EDU

	Abstract

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Abstract Gax is a homeobox-containing gene that has been detected in adult cardiovascular tissues and exhibits a growth arrest–specific pattern of expression in cultured vascular myocytes. To study the regulation of gax during development, we performed immunohistochemistry and in situ hybridization on mouse embryos. Gax was present in mesodermally and, as with other homeobox genes, neuroectodermally derived tissues. Early mesodermal protein expression was limited to the lateral plate and somitic mesoderm. Gax in the cardiac muscle lineage exhibited a biphasic pattern of expression. Expression was prominent in the heart tube of the earliest cardiomyocytes and remained prominent through the looping stage (day 12.5 post coitum [pc]) but fell below the threshold of detection in atria and ventricles by day 13.5 pc. At day 15.5 pc, Gax protein was again detectable but restricted to cells within the compact layer of the ventricular myocardium. Gax expression was also noted in smooth muscle cells as early as day 9.5 pc. In the skeletal muscle lineage, Gax protein was expressed at the onset of somitogenesis before the expression of the myogenic basic helix-loop-helix and MEF2/RSRF family proteins. Subsequently, it was noted at day 9.5 pc in premyogenic cells migrating into head, trunk, and limb buds. Gax was detected in myotomes, premuscle masses, and mature muscle groups. These data suggest an important developmental role for Gax in all muscle lineages.

Key Words: homeobox gene • Gax • cardiac muscle • smooth muscle • skeletal muscle

	Introduction

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As a homeobox-containing gene, gax is unique because its expression is rapidly downregulated in vascular myocytes after mitogen stimulation in vitro and after acute vessel wall injury in vivo.^{1 2} The expression properties of gax resemble those of the gas and gadd families of genes, some of which encode regulators of cell growth.^{3 4} Gax downregulation in response to mitogen stimulation is dose dependent and correlates with enhanced DNA synthesis. Conversely, expression is upregulated by conditions that favor differentiation and cell-cycle arrest. These findings indicate that gax may coordinate growth and differentiation in vascular smooth muscle cells. Gax also displays a tissue-specific pattern of expression in adults (lung, heart, and vascular smooth muscle), suggesting a possible role in the development of the cardiovascular system.^{1 5}

During development, homeobox transcription factors control cell lineage commitment and differentiation by regulating genes necessary for the mature phenotype. Cardiac, smooth, and skeletal muscles are mesodermal derivatives, but each arises from a different location in the mouse embryo. Cardiac muscle, the first muscle lineage to differentiate and become functional, is derived from lateral plate mesoderm. Between 7.5 and 8.0 days pc, two cardiac primordia fuse at the midline to form a simple contractile tube. Among the transcription factors that may regulate this process are Nkx-2.5/Csx, a homeobox-containing gene,^{6 7 8} GATA-4,⁹ MEF2C,¹⁰ MEF2B,¹¹ dHAND, and eHAND.¹² Similarly, most smooth muscle cells are derived from lateral plate mesoderm, but some vascular smooth muscle cells arise from the neural crest.^{13 14 15} Little is known about transcriptional regulators that are essential for smooth muscle differentiation. MEF2/RSRF family members are expressed in smooth muscle,^{16 17} as is gax.^{1 2} Furthermore, a functional MEF2 site occurs in the gax promoter.¹⁸ Skeletal muscle cells of the trunk and limbs arise from the somites, but some head muscles arise from regions rostral to the first somite, such as the prechordal plate.¹⁹ Skeletal muscle determination and differentiation are regulated by the MyoD family of bHLH transcription factors²⁰ and by the MEF2 family of nuclear factors.^{10 11}

Gax expression has been detected in adult cardiovascular tissues, but its embryonic pattern of expression has not been thoroughly described. Therefore, as a first step toward understanding the role of Gax during embryogenesis, we performed a detailed analysis of its spatial and temporal pattern of expression between 7.5 and 17.5 days pc using immunohistochemistry and in situ hybridization. In addition to its neuroectodermal expression, we report a mesodermal expression pattern that includes the early myocytes of the cardiac, skeletal, and smooth muscle lineages.

