Development of the mammalian axial skeleton
requires signaling through the Gαisubfamily
of heterotrimeric G proteins
Nicholas W. Plummera,1, Karsten Spicherb, Jason Malphursa, Haruhiko Akiyamac, Joel Abramowitza, Bernd Nürnbergd,
and Lutz Birnbaumera,1
aLaboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health/Department of Health and Human Services,
Durham, NC 27709;bFederal Institute for Drugs and Medical Devices (BfArM), D-53175 Bonn, Germany;cDepartment of Orthopaedics, Kyoto University, Kyoto
606-8507, Japan; anddDepartment of Pharmacology and Experimental Therapy, Institute for Experimental and Clinical Pharmacology and Toxicology,
Eberhard Karls University, and Interfaculty Center of Pharmacogenomic and Drug Research, 72074 Tübingen, Germany
Contributed by Lutz Birnbaumer, November 16, 2012 (sent for review October 9, 2012)
129/SvEv mice with a loss-of-function mutation in the heterotri-
meric G protein α-subunit gene Gnai3 have fusions of ribs and
lumbar vertebrae, indicating a requirement for Gαi(the “inhibi-
tory” class of α-subunits) in somite derivatives. Mice with muta-
tions of Gnai1 or Gnai2 have neither defect, but loss of both Gnai3
and one of the other two genes increases the number and severity
of rib fusions without affecting the lumbar fusions. No myotome
defects are observed in Gnai3/Gnai1 double-mutant embryos, and
crosses with a conditional allele of Gnai2 indicate that Gαiis spe-
cifically required in cartilage precursors. Penetrance and expressiv-
ity of the rib fusion phenotype is altered in mice with a mixed
C57BL/6 × 129/SvEv genetic background. These phenotypes reveal
a previously unknown role for G protein-coupled signaling path-
ways in development of the axial skeleton.
mouse|thoracic|sternum|lateral plate mesoderm
couple a wide variety of cell-surface receptors to intracellular
effectors, such as ion channels and enzymes (1–3). The complex
signal-transduction activity of these widely expressed proteins
has long been studied at the biochemical and cellular level, but
their role in development of whole organisms is less well un-
derstood. The “inhibitory” class of α subunits (Gαi), originally
named for its ability to inhibit adenylyl cyclase activity, is enco-
ded by the Gnai1, Gnai2, and Gnai3 genes. The three Gαisub-
units share 85–95% amino acid sequence identity, and they form
a subfamily with the neuronal α-subunit (Gαo/Gnao), the trans-
ducin α-subunits expressed in rod (Gαt-r/Gnat1) and cone cells
(Gαt-c/Gnat2), and gustducin (Gαgust/Gnat3) expressed in taste
buds. The Gαigenes are linked in pairs with the transducin and
gustducin genes on mouse chromosomes 3, 5, and 9. This link-
age, together with their sequence homology, suggests that these
subunits evolved from an ancestral G protein gene by a tandem
duplication followed by two block duplications (3). A Gαi
ortholog is present in Drosophila, and identification of transducin
genes in the lamprey genome indicates that the initial duplica-
tion to form an ancestral Gαiand Gαtgene predates the evolution
of gnathostomes (4).
Targeted loss-of-function mutations of all three Gαi genes
have been generated in mice, and the resulting phenotypes in-
dicate that Gnai1 and Gnai2 have gene-specific functions in
a wide variety of tissues: loss of Gnai1 affects long-term memory
(5), and Gnai2 knockout mice spontaneously develop an in-
flammatory bowel disease resembling ulcerative colitis (6) and
have altered heart rate dynamics (7). Initial analyses of Gnai3
knockout mice did not reveal an associated phenotype (8, 9), but
more recently Gnai3 has been shown to be required for insulin-
mediated regulation of autophagy in hepatocytes (10). Com-
parison of Gnai2 knockouts and Gnai3/Gnai1 double-knockouts
he heterotrimeric G protein α-subunits, encoded by 16 paral-
ogous genes in humans and mice, are cytoplasmic proteins that
suggests that the three Gαiproteins may also have both over-
lapping and gene-specific roles in the response of macrophages
and splenocytes to bacterial infection (11). Here, we demon-
strate that Gnai3 expression in sclerotomal derivatives is re-
quired for normal patterning of the axial skeleton. Gnai1 and
Gnai2 partially compensate for loss of Gnai3, and the phenotype
is dependent on genetic background.
