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@OPAL.TUFTS.EDU
Abstract
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
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 Nkx2.5/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
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.
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
View larger version (107K): 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
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):
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.
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
View larger version (151K): 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):
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).
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 larger version (128K):
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.
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
View larger version (164K): 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 ).
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).
View larger version (151K): 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
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
bHLH
gax
Mox-2
pc
SRF
TBS-T
=
=
=
=
=
=
=
-hemaglutinin antibody
basic helix-loop-helix
growth-arrest homeobox
mesoderm/mesenchyme homeobox
post coitum
serum response factor
Tris-buffered saline with Tween 20
Acknowledgments
This study was supported by National Institutes of Health grants AR-40197 and HL50692 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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