Download Gene trap insertion into a novel gene expressed during mouse limb

DEVELOPMENTAL DYNAMICS 212:318–325 (1998)
Gene Trap Insertion Into a Novel Gene Expressed
During Mouse Limb Development
ANDRÉ PIRES-DASILVA AND PETER GRUSS*
Department of Molecular Cell Biology, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany
ABSTRACT
Gene trapping is a useful method
to identify new genes involved in development.
Here we describe the spatiotemporal expression
of a gene identified in a gene-trap screen. This
gene is first expressed at 9.5 days postcoitum
(E9.5) in the forelimbs and in the branchial arches
region. At E11.5, expression was detected in the
stomach, genital bud, and pharyngeal epithelium. At later stages, expression includes the hair
follicles, whereas the expression in the stomach
and pharynx disappears. We performed 58-rapid
amplification of cDNA ends (RACE) to amplify
and clone a partial cDNA of the endogenous
sequence fused to the lacZ reporter gene. The
sequence did not reveal any similarity to known
sequences and was named paddy. The expression
pattern suggests multiple roles during limb development. The early phase of expression, for instance, correlates with anteroposterior (A/P) regionalization. In contrast to other molecules
involved in A/P polarization, paddy expression
fades away distally as the bud elongates. This
suggests that expression of paddy in late stages
does not depend on apical ectodermal ridge (AER)
and zone of polarizing activity (ZPA) signaling
and is probably involved in posterior determination in more proximal regions of the limb.
Dev. Dyn. 1998;212:318–325. r 1998 Wiley-Liss, Inc.
Key words: hair follicles; mouse embryogenesis;
lacZ; apical ectodermal ridge
INTRODUCTION
The screening for mutants in Drosophila and Caenorhabditis elegans has greatly enhanced our knowledge of molecules involved in the control of development (Nusslein-Volhard and Wieschaus, 1980; Meneely
and Herman, 1979). In vertebrates, however, such large
screenings have been hampered by the difficulty of
identifying the gene when only the mutant is available.
An alternative way to identify new genes and their
associated mutant phenotypes in mouse is gene trapping (Gossler et al., 1989). Gene trapping is based on
the random integration of a reporter gene in the
genome, which allows the detection of the expression
pattern of the endogenous gene by a simple histochemical procedure. The identification of the ‘‘trapped gene’’
is performed by using a polymerase chain reaction
r 1998 WILEY-LISS, INC.
(PCR) method that allows cloning of the exon just
upstream of the reporter gene (Frohman et al., 1988).
In this paper, we report a gene-trap event that shows
expression in the limb. The limb has been the subject of
studies for decades in the chick, because it is readily
accessible to experimental manipulations in ovo. Many
genes that are involved in limb development have been
identified by homology screening using Drosophila
probes (for review, see Tickle 1994). Such an approach
has proved to be very fruitful, because many signaling
pathways are highly conserved across phyla (Gaunt,
1997). The identification of vertebrate-specific genes,
however, has been very limited. Gene trapping allows a
large number of genes and their corresponding expression patterns to be obtained in a relatively short time.
Thus, this strategy is well suited for screening for novel
genes involved in vertebrate limb development.
The limb develops along three axes: a proximodistal
(Pr/Di) axis that runs between the shoulder and the tips
of the digits, an anteroposterior (A/P) axis that extends
(e.g., the hand between the little finger and the thumb),
and a dorsoventral (D/V) axis that extends from the
back of the hand to the palm. Patterning along these
axes is determined very early in vertebrate development, even before the limb bud is recognizable. Both
ectodermal and mesodermal components are actively
involved in maintaining the limb axial polarity. The
ectodermal sheath around the limb controls the D/V
axis, whereas its most distal part, the apical ectodermal
ridge (AER), is required for outgrowth and Pr/Di regionalization (Saunders, 1948; MacCabe et al., 1974). Patterning along the A/P axis is established by a series of
reciprocal interactions between the AER and the region
of posterior mesoderm known as the zone of polarizing
activity (ZPA; Saunders and Gasseling, 1968; Laufer et
al., 1994).
Recently, progress has been made in characterizing
genes that are involved in limb development. The AER,
for instance, can be substituted functionally by ectopic
administration of members of the fibroblast growth
factor (FGF) family (Niswander et al., 1993; Fallon et
al., 1994). Regionalization along the A/P axis, however,
is less clear. The role of molecules distributed along this
axis, such as sonic hedgehog (shh), retinoic acid, and
*Correspondence to: Peter Gruss, Department of Molecular Cell
Biology, Max Planck Institute of Biophysical Chemistry, Am Fassberg
11, D-37077 Göttingen, Germany.