	Materials and Methods

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Preparation of Antibodies
Polyclonal IgG antibodies to the Gax protein were generated at Babco by immunizing rabbits against KLH-coupled peptides encoded for in the C-terminal (-Gax 8.1) (amino acids SDHSSEHAHL) and N-terminal (-Gax 2.1) (amino acids LRSPHATAQGLH) regions of the rat Gax protein. Specific reactivity was determined in comparison with that of the rabbit prebleeds. Antibodies were then purified over ImmunoPure protein A columns (Pierce). Further characterization of these antibodies was performed by immunohistochemistry using transiently transfected A10 cells and Western blot analysis of C3H10T1/2 fibroblasts.¹⁸

Preparation of Embryos
Embryos were obtained by the natural matings of C57B1/6JxC57B1/6J mice (Jackson Laboratories, Bar Harbor, Me) as previously described.²¹ The day of vaginal plug detection was designated as day 0.5 pc. Mice were euthanized by cervical dislocation, and embryos from 7.5 to 17.5 days pc were removed. Embryos 12.5 days pc and older were anesthetized with ethyl ether. Embryos were then fixed in methyl Carnoy's fixative for immunocytochemistry or 4% paraformaldehyde for in situ hybridization. Embryos were dehydrated through graded alcohols and paraffin-embedded. Eight-micron sections were analyzed.

Western Blot Analysis
Whole-cell protein extracts from embryonic cardiomyocytes at various ages were a generous gift of Dr Loren Field (Krannert Institute, Indianapolis, Ind). Thirty micrograms of protein per time point was separated on a 7.5% polyacrylamide gel under denaturing conditions and electroblotted on nitrocellulose membranes. The membrane was blocked with 5% nonfat dry milk in TBS-T and then probed with 4 to 7 µg/mL of the -Gax 2.1 and -Gax 8.1 antibodies for 30 minutes at room temperature. After incubation with primary antibody, the blot was washed three times in TBS-T, followed by incubation for 20 minutes with 1:4000 of goat anti-rabbit horseradish peroxidase antibody (Amersham) in 5% milk/TBS-T at room temperature. The blot was again washed in PBS, and antigen-antibody complexes were visualized after incubation for 1 minute with an enhanced luminescence reagent (Amersham) at room temperature, followed by exposure to Kodak XAR-5 film.

Immunohistochemistry
Immunohistochemistry was performed according to a modified version of the method of Schatteman et al.²² Fifteen complete embryos were serially sectioned for immunohistochemical analysis. In addition, individual sections from one to three embryos were assessed at several time points, including at least two embryos for each day between 12.5 and 15.5 days pc. Briefly, embryonic sections were deparaffinized through xylenes, rehydrated through graded ethanols, and then rinsed in 0.1 mol/L sodium phosphate buffer, pH 7.5 (PBS). All incubations were performed at room temperature unless otherwise indicated. Sections were next blocked for 1 hour with 4% goat serum (GIBCO-BRL) in PBS and then quenched in 0.3% hydrogen peroxide in PBS for 30 minutes. Sections were incubated with -Gax 8.1, -Gax 2.1 (1:3000 in 4% goat serum in PBS), or preimmune serum control (1:3000 in 4% goat serum in PBS) for 1 hour and then at 4°C overnight. After a washing in PBS, sections were incubated for 30 minutes with a biotinylated goat anti-rabbit IgG (1:200 dilution in 4% goat serum in PBS; Vector Laboratories Inc). After the sections were washed in several changes of PBS, streptavidin/horseradish peroxidase (1:800, Vector Laboratories Inc) was added and incubated for 1 hour. This was followed by repeated washes in PBS and a single wash in 0.1 mol/L Tris, pH 7.6. 3,3'-Diaminobenzidine (Sigma) was used as the chromagen, and then the slides were counterstained with hematoxylin. Some sections were also analyzed without counterstain. Control sections incubated with preimmune serum or with secondary antibody alone were also analyzed. Peptide competition was performed by preincubating -Gax 8.1 antibody with a 100 times mass excess of its immunogenic peptide (SDHSSEHAHL) for 90 minutes before immunohistochemistry.

In Situ Hybridization
In situ hybridization was performed according to Lyons et al²³ using [³⁵S]UTP-labeled RNA probes prepared from a 195-bp segment that spanned both the coding (154-bp) and 3' untranslated (41-bp) regions of the rat gax gene (nucleotides 953 to 1148) that had been subcloned into the pBluescript SK^- transcription vector (Stratagene Inc). In situ localization was confirmed using two other probes from nucleotides 197 to 382 and 455 to 685 (data not shown). All probes excluded both the homeobox region and CAX repeat. Antisense and sense probes were transcribed using T3 and T7 RNA polymerases (Promega). The probe for MyoD mRNA corresponds to the 3' terminal 1000 nucleotides as described in Sassoon et al.²⁴ Probes were hydrolyzed to an average size of 150 bases, and ethanol was precipitated before scintillation counting of a 1-mL sample.