Skeletal Defects in Gnai3−/−Mice. Inbred 129/SvEv mice that are
homozygous for a targeted loss-of-function mutation in the
Gnai3 gene (12) are viable and fertile, but staining of skeletons
revealed an unexpected phenotype: 95% of 129/SvEv-Gnai3−/−
mice have fusions of the cartilaginous portion of the distal ribs
(Fig. 1B and Table 1, first row). These fusions involve any of the
true ribs (those ribs that articulate with the sternum) but usually
do not affect the false ribs, the ends of which are free in the body
wall. The proximal bony portions of the ribs appear normal, and
in all cases the normal complement of ribs is present. The single
animal that lacked rib fusions had small triangular outgrowths of
cartilage at the distal end of the second rib pair where they join
the sternum. One animal had an eighth rib (first false rib) with
an ectopic connection to the sternum, but other than that,
contacts between ribs and sternum appeared normal in all of the
mice. In fetuses stained with Alcian blue, the rib fusions are
visible as early as embryonic day (E) 14.5, suggesting that they
occur as the ribs develop and are not caused by later overgrowth
In the lumbar region, we observed deformation or partial fu-
sion of one or more vertebral bodies in 9 of 10 Gnai3−/−pups
(Fig. 2). In 7 of 10 pups, lumbar abnormalities consisted of
a small “bridge” of bone connecting the bodies of two or three
adjacent vertebrae (Fig. 2B, arrow), and in 2 of 10 pups the
deformed vertebrae have pointed outgrowths that do not actually
fuse. Bony lumbar fusions were not observed in wild-type and
heterozygous pups, but one of eight wild-type and three of nine
heterozygotes had deformed vertebrae. The frequency of bony
lumbar fusions in Gnai3−/−mice is statistically significant com-
pared with heterozygotes (P < 0.01, Fisher’s exact test).
Because skeletal abonormalities have not been reported for
a different Gnai3 knockout allele that was generated on a C57BL/6
background (8), we investigated whether genetic background
can modify the rib fusion phenotype. In the F2 generation of
a C57BL/6J × 129/SvEv intercross we observed reduction in
Author contributions: N.W.P., K.S., J.A., B.N., and L.B. designed research; N.W.P., K.S., J.M.,
and J.A. performed research; H.A. contributed new reagents/analytic tools; N.W.P., J.A.,
B.N., and L.B. analyzed data; and N.W.P. and L.B. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence may be addressed. E-mail: email@example.com or plummer@
| December 26, 2012
| vol. 109
| no. 52www.pnas.org/cgi/doi/10.1073/pnas.1219810110
penetrance and expressivity of the rib fusion phenotype. Less
than 40% of B6129F2-Gnai3−/−mice had rib fusions (Table 1),
and in the animals with fusions, the average number was reduced
from 2.7 to 1.3 (P < 0.001, unpaired t test). We observed lumbar
defects in only 13 of 61 mice, a significant reduction relative to
the inbred 129/SvEv background (P < 0.01, Fisher’s exact test).
However, the fusions in several B6129F2-Gnai3−/−mice were
more extensive than those observed in any of the 129/SvEv-
Gnai3−/−mice (Fig. 2C). Whole-genome SNP genotyping of 35
B6129F2-Gnai3−/−mice (18 with rib fusions, 17 without) did not
reveal a major locus associated with presence or absence of rib
fusions, suggesting that these axial defects are modified by
multiple loci acting additively.
Effects of Gnai1 and Gnai2 Mutations. The amino acid sequences
encoded by the mouse Gnai1 and Gnai2 genes are, respectively,
94% and 85% identical to Gnai3. Given this degree of similarity,
it would not be surprising if the three proteins have some over-
lapping function. We intercrossed Gnai1, Gnai2, and Gnai3
knockout mice to investigate whether the other Gαigenes also
contribute to skeletal development (Table 1).
In Gnai3−/−mice with either heterozygous or homozygous loss
of Gnai1, the rib fusions are significantly more severe, frequently
involving false ribs as well as true ribs (Figs. 1 C–D and 3). In
addition to rib fusions, we observed asymmetric contacts with the
sternum and fusions of sternebrae in the double mutants (Fig.
1D and Table 1). Lumbar fusions were not noticeably more se-
vere in Gnai3−/−Gnai1−/−compared with Gnai3−/−mice.
Complete loss of both Gnai3 and Gnai2 is lethal before E10
(10), but mice with the genotype Gnai3−/−Gnai2+/−are viable
and have rib fusions equivalent in severity to Gnai3−/−Gnai1−/−
(Figs. 1E and 3). Animals with the genotype Gnai1−/−Gnai2−/−
had no rib fusions, but one had an eighth rib ectopically con-
nected to the sternum (Fig. 1F). This phenotype was also ob-
served in one Gnai3−/−Gnai2+/−mouse and one Gnai3−/−
mouse. Taken together, these data indicate that all three Gαi
genes participate in skeletal development, but Gnai3 is the most
important; rib fusions were never observed in any animal that
was not homozygous for loss of Gnai3 (Table 1).