Received 11 September 1997; Accepted 17 December 1997
Paddy EXPRESSION IN THE LIMB
Hox genes, is not fully understood (Noji et al., 1991;
Dolle et al., 1993; Davis and Cappecchi, 1994; Chiang et
al., 1996). Thus, characterization of additional molecules is required to fully understand the mechanisms
of limb development.
Here, we describe a gene-trap insertion into a novel
gene, paddy, that has an expression pattern that
correlates with multiple roles during limb development
and that is expressed in restricted mesodermal domains along the limb axes, which are probably involved
in A/P and D/V differentiation processes of the limb pad.
The patterns of expression in other tissues, such as the
hair follicles, stomach, genital bud, and pharynx, are
also discussed.
RESULTS
Mouse embryonic stem (ES) cells were electroporated
with the internal ribosome entry site (IRES)-bGeo
vector (see Experimental Procedures) and screened
initially for their pattern of expression in vitro in an
undifferentiated state. Of approximately 100 G418resistant ES colonies, 80 stained for b-galactosidase
(b-gal) activity. The pattern of expression was highly
variable among the different ES cell clones maintained
in culture. Clones that had fewer than 30% of their cells
stained with lacZ were further processed. Twenty of
these clones were selected for morula aggregation to
generate chimeras. Nine of them were found to contribute to the germ line. Embryos were analyzed for b-gal
staining from embryonic day 8.5 (E8.5) to E16.5. One
mouse line presented a very restricted expression pattern during embryogenesis.
319
ment by about 1 day, has an expression pattern similar
to that seen in the forelimb.
D/V of lacZ expression starts at E13.5 (Fig. 1F–I). At
this time, mesenchymal cells lining the ventral epithelium of the limb are labelled. No expression is observed
in chondrification centers. In subsequent stages of
development, expression localizes to the limb pads,
which are small elevations in the plantar surface of the
hind limb and the palmar surface of the forelimb (Fig.
1J–L). The expression in the forelimb is first observed
in the metacarpal region and in the metacarpophalangeal joints at E14.5; later (E15.5), it includes the five
digital pads. Tangential sections revealed that staining
was restricted to the dermal component of the pad.
Hair Follicles
Hair follicles, like the limbs, require epitheliomesenchymal interactions to develop (for review, see Hardy,
1992). The first step of development is a thickening of
the epithelium, which is thought to induce a mesenchymal condensation of underlying dermal cells. This
condensation, called dermal papilla, promotes ingrowth
of the epidermis and subsequent proliferation of the
adjacent epithelial cells that, later, will form the hair
itself.
Hair follicles develop in a rostral-to-caudal sequence.
The first follicles to appear, those of the vibrissae, are
seen at E12.5. LacZ expression is observed in mesenchymal cells that condense to form the dermal papilla (Fig.
2A,B). Pelage hair follicles appear in E14.5 embryos,
with the same lacZ expression pattern as that seen in
the vibrissae follicles.
Stomach, Genital Bud, and Branchial Arc
Expression in the Limbs
LacZ expression corresponding to paddy activation is
first observed at E9.5, when the forelimb bud becomes
visible (Fig. 1A,B). Blue staining is restricted to the
mesenchymal layer of the bud. Staining is not observed
either in the anterior one-third of the limb or in the
ectoderm. As the bud elongates, paddy expression is
restricted to the posterior half of the limb (Fig. 1C–H).
In late limb development, expression includes anterior
domains (Fig. 1J), culminating in 5-bromo-4-chloro-3indolyl B-D-galactopyranoside (X-Gal) staining in restricted cells along the whole A/P axis (Fig. 1K). Interestingly, a gradient of expression can be observed along
the A/P and Pr/Di axes at E9.5–11.5 (Fig. 1B,C,E). The
most posterior domain, which includes the ZPA, has the
strongest staining.