Tissue sections were deparaffinized through xylenes, dehydrated through graded alcohols, washed in PBS, and then fixed in 4% paraformaldehyde for 20 minutes. After an additional wash and digestion for 7.5 minutes with 20 µg/mL proteinase K (Boehringer-Mannheim) at room temperature, sections were fixed in 4% paraformaldehyde for 5 minutes and then treated with freshly prepared 0.1 mol/L triethanolamine containing 0.25% acetic anhydride. ³⁵S-labeled RNA probe (50 000 cpm/µL) in hybridization buffer (50% formamide, 10% dextran sulfate, 1x Denhardt's solution, 0.4 mol/L SSPE, 1 mmol/L EDTA, 50 mmol/L dithiothreitol, and 1 mg/mL tRNA) was added to each slide. Except for Fig 7G (with hybridizations performed at 45°C), all hybridizations were performed in a humidified chamber at 50°C for 16 hours. Slides were washed with 5x SSC with 10 mmol/L dithiothreitol at 50°C for 30 minutes and 50% formamide in 4x SSC for 20 minutes at 65°C before treatment with a 20 µg/mL RNase A solution (Boehringer-Mannheim). They were then passed through high-stringency washes of 2x SSC for 10 minutes at 37°C and 0.1x SSC for 10 minutes at 37°C before dehydration with ethanol and air drying. Slides were then dipped in photographic emulsion (NTB-2, Kodak), dried, and then exposed in the dark for 1 to 2 weeks. These were then developed (D-19 developer and fixer, Kodak) at 16°C (according to the manufacturer's instructions) and counterstained with toluidine blue. Results were analyzed using bright-field and dark-field optics of a Zeiss Axiophot microscope.

View larger version (107K): [in this window] [in a new window]   Figure 7. Gax mRNA colocalizes with the protein. A and B, The gax (A) and the MyoD (B) probes were hybridized to serial transverse sections of 10.5-day-pc neural tube (n) and one somite. Solid arrows indicate myotome. (The bright object at the left of the neural tube in panel A is a refractile piece of debris. The plane of section was oblique, because of the embryo curvature, so only one differentiating somite was sectioned.) C, Dark-field micrograph of a transverse section of a 12.5-day-pc forelimb bud shows gax gene transcripts at the edges of three developing digital cartilages (c). D, In a parasagittal section of an 11.5-day-pc embryo, gax mRNAs are detected in smooth muscle of the stomach (s) and in myotomes (m). E, Hybridization of the sense control gax probe to a section serial to that in panel D shows the background level of silver grains. F, Gax gene transcripts are detected in skeletal muscle in a transverse section of a 15.5-day-pc forelimb. Arrows point to muscle groups. c indicates cartilage. G, In the 9.5-day-pc embryo, gax mRNA is detected along a gradient within the neural tube (n) and in the second and third brachial arches (ba). H, In a parasagittal section of 14-day-pc ribs (r) and lung and diaphragm (open arrow), gax is detected in lung mesenchyme, skeletal muscle, and surrounding cartilage (arrows). li indicates liver. I, Gax mRNAs are detected in tongue (t) and sinus (si) epithelium (arrow) of a 13.5-day-pc embryo. j indicates jaw. Bar=200 µm (A through E, G) and 400 µm (F, H, and I).

	Results

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Characterization of Antibodies
Two anti-peptide antibodies were generated against separate Gax protein coding regions (Fig 1A). Initial characterization of these serum-derived antibodies was performed via immunohistochemistry and Western blot analysis. Immunohistochemical analysis using the -Gax 8.1 and -Gax 2.1 antibodies revealed nuclear staining in a subset of A10 cells (5%) that were transiently transfected with the pCGN-Gax expression vector (Fig 1B) that produces an N-terminal fusion between the hemaglutinin epitope and the Gax polypeptide.²⁵ A10 cells transfected with the empty pCGN vector did not stain positively with either antibody. Western blot analyses, with either the -Gax 8.1 or -Gax 2.1 antibodies, detected a single band corresponding to the recombinant fusion protein in C3H10T1/2 fibroblasts transfected with pCGN-Gax (Fig 1C). This band was absent in extracts obtained from cells transfected with the pCGN vector alone. An antibody specific for the hemaglutinin epitope (-HA) of the Gax fusion protein also detected a single band of identical electrophoretic mobility for cells transfected with pCGN-Gax. Further evidence of antibody specificity was provided by the abrogation of Western blot signal with preincubation of the anti-Gax antibodies with the corresponding immunogenic peptides (data not shown). Unless otherwise indicated, the immunohistochemical analyses depicted below were performed with the -Gax 8.1 antibody because of its superior signal-to-background stain in tissue sections. However, comparisons of 100 sections from various developmental time points revealed similar patterns of Gax distribution with either antibody. Embryonic sections incubated with preimmune serum or with secondary antibody alone showed no staining, and the signal was abrogated by competition with a 100-fold mass excess of immunogenic peptide in all regions except the renal distal tubule (data not shown).