GαiIs Required in Rib Precursors. During vertebrate development,
somites differentiate into dermotome, myotome (which gives rise
to the intercostal muscles), and sclerotome (which gives rise to
the ribs and spinal column) (13). Growth of ribs is controlled by
a signaling cascade initiated by Hox gene expression in myotome
of the thoracic region and transmitted to the sclerotome by
PDGF and FGF signaling (14–16). In mice with mutations that
disrupt expression of myotome-specific genes required in this
signaling pathway, rib fusions are preceded by disorganization
and fusion of developing intercostal muscles, with initial myo-
tome defects visible by E10.5–E11.5 (17, 18). Therefore, the rib
fusions in Gnai3 mutant mice could reflect a requirement for Gαi
in either developing musculature or skeleton.
We used a muscle-specific Myog (myogenin) probe to reveal
morphology of developing intercostal muscles in wild-type and
Gnai3−/−Gnai1−/−embryos at E11.5 and E12.5. The staining
pattern in wild-type and mutant embryos was indistinguishable at
both developmental stages (Fig. 4 A–D). At E12.5, a Sox9 probe
revealed what may be the initial stages of fusion of the rib pri-
mordia; in three of five mutant embryos, the distal ends of the
Sox9-expressing domains appeared broader than the equivalent
regions of four wild-type embryos, and in several places adjacent
domains were in contact (Fig. 4F, arrowhead). The lack of ob-
vious myotome defects during this period suggested that the
skeletal defects in Gnai3−/−mice are not secondary to a re-
quirement for Gαiin muscle.
Because mutation of Gnai1 or Gnai2 increases the severity of
rib fusions in Gnai3−/−mice, we reasoned that a conditional
allele of Gnai2 could be used as an independent test of the tis-
sue-specific origin of the rib fusion phenotype. We made a con-
ditional allele of Gnai2 (Fig. 5) in which loxP sites flank exons
2, 3, and 4, and we used a Myog-cre transgene (19) to drive Cre
expression in skeletal muscle precursors and a Sox9creknock-in
allele (20) to drive Cre expression in cartilage precursors, in-
cluding the sclerotomal cells that give rise to the ribs. In mice that
have the genotype Gnai3−/−Gnai2flx/+or Gnai3−/−Gnai2flx/flx,
severity of the rib phenotypes should be increased by expression
of Cre recombinase in the critical tissue. The Myog-cre transgene
had no effect, but the Sox9creknock-in significantly increased the
severity of rib fusions in the double-mutant mice (Fig. 6).
These results confirm that the rib fusion phenotype is caused
by loss of Gαiin cartilage. The reduced penetrance of the rib
images show the sternum with true ribs attached. False ribs that are not
involved in any fusions have been removed. (A) Gnai3+/−, 19-wk-old, show-
ing normal morphology of sternum and true ribs. (B) Gnai3-/−, 19-wk-old,
with fusions of the cartilaginous portion of four rib pairs. The fusions involve
true ribs only. Although there is the normal number of symmetric contacts of
ribs and sternum, the sternum itself is distorted. (C) Gnai3−/−Gnai1+/−, 22-
wk-old, with fusion of nine rib pairs. On the right side, the first false rib is
fused to the seventh true rib (blue arrowhead). (D) Gnai3−/−Gnai1−/−, 41-wk-
old, with fusions involving all but one of the true ribs and one false rib (blue
arrowhead). Rib-sternum contacts are asymmetric (black arrowhead), and
the second and third sternebrae are fused (red arrowhead). (E) Gnai3−/−
Gnai2+/−, 4-wk-old, with fusions of ten rib pairs, including fusion of one false
rib (blue arrowhead). The eighth rib on the right side is connected to the
sternum (gray arrowhead). (F) Gnai1−/−Gnai2−/−, 5-wk-old, with an eighth
rib connected to the sternum (gray arrowhead) but no rib fusions.
Rib and sternum defects in mice with mutations of Gαigenes. The
Plummer et al.PNAS
| December 26, 2012
| vol. 109
| no. 52
phenotype in Gnai3−/−Gnai2flx/flxmice relative to 129/SvEv-
Gnai3−/−, and the similarity of the Gnai3−/−Gnai2flx/flxSox9cre/+
mice to 129SvEv-Gnai3−/−Gnai2+/−mice, is likely because of
C57BL/6 alleles of modifier genes contributed by the Sox9creand
Tg(Myog-cre) mice. Number and severity of the lumbar vertebral
fusions was not increased in Gnai3−/−Gnai2flx/flxSox9cre/+, again
indicating that this phenotype is dependent primarily on loss
The restriction of rib fusions in Gnai3−/−mice to the cartilagi-
nous, distal portion of the ribs and the apparently normal de-
velopment of the bony proximal ribs suggest that the primaxial/
abaxial classification system for somitic development (21, 22)
may provide insight into Gαi function. Unlike the epaxial/
hypaxial classification, which originally defined somite-derived
muscle on the basis of innervation by dorsal or ventral branches
of the spinal nerves and was later extended to embryonic position
relative to the notochord (23), the primaxial/abaxial classification
is based on interaction between migrating somitic cells and the
lateral plate mesoderm (LPM). The primaxial domain, close to
the body axis, consists solely of somitic cells, but in the abaxial
domain somite derivatives migrate away from the axis and dif-
ferentiate in close proximity to tissue derived from the LPM. The
LPM forms the connective tissue that surrounds and penetrates
the somite-derived bone and muscle of the abaxial domain.