A dynamic pattern of expression is also observed
along the Pr/Di axis. Up to E10.5, no polarization of
expression is observed. In later stages, however, cells
underlying the AER become progressively negative for
X-Gal staining up to the proximal half of the hand/foot
plate at E12.5 (Fig. 1H). The proximal expression
domain extends to the axilla region until E13.5; later, it
remains only in the autopod and the anterior part of the
zeugopod. The hind limb, which is delayed in develop-
At E9.5, lacZ expression appears at the level of the
third and fourth branchial arches (Fig. 1A). The iden-
Fig. 1. (Overleaf.) LacZ expression of paddy in the limb in embryos
9.5–15.5 days postcoitum (E9.5–E15.5). B, C, E, H, J, and K show limbs
that are oriented with proximal to the left, distal to the right, anterior on the
top, and posterior on the bottom of the photomicrograph. A: Whole-mount
of an E9.5 embryo exhibiting expression in the branchial arch region
(arrowhead) and in the forelimb. B: Higher power magnification of the
E9.5 forelimb showing expression restricted to the mesodermal layer.
Note that the most anterior region of the limb is negative for lacZ. C: E10.5
forelimb whole-mount showing staining localized predominantly in the
posterior half of the limb. D: At E11.5, expression includes the stomach. E:
Sagittal section along an E11.5 limb showing strong expression in the
region overlapping with the zone of zone of polarizing activity (ZPA). No
expression is observed in the apical ectodermal ridge (AER). F: Cross
section of an E11.5 limb. There is no sign of dorsoventral (D/V)
polarization of the lacZ expression. G: Whole-mount of E12.5 embryo
showing staining in whisker follicles and limbs. H: The lacZ expression at
E12.5 is detected in a more proximal region of the limb compared with
earlier stages. I: At E13.5, staining is polarized along the D/V axis and is
restricted to the mesodermal cells lining the ventral surface of the limb. J:
Ventral view of an E14.5 limb. K: Ventral view of an E15.5 limb showing
5-bromo-4-chloro-3-indolyl B-D-galactopyranoside (X-Gal) staining in the
limb pads. L: Cross section of an E15.5 limb with lacZ expression in the
ventral dermis. a, Anterior; p, posterior; d, dorsal; v, ventral; pr, proximal;
di, distal; st, stomach; ect, ectoderm. Scale bars 5 500 µm in A,D,G–L,
150 µm in B,C,E,F.
A
B
a
C
ect
di
pr
p
D
E
AER
a
F
v
ZPA
st
G
d
p
pr
I
H
v
d
di
J
d
L
K
v
Figure 1.
A
B
C
D
E
F
Fig. 2. Paddy is expressed in the hair follicles (A,B), stomach (C),
pharynx (D), and genital bud (F,H). A: Section of an E12.5 embryo
showing X-Gal staining in the condensing mesenchymal cells underlying
the epithelium of the hair vibrissae. B: Later, at E13.5, expression is
detected exclusively in the dermal papilla. C:. Blue staining is observed in
the pyloric region of the stomach at E11.5 D: E11.5 sagittal section
showing signal in the epithelium lining the pharynx. E: Genital bud of an
E12.5 embryo with lacZ expression in mesenchymal cells at the tip of the
bud. F: In later stages (E13.5), expression continues in the tip. Scale
bars 5 150 µm.
322
PIRES-DASILVA AND GRUSS
Fig. 3.
Sequence obtained by 58-rapid amplification of cDNA ends (RACE) of paddy limb RNA.
tity of these cells was not determined. At around E10.5,
expression of lacZ begins to be detected in parts of the
digestive system. More specifically, staining is detected
in the pyloric region of the developing stomach (Fig. 2C)
and in the epithelium of the midgut (not shown). Later
in development, at E11.5, blue staining is observed in
the epithelium lining the pharynx (Fig. 2C). In no other
stage is staining detected in this region.
Expression in the genital bud starts at E10.5 and
extends until E13.5 (Fig. 2E,F). LacZ expression is
observed exclusively in the mesodermal component of
the bud. X-Gal staining is detected in more distal
regions as the genital bud develops.
58-Rapid Amplification of cDNA Ends Sequence
Total RNA of heterozygous E12.5 limbs was prepared
to amplify 58 sequences flanking the lacZ gene. A 258
base pair (bp) fragment was obtained that did not show
any similarity to the sequences described previously
(Fig. 3), and the open reading frame (ORF) that was
found did not show similarity to any protein domain.
Therefore, it is very likely that this sequence derives
from an uncharacterized gene.