View larger version (59K): [in this window] [in a new window]   Figure 1. Antibody characterization and cRNA probe structures. A, Schematic of the gax gene with location and sequence of antibodies within the 5' region and 3' termini indicated above the gene. Below the gene are the representations of the three cRNA probes generated for in situ hybridization (for details, see "Materials and Methods"). TNR indicates trinucleotide repeat; HD, homeodomain. B, Immunostaining of A10 cells transfected with either pCGN-Gax or pCGN control vector. C, Western blot analysis of C3H10T1/2 cells transfected with the pCGN-Gax expression vector (G) or vector alone (control [C]). Shown is the recognition of the Gax fusion protein by -HA and by the -Gax 8.1 (-8.1) or -Gax 2.1 (-2.1) antibodies.

Expression of Gax in Mesodermal Derivatives
Gax protein expression was noted in derivatives of the lateral plate (cardiomyocytes, visceral and vascular smooth muscle, and stroma of the lung and kidney) and somitic (skeletal muscles of the trunk and limbs) mesoderm. Muscles and connective tissue of the head and neck also prominently expressed Gax protein. No expression of Gax was detected in the notochord, intermediate mesoderm, or the portion of the lateral plate mesoderm from which the adrenal cortex, hematopoietic system, and lymphatic system are derived (data not shown).

Expression of Gax in Lateral Plate Mesoderm Derivatives
Heart. By days 7.5 to 8.0 pc (presomite stage), Gax protein was noted in the lateral plate mesoderm but was absent in cells surrounding the intraembryonic coelomic cavity, which becomes the pericardial cavity (data not shown). Gax protein was noted in the early heart tube (days 8 to 8.5 pc) (Fig 2A) and rudimentary segmented heart, becoming prominent in the bulbus cordis, primitive ventricle, common atrial chamber, and proximal portion of the horns of the sinus venosus by embryonic day 10.0 pc (Fig 2B). By day 12.5 to 13.0 pc, Gax protein expression appeared to peak (Fig 2C and 2D). By day 13.5 pc, Gax protein was undetectable by immunohistochemistry but again readily detectable by day 15.5 pc in the nuclei of some cells within the compact layer of the ventricle (Fig 2E and 2F). This expression pattern within the ventricular myocardium persisted through day 17.5 pc (data not shown). Gax was not detected at any point during cardiogenesis in the endocardium or epicardium or in the region of the endocardial cushions or interatrial septa.

View larger version (151K): [in this window] [in a new window]   Figure 2. Gax expression in the developing heart. A, At 8.0 days pc, Gax protein was detected in most cardiomyocytes (h). hf indicates headfold; f, foregut; b, red blood cell; and d, decidua. B, At 10.0 days pc, Gax was present but noticeably absent from the endocardium (en) (data shown using the -Gax 2.1 antibody, with similar results using the -Gax 8.1 antibody). bc indicates bulbus cordis; v, ventricle; and tr, trabeculae. C, At 12.5 days pc, Gax was detected in the atrium (a) and in the trabeculae (tr) and developing compact layer of the ventricles (cl) but not in the endocardial cushions. ao indicates aorta; or, outflow ridge; and vs, ventricular septum. D, Higher power view is shown of the ventricle and trabeculae (tr) of a 12.5-day-pc heart. cl indicates compact layer; vs, ventricular septum. E, Low power view is shown of a 15.5-day-pc embryo with nuclear Gax expression in compact layer of the heart. es indicates esophagus; t, thymus; a, atrium; v, ventricle; vs, ventricular septum; lu, lung; di, diaphragm; and li, liver. F, Another 15.5-day-pc embryo is shown. p indicates pericardium; v, ventricle. Note the Gax expression in the nuclei of the ventricular compact layer (arrows). In panels A, C, and D through F, sections were immunostained with the -Gax 8.1 antibody, and in panel B, the section was immunostained with the -Gax 2.1 antibody. Relative magnifications are as follows: x4 (E), x10 (C), and x20 (A, B, D, and F).

The biphasic expression of gax transcripts was also detected by in situ hybridization (Fig 3A to 3C). At day 12.5 pc, gax mRNA expression was detected in both the atria and ventricles, but by day 14 pc, the level of in situ signal had dropped close to background, whereas a strong signal was still detected in the lung. By day 15.5 pc, gax mRNA was once again detectable, limited to the compact layer of the ventricle. Immunohistochemical analyses, performed with the -Gax 8.1 or -Gax 2.1 antibodies without counterstain, revealed appreciable perinuclear/cytoplasmic staining in separate 8.0- to 8.5-pc embryonic hearts (Fig 3D and 3E). However, prominent nuclear signal was also detected in cardiomyocytes from day-15.5-pc hearts (Fig 3F and 3G). To test whether the same protein was recognized by both antibodies in the early versus late embryonic cardiomyocytes, Western blot analyses were performed on day-12-pc hearts, chosen because of relative tissue abundance and Gax's appreciable cytoplasmic/perinuclear expression (Fig 2D) and 15- and 18-day-pc hearts because of the presence of appreciable nuclear signal at these time points (Fig 3F and 3G and data not shown). In embryonic day-12, -15, and -18 hearts, both antibodies recognized bands of identical electrophoretic mobility, corresponding to the predicted molecular weight of endogenous Gax¹ (Fig 3H).