Analysis of chick development indicates that the spinal column
and vertebral ribs are primaxial, but the sternal ribs are abaxial
and dependent on bone morphogenetic protein (BMP) signaling
from the LPM (24). One part of the axial skeleton, the sternum,
is derived directly from the LPM, not somites (25, 26). Mammals
do not have distinct, ossified sternal ribs, but the distal, carti-
laginous portion of the ribs appears to be analogous to the
sternal ribs of birds. Analysis of the Tg(Prrx1-cre)1Cjt transgenic
mouse, which labels the LPM, suggests that the distal, cartilag-
inous portion of the first rib is abaxial, but the remaining ribs are
surrounded by somite-derived periosteum, technically making
them primaxial (26). The distal portions of those ribs are nev-
ertheless in close proximity to LPM-derived connective tissue,
the sternum, and to the distal portion of intercostal muscles,
which are labeled by the Prrx1-cre transgene. Although the ver-
tebrae are definitively primaxial, the lateral surfaces of the
lumbar vertebrae are attached to an abaxial muscle, the psoas
major (26). Thus, all three of the regions in which we observe
axial skeletal fusions in the Gαimutant mice (ribs, sternum, and
lumbar vertebrae) are either LPM-derived or develop close to
Consistent with a hypothesis that Gαiis required for trans-
duction of signals from LPM to rib primordia, fusions of the
distal rib cartilage almost identical to those of Gnai3−/−mice are
seen in Bmp7−/−knockout mice (27) and in Bmp4+/−Bmp7+/−
Table 1. Thoracic skeletal defects in Gαimutant mice
Genetic backgroundGenotype No. examined*
Asymmetric sternal contacts (%) True ribs (%) False ribs (%)Sternebrae (%)
*Analysis includes adults, neonates, and E14.5 fetuses.
†Percentages are for number of animals exhibiting the trait, not number of ribs or sternebrae affected.
‡Three E14.5 fetuses not included in this calculation because the sternum was not ossified.
5-d-old, showing wild-type morphology of the second through fifth lumbar
vertebrae. (B) 129/SvEv-Gnai3−/−, 4-d-old, with partial fusion of the third
and fourth lumbar vertebrae. (C) B6129F2-Gnai3−/−, 5-d-old, with fusion of
the second, third, fourth, and fifth lumbar vertebrae. The fusion of the third
and fourth vertebrae is more extensive than that seen in any 129/SvEv-
Gnai3−/−mouse. (Magnification: 10×.)
Fusion of lumbar vertebrae in Gnai3−/−mice. (A) B6129F2-Gnai3+/+,
129/SvEv-Gnai3−/−mice. Each datapoint on the scatter plot represents an
individual mouse. Black horizontal lines indicate mean number of rib fusions
for a genotype. ****P < 0.0001, unpaired t test.
Mutation of Gnai1 or Gnai2 increases the number of rib fusions in
| www.pnas.org/cgi/doi/10.1073/pnas.1219810110Plummer et al.
compound heterozygotes (28). Both Bmp4 and Bmp7 are
expressed by the LPM (29, 30), and in the chick, Bmp4 is re-
quired for growth of somitic cells into the LPM domain (24).
There is little evidence that BMP signaling requires hetero-
trimeric G proteins, but sonic hedgehog (Shh) signaling appears
to alter the response of sclerotome cells to BMP and is required
for chondrogenesis (31). Hedgehog signaling in Drosophila has
been linked to heterotrimeric G proteins through Smoothened
(Smo), which couples to Gαi(32, 33), and Shh-induced pro-
liferation of rat cerebellar granule cell precursors is reduced by
in Gnai3−/−mice result from defects in an interaction between
hedgehog and BMP signaling pathways, disruption of hedgehog
signaling in chondrocytes might be expected to resemble loss of
Gαi. However, neither chondrocyte-specific overexpression of
Shh nor conditional knockout of Smo or the hedgehog recep-
tor Ptch1 in chondrocytes closely resemble the Gnai3 knockout
Other signaling pathways involved in skeletal development
and dorsal-ventral patterning that plausibly could require Gαi
activity include Wnt and PDGF. Frizzled proteins, the receptors
for Wnt, are putative G protein-coupled receptors (38), although
they have not been specifically linked to Gαi. Negative regulators
of Wnt signaling include Axin1, which may modulate hetero-
trimeric G-protein activity through its regulator of G-protein
signaling domain. Mutation of Axin1 in the spontaneous mouse
mutant Fused results in fusions of both vertebrae and ribs, but
the rib fusions tend to involve the proximal ribs close to the spine
phological defects of myotome. (A) Wild-type embryo, E11.5, side view of
trunk between the limb buds. In situ hybridization with a Myog probe
reveals morphology of the developing intercostal muscles. (B) Gnai3−/−
Gnai1−/−embryo, E11.5, Myog probe. Intercostal muscle morphology is in-
distinguishable from that of the wild-type embryo. (C) Wild-type, E12.5, Myog
probe. (D) Gnai3−/−Gnai1−/−, E12.5, Myog probe. (E) Wild-type, E12.5. In situ
hybridization with a Sox9 probe reveals rib primordia. (F) Gnai3−/−Gnai1−/−,
E12.5, Sox9 probe. Arrowhead indicates possible fusion of rib primordia.