View larger version (0K): [in this window] [in a new window]   Figure 3. A through C, Gax mRNA expression is biphasic in developing cardiac myocytes. A, A frontal section through a 12.5-day-pc heart reveals gax gene transcripts in atrial (a) and in both compact (arrow) and trabecular (tr) myocytes of the ventricle. f indicates forelimb; or, outflow ridge; and vs, ventricular septum. B, In a parasagittal section of the 14-day-pc embryo, the level of in situ signal has dropped close to background level in the atrium (a) and ventricle (v, arrow points to compact layer) but is still detectable in the lung (l). li indicates liver. C, In a transverse section of a 15.5-day-pc embryo, gax mRNAs are once again detectable in the compact layer (arrow) of the ventricle (v) but not in atrial (a) myocytes. These mRNAs are also expressed in smooth muscle of the esophagus (e) and lung (l). Red blood cells (b) in the atrium and pulmonary artery (pa) are refractile under dark-field illumination. r indicates rib. Bar=350 µm. D through G, Immunohistochemical analyses of Gax protein in the developing heart at early and late time points using -Gax 8.1 or -Gax 2.1 antibodies. Predominantly cytoplasmic/perinuclear staining is seen at higher magnification in day-8 to -8.5-pc embryonic myocardium with either the -Gax 8.1 (D) or -Gax 2.1 (E) antibodies. Marked nuclear staining within the compact layer of the ventricle (cl) was apparent at day 15.5 pc with either antibody (F and G). tr indicates trabeculae. H, Western blot analyses of Gax protein expression in extracts from embryonic heart using -Gax 8.1 or -Gax 2.1 antibodies. Western blot analysis with the -Gax 2.1 or -Gax 8.1 antibodies against protein extracts from embryonic mouse hearts at approximate embryonic days 12, 15, and 18 (E12, E15, and E18, respectively) revealed a single band, corresponding to the predicted molecular weight of the endogenous Gax protein. A third antibody, raised against a peptide from a distinct region of the Gax protein, also detected an identical electrophoretic band pattern (data not shown).

Smooth muscle. Gax protein expression was detected in the embryonic hindgut by day 9.5 pc (Fig 4A). Protein expression was clearly apparent in two layers of the stomach by day 12.5 pc, a well-defined inner (submucosal) and a more diffuse external layer, which extended from the stomach to the distal intestine (Fig 4B). By day 15.5 pc, Gax expression appeared more discretely organized and in later embryos was present in two clearly distinct layers, the muscularis submucosa and muscularis externa (Fig 4C). Gax expression in the submucosa appeared to be predominantly nuclear, whereas the staining was more diffuse in the muscularis externa (Fig 4D). Other sites of visceral smooth muscle that expressed Gax included the posterior pharynx and esophagus (Figs 2E and 6F), the diaphragm (Fig 2E), and the bladder (data not shown). Gax expression was present within the mesenchyme surrounding the tubules and glomeruli in the kidney by day 12.5 pc (data not shown) in a pattern consistent with mesangial and juxtaglomerular cells, which have contractile and proliferative properties similar to smooth muscle cells. Gax protein expression was also detected within the lung and was restricted to the mesodermally derived mesenchyme (data not shown), which expresses smooth muscle markers.²⁶ At no point during development was Gax detectable within the endodermally derived bronchi or respiratory epithelium of the lung. Expression of Gax was detected in the embryonic vasculature of several organs including thymus (Fig 2E) and kidney (data not shown). With the exception of umbilical vessels, Gax was not readily detectable in larger blood vessels at early developmental time points but was detectable within the maternal component of the decidua (data not shown). Of note, Gax protein has been detected in the smooth muscle of adult human aorta, vasa vasorum, saphenous veins, and internal mammary arteries (H.A. Skopicki and K. Walsh, unpublished data, 1996).

View larger version (128K): [in this window] [in a new window]   Figure 4. Gax expression in visceral smooth muscle. A and B, Gax protein was detected in the 9.5-day-pc hindgut (hg) (A) and the 12.5-day-pc stomach (B). n indicates neural tube; s, somite; st, stomach; and k, kidney. C, Gax immunostain of the 15.5-day-pc stomach delineates the muscularis mucosa (arrow) and muscularis externa (arrowhead). st indicates stomach. D, High-power view of the muscularis mucosa (mm) and muscularis externa (me) shows staining for Gax at 15.5 days pc. Relative magnifications are as follows: x10 (B), x20 (A and C), and x40 (D). Immunostaining was performed with the -Gax 8.1 antibody.

View larger version (164K): [in this window] [in a new window]   Figure 6. Gax expression in developing limb, head, and neck muscle. A through C, Gax was detected in the nuclei of limbs of 12.5-day-pc (A), 13.5-day-pc (B), and 17.5-day-pc (C) embryos. dist indicates distal; prox, proximal; li, liver; r, ribs; d, digit; and skm, skeletal muscle. D, Gax was detected in the cochlea (c) and extraocular muscles (eo), among other skeletal muscles of the head. w indicates whisker follicles; skm, skeletal muscle; and r, ribs. E and F, Gax protein was also found within the olfactory epithelium (si) and tongue (t) (E) and in the proximal portion of the esophagus (arrow) (F). w indicates whisker follicles; mc, mesodermal condensation; r, ribs; v, ventricle; and a, atrium. G, External facial muscles and whiskers expressing Gax protein at 15.5 days pc are shown. ls indicates lens; rt, retina; skm, skeletal muscle; and w, whisker follicles. In panels A, B, and D through G, sections were immunostained with the -Gax 8.1 antibody, and in panel C, the section was immunostained with the -Gax 2.1 antibody. Relative magnification x4 for all.

Gax Expression in Somites and Skeletal Muscle
Gax protein was detected in the first somites around day 8.0 pc, and this signal intensified with mesodermal condensation (Fig 5A and 5B). During somite differentiation, Gax was prominently and uniformly expressed throughout the dermatomyotome and sclerotome (Fig 5C). Gax expression remained high during migration of cells from somites into the trunk around day 9.0 pc (Fig 5D). Neither myogenin nor MyoD protein was present in the migrating myoblasts of adjacent sections at this time point (data not shown; see also Reference 27²⁷ ). Gax protein was noted in myoblasts surrounding the early chondrification centers of ribs and vertebrae (Fig 5E). Gax expression was present in lateral somites migrating into the developing limbs as early as day 9.5 pc (Fig 5F). By days 12.5 to 15.5 pc, both the forelimbs and hindlimbs displayed expression at the periphery of the limb buds and in developing muscle masses (Fig 6A to 6C). Gax protein in the branchial arches was apparent by day 9.5 pc (data not shown), and several head and neck muscles, including the tongue, jaw, and extraocular muscles, had clear evidence of Gax expression by days 10.5 to 15.5 pc (Fig 6D and 6G). High levels of Gax protein were also noted in the region surrounding, but not within, whisker follicles (Fig 6D, 6E, and 6G).

View larger version (151K): [in this window] [in a new window]   Figure 5. Gax expression in somites (s), body wall, and developing limbs. Gax was detected in somitic cells in 8.0-day-pc (A), 8.5-day-pc (B), 9.0-day-pc (C), and 9.5-day-pc (D) embryos. Gax was detected in myoblasts (arrows) and skeletal muscles of the trunk at day 11.5 pc (E) and premyogenic cells migrating into the limb bud (lb) at 9.5 days pc (F). n indicates neural tube; d, dermatomyotome; m, migrating cells; a, aorta; g, gut; v, vessels; li, liver; and r, ribs. Relative magnifications are as follows: x10 (E and F) and x20 (A, B, C, and D). Immunostaining was performed with the -Gax 8.1 antibody.

Neurectodermal Derivatives Express Gax
As with most homeobox genes, a neuroectodermal distribution of Gax protein was detected. Expression of Gax in the central nervous system was noted by day 8.0 pc (Figs 2A and 5A), extending from the ventricles of the developing brain to the neural tube (in the ventricular zone of the brain and ependymal [proliferating] and mantle layers of the neural tube) (data not shown). Derivatives of the neural tube expressing Gax included the retina (Fig 6G) and olfactory epithelium (Fig 6E), and neural crest derivatives that express Gax included the cranial ganglia, adrenal medulla, and dorsal root ganglia (data not shown).