(Magnification: A and B, 11×; C–F, 10×.)
Rib fusions in Gnai3−/−Gnai1−/−mice are not associated with mor-
the region of the wild-type Gnai2 gene containing the targeted exons 2, 3,
and 4 (blue) used to construct the targeting vector. Targeting vector: depicts
the portion of the targeting vector used to target the Gnai2 locus. Floxed
allele: depicts the structure of the targeted allele after Cre-mediated exci-
sion of the PGK-Neo cassette. Deleted allele: depicts the structure of the
disrupted allele from which exons 2, 3, and 4 have been removed by the
action of Cre recombinase. The position of key restriction endonuclease sites
and the location of genotyping primers A and B are indicated. Rectangles,
exons included in targeting vector; heavy red line, intronic sequence in-
cluded in the targeting vector; PGK-Neo, neomycin selection cassette; dt,
diptheria toxin selection cassette. (B) PCR analysis of mouse genomic DNA
using primers A and B. All Gnai2 genotypes produce PCR products of the
expected sizes indicated in Fig 4A. WT, DNA from wild-type mouse; flx/flx,
DNA from mouse homozygous for the floxed allele;−/−, DNA from mouse
homozygous for the deleted allele which exons 2–4 have been excised by
a Cre transgene driven by the ubiquitous Sox2 promoter. (C) (Upper) Dia-
gram of the wild-type intron/exon organization of the Gnai2 gene. Locations
of LoxP sites in the floxed allele and RT-PCR primers are indicated. (Lower)
Diagram of the deleted allele. Deletion of exons 2–4 by Cre recombinase is
predicted to result in a frame-shift and premature stop codon in exon 5. The
lengths of the depicted amplicons include the primers. Black boxes, coding
sequence; open boxes, untranslated exon sequence; *, stop codon. (D) RT-
PCR analysis of brain RNA from a wild-type and a Gnai2−/−mouse. Se-
quencing of the RT-PCR products confirmed splicing from exon 1 to exon 5
and the presence of a premature stop codon in mRNA transcribed from the
Conditional allele of Gnai2. (A) Targeting strategy. WT allele: depicts
Plummer et al.PNAS
| December 26, 2012
| vol. 109
| no. 52
(39). As described above, PDGF is involved in signaling from
myotome to sclerotome during rib development, and activation
of PDGFRα, a receptor tyrosine kinase, on sclerotome cells
leads to altered cell migration via a PI3 kinase-Akt pathway (40).
Gαi3is known to regulate migration of HeLa cells by an Akt-
dependent pathway (41), and a growing body of data indicates
that heterotrimeric G proteins can function within receptor ty-
rosine kinase signaling pathways (42–44).
We have identified a previously undescribed requirement for
heterotrimeric G proteins in skeletal development. The specifics
of the rib fusion phenotype suggest that Gαiis required at the
interface between somitic and lateral plate mesoderm. Addi-
tional crosses between the Gnai3 mutants and mice with targeted
mutations in some of the genes mentioned above (e.g., Smo,
Axin1, or Pdgfra) may reveal the pathway in which Gαipartic-
ipates. Mapping of the genes involved in modifying the rib fusion
phenotype in C57BL/6 may also be informative, possibly identi-
fying other members of the signaling cascade.
Materials and Methods
Mice. Targeted mutations of the Gnai1, Gnai2, and Gnai3 genes were pre-
viously described (12, 45). A colony of 129/SvEv-Gnai3tm1LbiGnai1tm1Drsmice
was maintained by intercrossing homozygotes (hereafter indicated Gnai3−/−
Gnai1−/−). To generate other genotypes, a Gnai3−/−Gnai1−/−mouse was
crossed to a wild-type 129/SvEv mouse, and the offspring were intercrossed.
The 129/SvEv- Gnai3−/−Gnai1+/+and 129/SvEv-Gnai3+/+Gnai1−/−sublines
were established by intercrossing homozygotes. The 129/SvEv-Gnai2tm1Uru
mouse colony was maintained by intercrossing heterozygotes (Gnai2+/−).
A conditional allele of Gnai2 (Gnai2flx) with loxP sites upstream of exon 2
and downstream of exon 4 was generated by homologous recombination in
129/SvEv embryonic stem cells. Chimeric mice derived from the targeted cells
were crossed with 129/SvEv mice, and offspring were intercrossed to pro-
duce an inbred 129/SvEv-Gnai2flx/flxhomozygous line. DNA genotyping and
RT-PCR of brain RNA from mice homozygous for the targeted allele and
hemizygous for the Tg(Sox2-cre)1Amc transgene (46), which drives ubiqui-
tous expression of Cre recombinase in the early embryo, were used to con-
firm recombination of the loxP sites and deletion of exons 2–4 in the
presence of Cre recombinase (Fig. 4). Genotyping primers were A (5′-
GTGGTAAGCCTGTGTTTGTGAGAG) and B (5′-GGAGCCTGGACTTTGCTTCT-
GACC). Primers for RT-PCR were F1 (5′-TGCACCGTGAGCGCCGAGGACAAG),
F2 (5′-ACCTGAATGATCTGGAGCGCATTG), R1 (5′-CTAACAGAAGCAACTTCA-
CCTCCC), and R2 (5′-TCAAGGCGACACAGAAGATGATGG). Tg(Myog-cre)1Eno,
and Sox9tm3(cre)Crmmice were previously described (19, 20).
For the B6 × 129 intercross, C57BL/6J mice were purchased from the
Jackson Laboratory. SNP genotyping was performed by the Mutation Map-
ping and Developmental Analysis Project of Brigham and Women’s Hospital,
Harvard University. All animal experiments were performed with approval
of the National Institute of Environmental Health Sciences Institutional
Animal Care and Use Committee.
Skeleton Staining and Analysis. Skeletons of neonatal and adult mice were
stained with Alcian blue and Alizarin red (47). Mouse fetuses at E14.5 were
stained with Alcian blue, as described previously (48), except that the fetuses
were skinned and eviscerated after fixation. Two or three complete skel-
etons of adult mice were stained for each genotype, and thereafter only the
rib cage was stained. The skeletons of pups and fetuses were stained intact.
Stained skeletons were photographed with a Coolpix 995 digital camera
(Nikon). For scoring the number of rib fusions, fusion of one pair of ribs was
counted as one fusion, and fusion of three consecutive ribs was counted as
two fusions. Statistical analyses and graphing were performed using
GraphPad Prism and QuickCalcs software (GraphPad Software).
In Situ Hybridization. The template for the Sox9 riboprobe has been described
previously (49). Templates for sense and antisense myogenin (Myog) ribop-
robes were produced from a cDNA clone (IMAGE: 6508229, GenBank:
BC068019) by PCR with the T7 promoter sequence incorporated in either the
forward or reverse primer. Primers to generate template for the antisense
riboprobe were: 5′-GGCCAGTGGCAGGAACAAGC (Myog, forward) and 5′-
reverse). Primers to generate template for the sense control probe were: 5′-
forward) and 5′-GGAATTCGAGGCATATTATG (Myog, reverse). Digoxigenin-
labeled riboprobes were synthesized from these templates using the DIG
RNA Labeling Mix (Roche Applied Science).
Embryos for whole-mount in situ hybridization were fixed overnight at
4 °C in 4% (wt/vol) paraformaldehyde dissolved in PBS, pH 7.4 (137 mM NaCl,
2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4). After fixation, the embryos
were dehydrated and stored in 100% methanol at −20 °C. In situ hybrid-
ization was performed as previously described (50) with the following
modifications: PBS with Tween-20 contained 1% Tween-20 rather than
0.1%. Hybridization buffer contained 100 μg/mL sheared salmon sperm
DNA. The embryos were not treated with RNase A after probe hybridization.
After incubation with antidigoxygenin antibody (Roche Applied Science),
embryos were washed overnight in Tris buffered saline (140 mM NaCl, 2.7
mM KCl, 25 mM Tris-HCl, pH7.5) with 1% Tween-20 at 4 °C. Hybridized probe
was visualized with BM Purple Reagent (Roche Applied Science).
ACKNOWLEDGMENTS. The authors thank Tom Sliwa for animal husbandry,
Mitzie Walker for assistance with genotyping, and Jennifer Moran for SNP
analysis. The template for the Sox9 in situ probe was generously provided by
Masahiro Iwamoto (Thomas Jefferson University), and the Tg(Myog-cre)
1Eno mice were generously provided by Eric Olson (University of Texas
Southwestern Medical Center). This research was supported by the Intramu-
ral Research Program of the National Institutes of Health, National Institute
of Environmental Health Sciences (Project Z01-ES-101643).
1. Downes GB, Gautam N (1999) The G protein subunit gene families. Genomics 62(3):
2. Wettschureck N, Offermanns S (2005) Mammalian G proteins and their cell type
specific functions. Physiol Rev 85(4):1159–1204.
3. Wilkie TM, et al. (1992) Evolution of the mammalian G protein alpha subunit multi-
gene family. Nat Genet 1(2):85–91.