Localization of Gax Gene Transcripts
In situ hybridization was used to examine whether gax mRNA could be detected in the same tissues as the protein. Fig 7 presents a summary of the in situ hybridization results. Fig 7A shows gax expression in differentiating somites. Gax was detected in more cells than the myotome, which was labeled by a probe for MyoD (Fig 7A and 7B). Fig 7C demonstrates gax expression in developing limbs, and Fig 7D demonstrates gax expression both in skeletal muscle cells in the developing myotomes and in the differentiating visceral smooth muscle of the stomach. The level of background grains in these experiments was very low (Fig 7E). Fig 7F shows gax expression in skeletal muscles at 15.5 days pc. Fig 7G demonstrates gax expression in the branchial arches and neural tube caudal to the regions seen in Fig 7A and 7B. Fig 7H shows gax expression in lung mesenchyme. Fig 7H also shows gax expression in intercostal muscle, in the diaphragm, and around cartilage. Fig 7I shows gax expression in tongue, in nasal sinus epithelium, and around the oral cavity. Gax gene transcripts were also detected surrounding the inner ear (Fig 7I).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study reports the embryonic expression pattern of the homeobox gene, gax. Mesodermal expression of Gax includes all three muscle lineages. Neuroectodermal expression includes both the central and peripheral nervous systems and the neural crest derivatives, such as cranial ganglia and dorsal root ganglia. Throughout development, gax mRNA was detected in the same tissues as Gax protein. The results reported here, obtained by in situ hybridization and immunohistochemistry, are in contrast to a previous report on this gene (referred to as Mox-2), which noted only expression in somites that became restricted to the sclerotome (Candia et al²⁸ ). The reasons for the differences between our results and those of Candia et al are not clear. Likely explanations include the greater sensitivities and specificities of the probes used in the present study. For example, Candia et al used only in situ hybridization to detect Mox-2 gene expression and noted a low level of expression even after 3 to 4 weeks of exposure to photographic emulsion. The cRNA probes in the present study were shorter, excluded the trinucleotide repeat and homeodomain, and required only 1- to 2-week exposures to photographic emulsion. Moreover, in our hands, the use of polyclonal antibodies was more sensitive than the in situ technique.

Gax in Cardiac Muscle Development
Gax expression in the developing heart was detected in cardiomyocytes but not in endocardial or pericardial structures. Moreover, Gax was not present in cells contributing to the endocardial cushions or the outflow tracts. Gax expression in cardiomyocytes was biphasic. Gax protein was detected very early in cardiac development, being clearly evident by day 8.0 pc in the heart tubes, and its expression appeared to intensify in both atrial and ventricular myocytes past day 12.5 pc. However, by day 13.5 pc, the levels of Gax protein were below the limit of immunohistochemical detection. This drop in Gax protein expression correlated with a decrease in gax mRNA expression. By day 15.5 pc, Gax protein and mRNA were again detectable but limited to cells within the compact layer of the ventricle.

Appreciable perinuclear/cytoplasmic Gax signal was detected with either of two antibodies in early embryonic cardiomyocytes. In addition to early cardiomyocytes, notable cytoplasmic/perinuclear expression was also apparent in neuroblasts, cells of the adrenal medulla, and some visceral smooth muscle. Thus, it is tempting to speculate that Gax may accumulate in the cytoplasm (in response to a nuclear exclusion mechanism) in anticipation of precise developmental time points when it will be required for transcriptional regulation within the nucleus. A similar regulatory mechanism has been proposed for the Drosophila homeodomain protein Extradenticle.²⁹ Alternatively, Gax may have regulatory functions within the cytoplasm, as has been recently described for the bicoid homeoprotein, which inhibits caudal translation by binding, via its homeodomain, to a site in the 3' untranslated region of the caudal transcript.^{30 31} A number of other transcription factors, including MyoD and SRF, are also regulated at the level of import into the nucleus.^{32 33}

Gax in Smooth Muscle Differentiation
The embryonic origins of smooth muscle are less well defined than those for striated muscle. A population of vascular smooth muscle cells is derived from neural crest, but other vascular smooth muscle cells have an origin independent of neural crest.^{14 15} Visceral smooth muscle cells arise from the lateral plate mesoderm and from local mesenchyme within developing organs, apparently through inductive processes.¹³ It has been difficult to identify smooth muscle cells during embryonic development because of a lack of specific markers. Smooth muscle myosin heavy chain appears to be a marker for these cells in the mouse embryo.³⁴

Our observations suggest that Gax may be an early marker for visceral smooth muscle differentiation. We detected Gax protein in the gut primordium by day 9.5 pc, which is 3 days before the detection of smooth muscle myosin heavy chain mRNA.³⁴ SM22 mRNA, although present at day 9.5 pc in the vascular smooth muscle, is not expressed in visceral smooth muscle until 13.5 pc.³⁵ Like Gax, SM22 is also expressed in cardiac and skeletal muscle during development. The expression of Gax in the developing gut is also similar to that of Nkx-2.5/Csx and other homeobox genes.^{8 36 37 38} Although the null mutation of Nkx-2.5 shows no gastrointestinal abnormalities (perhaps because embryos are not sufficiently developed when they die in utero), disruption of the Drosophila homeobox gene tinman, a homologue of Nkx-2.5, results in the absence of visceral smooth muscle of the midgut.^{6 39} The expression of Gax in the renal and pulmonary stroma is consistent with the detection of Gax in cells of the smooth muscle lineage. As has been noted by several investigators, the renal cortex contains contractile cells (juxtaglomerular and mesangial cells) that are similar in phenotype to smooth muscle.⁴⁰ In fact, gax mRNA expression has previously been detected in adult mesangial cell culture.¹ Moreover, muscle cell markers such as -actin and smooth muscle myosin have been described in the developing lung, where Gax is also expressed.^{26 41}