4. Muradov H, Kerov V, Boyd KK, Artemyev NO (2008) Unique transducins expressed in
long and short photoreceptors of lamprey Petromyzon marinus. Vision Res 48(21):
5. Pineda VV, et al. (2004) Removal of G(ialpha1) constraints on adenylyl cyclase in the
hippocampus enhances LTP and impairs memory formation. Neuron 41(1):153–163.
6. Rudolph U, et al. (1995) Ulcerative colitis and adenocarcinoma of the colon in G
αi2-deficient mice. Nat Genet 10(2):143–150.
7. Zuberi Z, Birnbaumer L, Tinker A (2008) The role of inhibitory heterotrimeric G pro-
teins in the control of in vivo heart rate dynamics. Am J Physiol Regul Integr Comp
8. Jain M, et al. (2001) Targeted inactivation of Galpha(i) does not alter cardiac function
or beta-adrenergic sensitivity. Am J Physiol Heart Circ Physiol 280(2):H569–H575.
in cartilage. (A) Scatter plot showing the distribution of rib fusions in mice of
different genotypes. Statistically significant increase in the number of
fusions is seen in Gnai3−/−Gnai2flx/+Sox9cre/+and Gnai3−/−Gnai2flx/flxSox9cre/+
mice. Horizontal lines indicate mean number of rib fusions. ***P < 0.001, un-
paired t test. (B) Gnai3−/−Gnai2flx/flxneonate with one rib fusion (black ar-
rowhead). (C) Gnai3−/−Gnai2flx/flxTg(Myog-Cre) neonate with one rib fusion
(black arrowhead). (D) Gnai3−/−Gnai2flx/flxSox9cre/+neonate with 10rib fusions.
Rib fusion phenotype of Gnai3−/−mice is enhanced by loss of Gnai2
| www.pnas.org/cgi/doi/10.1073/pnas.1219810110 Plummer et al.
9. Yang J, et al. (2002) Signaling through Gi family members in platelets. Redundancy
and specificity in the regulation of adenylyl cyclase and other effectors. J Biol Chem
10. Gohla A, et al. (2007) An obligatory requirement for the heterotrimeric G protein Gi3
in the antiautophagic action of insulin in the liver. Proc Natl Acad Sci USA 104(8):
11. Fan H, et al. (2005) Lipopolysaccharide- and Gram-positive bacteria-induced cellular
inflammatory responses: Role of heterotrimeric Galpha(i) proteins. Am J Physiol Cell
12. Jiang M, et al. (2002) Mouse gene knockout and knockin strategies in application to
alpha subunits of Gi/Go family of G proteins. Methods Enzymol 344:277–298.
13. Christ B, Huang R, Wilting J (2000) The development of the avian vertebral column.
Anat Embryol (Berl) 202(3):179–194.
14. Huang R, et al. (2003) Ventral axial organs regulate expression of myotomal Fgf-8
that influences rib development. Dev Biol 255(1):30–47.
15. Tallquist MD, Weismann KE, Hellström M, Soriano P (2000) Early myotome specifi-
cation regulates PDGFA expression and axial skeleton development. Development
16. Vinagre T, et al. (2010) Evidence for a myotomal Hox/Myf cascade governing non-
autonomous control of rib specification within global vertebral domains. Dev Cell 18
17. Laclef C, et al. (2003) Altered myogenesis in Six1-deficient mice. Development 130
18. Vivian JL, Olson EN, Klein WH (2000) Thoracic skeletal defects in myogenin- and
MRF4-deficient mice correlate with early defects in myotome and intercostal mus-
culature. Dev Biol 224(1):29–41.
19. Li S, et al. (2005) Requirement for serum response factor for skeletal muscle growth
and maturation revealed by tissue-specific gene deletion in mice. Proc Natl Acad Sci
20. Akiyama H, et al. (2005) Osteo-chondroprogenitor cells are derived from Sox9 ex-
pressing precursors. Proc Natl Acad Sci USA 102(41):14665–14670.
21. Burke AC, Nowicki JL (2003) A new view of patterning domains in the vertebrate
mesoderm. Dev Cell 4(2):159–165.
22. Nowicki JL, Takimoto R, Burke AC (2003) The lateral somitic frontier: Dorso-ventral
aspects of anterio-posterior regionalization in avian embryos. Mech Dev 120(2):
23. Spörle R (2001) Epaxial-adaxial-hypaxial regionalisation of the vertebrate somite:
Evidence for a somitic organiser and a mirror-image duplication. Dev Genes Evol
24. Sudo H, et al. (2001) Inductive signals from the somatopleure mediated by bone
morphogenetic proteins are essential for the formation of the sternal component of
avian ribs. Dev Biol 232(2):284–300.
25. Chen JM (1952) Studies on the morphogenesis of the mouse sternum. II. Experiments
on the origin of the sternum and its capacity for self-differentiation in vitro. J Anat
26. Durland JL, Sferlazzo M, Logan M, Burke AC (2008) Visualizing the lateral somitic
frontier in the Prx1Cre transgenic mouse. J Anat 212(5):590–602.
27. Luo G, et al. (1995) BMP-7 is an inducer of nephrogenesis, and is also required for eye
development and skeletal patterning. Genes Dev 9(22):2808–2820.
28. Katagiri T, Boorla S, Frendo JL, Hogan BLM, Karsenty G (1998) Skeletal abnormalities
in doubly heterozygous Bmp4 and Bmp7 mice. Dev Genet 22(4):340–348.
29. Mine N, Anderson RM, Klingensmith J (2008) BMP antagonism is required in both
the node and lateral plate mesoderm for mammalian left-right axis establishment.
30. Solloway MJ, Robertson EJ (1999) Early embryonic lethality in Bmp5;Bmp7 double
mutant mice suggests functional redundancy within the 60A subgroup. Development
31. Murtaugh LC, Chyung JH, Lassar AB (1999) Sonic hedgehog promotes somitic chon-
drogenesis by altering the cellular response to BMP signaling. Genes Dev 13(2):
32. Ogden SK, et al. (2008) G protein Galphai functions immediately downstream of
Smoothened in Hedgehog signalling. Nature 456(7224):967–970.
33. Philipp M, Caron MG (2009) Hedgehog signaling: Is Smo a G protein-coupled re-
ceptor? Curr Biol 19(3):R125–R127.
34. Barzi M, Kostrz D, Menendez A, Pons S (2011) Sonic Hedgehog-induced proliferation
requires specific Gα inhibitory proteins. J Biol Chem 286(10):8067–8074.
35. Long F, Zhang XM, Karp S, Yang Y, McMahon AP (2001) Genetic manipulation of
hedgehog signaling in the endochondral skeleton reveals a direct role in the regu-
lation of chondrocyte proliferation. Development 128(24):5099–5108.
36. Mak KK, Chen M-H, Day TF, Chuang P-T, Yang Y (2006) Wnt/β-catenin signaling in-
teracts differentially with Ihh signaling in controlling endochondral bone and syno-
vial joint formation. Development 133(18):3695–3707.
37. Tavella S, et al. (2004) Targeted expression of SHH affects chondrocyte differentia-
tion, growth plate organization, and Sox9 expression. J Bone Miner Res 19(10):
38. Schulte G, Bryja V (2007) The Frizzled family of unconventional G-protein-coupled
receptors. Trends Pharmacol Sci 28(10):518–525.
39. Reed SC (1937) The inheritance and expression of Fused, a new mutation in the house
mouse. Genetics 22(1):1–13.
40. Pickett EA, Olsen GS, Tallquist MD (2008) Disruption of PDGFRalpha-initiated PI3K
activation and migration of somite derivatives leads to spina bifida. Development
41. Ghosh P, Garcia-Marcos M, Bornheimer SJ, Farquhar MG (2008) Activation of Galphai3
triggers cell migration via regulation of GIV. J Cell Biol 182(2):381–393.
42. Kreuzer J, et al. (2003) Platelet-derived growth factor activates production of reactive
oxygen species by NAD(P)H oxidase in smooth muscle cells through Gi1,2. FASEB J
43. Marty C, Ye RD (2010) Heterotrimeric G protein signaling outside the realm of seven
transmembrane domain receptors. Mol Pharmacol 78(1):12–18.
44. Pyne NJ, et al. (2004) Experimental systems for studying the role of G-protein-coupled
receptors in receptor tyrosine kinase signal transduction. Methods Enzymol 390:
45. Rudolph U, Bradley A, Birnbaumer L (1994) Targeted inactivation of the Gi2α gene
with replacement and insertion vectors: Analysis in a 96-well plate format. Methods
46. Hayashi S, Lewis P, Pevny L, McMahon AP (2002) Efficient gene modulation in mouse
epiblast using a Sox2Cre transgenic mouse strain. Mech Dev 119(Suppl 1):S97–S101.
47. Peters PWJ (1977) Double staining of fetal skeletons for cartilage and bone. Methods
in Prenatal Toxicology, eds Neubert D, Merker H-J, Kwasigroch TE (Georg Thieme,
Stuttgart, Germany), pp 153–154.
48. Jegalian BG, De Robertis EM (1992) Homeotic transformations in the mouse induced
by overexpression of a human Hox3.3 transgene. Cell 71(6):901–910.
49. Tamamura Y, et al. (2005) Developmental regulation of Wnt/β-catenin signals is re-
quired for growth plate assembly, cartilage integrity, and endochondral ossification.
J Biol Chem 280(19):19185–19195.
50. Wilkinson DG (1992) In Situ Hybridization: A Practical Approach (IRL, Oxford).
Plummer et al.PNAS
| December 26, 2012
| vol. 109
| no. 52