Gax in Somites and Skeletal Muscle
Gax protein expression occurs with the earliest somite formation, and its expression persists as the somites differentiate into sclerotome and premyogenic cells migrate to form both body wall and limb myoblasts.^{42 43} Gax protein expression is present in somites before the expression of the first myogenic bHLH proteins.⁴⁴ Gax is also detected before the appearance of the mef2 family mRNAs.¹⁰ At later stages of skeletal muscle development, Gax is detected predominantly at the ends of developing muscle fibers, a localization that has been noted for mef2C⁴⁵ and mef2B¹¹ transcripts.

Gax expression in presumptive migrating myoblasts is similar to that of Pax-3, a mouse paired homeobox gene that labels the migratory cells of the dermatomyotome and developing limb buds.^{46 47} Splotch mice, which express a mutated Pax-3 gene, lack limb musculature. Gax expression in limb buds appears first proximally and then along the periphery of the limb, avoiding the apical ectodermal ridge. This is coincident with the migration pattern of committed myoblasts, which also follows a proximal to peripheral pattern and is unlike other homeobox genes, such as the Hox 4 genes, which are more concentrated distally and posteriorly, and undergo sequential activation at the limb's tip as it continues to grow.⁴⁸ Finally, Gax is also expressed in all skeletal muscles of the face, jaw, and neck.

Gax and MEF2
At a molecular level, the conserved features of cardiac, smooth, and skeletal muscle developmental programs are poorly understood. The MEF2 transcription factors represent one component of the regulatory mechanism controlling gene expression in each of these cell lineages (for review, see Reference 49⁴⁹ ). The identification of Gax in all three muscle types suggests that it may also participate in this common regulatory pathway during growth arrest and differentiation. Interestingly, Gax overlaps in its expression pattern with mef2 transcripts, particularly mef2B and mef2C, known regulators of gene transcription in all three muscle types.^{10 11 50} Similar to Gax protein expression, expression of mef2C mRNAs becomes evident in the precardiac mesoderm beginning at day 7.5 pc, returning to near background levels by day 13.5.¹⁰ The somitic expression of mef2 transcripts is similar to that of Gax, but mef2 transcripts are limited to the myotome. Interestingly, Gax and MEF2 are expressed in cells of neuronal lineage, which suggests that they may also have a role in neuron differentiation.⁵¹ The overlapping patterns of mef2 and Gax expression and the direct regulation of gax gene transcription by MEF2 noted previously¹⁸ indicate that MEF2 may participate in the regulation of Gax expression during embryogenesis. These data also suggest that the Gax homeoprotein may mediate some of the developmental functions of the MEF2 transcription factors.

In summary, these data demonstrate that Gax is expressed in all three muscle lineages. Gax protein expression occurs early in cardiogenesis with detection in the heart tube several days before cardiac looping. In addition, Gax is among the earliest proteins expressed in smooth muscle cells, appearing in the gut by embryologic day 9.5 pc. In the skeletal muscle lineage, Gax protein expression in somites occurs early, before the appearance of the myogenic bHLH proteins, and Gax is also expressed in migrating myoblasts and in mature skeletal muscle in all regions of the embryo. On the basis of its structure as a homeodomain protein, its early embryonic expression pattern, and its expression in muscle that coincides with the MEF2 transcription factors, Gax may play an important role in the differentiation pathway in myocytes of all three muscle lineages.

	Selected Abbreviations and Acronyms

 

-HA = -hemaglutinin antibody bHLH = basic helix-loop-helix gax = growth-arrest homeobox Mox-2 = mesoderm/mesenchyme homeobox pc = post coitum SRF = serum response factor TBS-T = Tris-buffered saline with Tween 20

	Acknowledgments

 
This study was supported by National Institutes of Health grants AR-40197 and HL-50692 to Dr Walsh and HL-02824 and HL-53354 to Dr Isner and by grants from the Muscular Dystrophy Association and the American Heart Association of Wisconsin to Dr Lyons. We thank Dr Loren Field for his generous gift of embryonic heart protein. Dr Skopicki thanks Dr Valentin Fuster for his guidance. We thank Bruce Micales, Marianne Kearney, and Jason Lowry for technical assistance, Danute Nitecki for design of the antigenic peptides, Maria Andrés for artwork, and Linda Whittaker for aid in the preparation of the manuscript.

Received May 16, 1996; accepted December 18, 1996.

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