The evidence that this amplified sequence corresponds to the cDNA of the trapped gene is supported by
the following data. First, Southern blot analysis using
an enzyme that cuts the gene-trap vector in only one
site shows that there is reporter gene integration in
only one locus (Fig. 4A). Second, reverse transcriptase
(RT)-PCR between the amplified sequence and the
gene-trap vector sequence shows a single band of the
expected size in RNA derived from the limbs but not
from tissues that are negative for X-Gal staining, e.g.,
the brain (Fig. 4B). Third, the sequence is spliced
correctly with the reporter gene, ruling out the possibility of a product derived from a nonspecific PCR amplification (data not shown).
Homozygous Mice Are Apparently Normal
Homozygous animals were identified by quantitative
Southern blot analysis. Preliminary data reveal that
they were born at the expected Mendelian ratio and did
not show any obvious morphological phenotype compared with their heterozygous or wild type litter mates.
DISCUSSION
Here, we present the characterization of a gene-trap
event with expression in the branchial arches, stomach,
limbs, hair follicles, and genital bud. The short 58-RACE
sequence available does not allow us to assign a possible function for the trapped gene, although its spatiotemporal pattern of expression suggests multiple roles
during development.
Expression is first detected at E9.5, when it is
restricted to the forelimbs and branchial arches. Later
in development, X-Gal staining is observed sequentially in hind limbs and stomach at E10.5, in vibrissae
follicles and genital bud at E11.5–E12.5, and in pellage
hair follicles at E14.5.
Paddy in Limb Development
The determination of limb asymmetry depends on a
series of epitheliomesenchymal interactions that occur
very early during limb development. Classical experiments have shown that inversion of the ectodermal
sheath along the D/V axis promotes concomitant mesoderm polarity inversion along the same axis (MacCabe
et al., 1974; Geduspan and MacCabe, 1987, 1989).
Recent studies have provided information about molecules that are important for dorsal determination.
Wnt-7a, for instance, is expressed in dorsal ectoderm
during early stages of mouse and chick limb development (Dealey et al., 1993; Parr et al., 1993). Its functional inactivation results in the transformation of
dorsal limb structures toward a ventral phenotype
(Parr and McMahon, 1995). Wnt-7a signaling activates
genes that encode transcription factors in dorsal mesoderm. One such gene is Lmx1, a member of the Lim
gene family (Riddle et al., 1995; Vogel et al., 1995).
Less is known, however, about the molecular cues
that determine ventral polarity. The functional inactivation of engrailed-1 (En-1) has shown that this gene is
required for limb ventralization (Loomis et al., 1996).
Molecules in the mesoderm component that respond to
En-1 signal, however, are not known. Paddy is expressed in both surfaces early in development, making
it unlikely that it has a role in early patterning
decisions. Its later expression (from E14.5 onward),
however, suggests a function that is related to the
differentiation process of the plantar/palmar surface of
the limbs. Paddy, then, is probably activated in response to signals from ventral mesenchyme, which then
pattern the ectoderm. Possible roles for the expression
in the pad dermis are the guidance of nerve fibers or the
elevation of the epidermal surface. To our knowledge,
Pax-9 is the only known gene with overlapping expres-
Paddy EXPRESSION IN THE LIMB
323
able to respecify the limb pattern when it is grafted to
more anterior positions (Saunders and Gasseling, 1968).
The biochemical nature of the molecule that confers
this activity is not yet known, although candidates are
available. For example, shh, which is expressed in the
ZPA region, is able to specify posterior identity when it
is expressed in anterior regions of the limb bud (Riddle
et al., 1993). The fact that paddy has a similar expression, although it occurs in a broader domain, suggests
that it has a role along this axis. It has been hypothesized that a gradient of positional information is
required to specify different identities (Wolpert, 1969).
Paddy is expressed in a graded manner, suggesting
that the level of expression is important for its function.
A reciprocal signaling cascade has been shown to
occur between the AER and the ZPA (Laufer et al.,
1994). Shh, which is expressed in the ZPA, is thought to
activate Fgf-4 in the AER. Further maintenance of shh
expression in the distal domain of the limb is then
controlled by Fgf-4. Consequently, the shh expression
domain is found subjacent to the AER as the limb grows
out distally. The continued shh expression in distal
regions may be important to activate and maintain the
expression of HoxD and Bmp genes, which then pattern
the digits. Paddy expression, however, does not seem to
depend on AER/ZPA signalling in late stages, because
its expression domain moves proximally rather than
distally as the limb develops. Thus, it can be hypothesized that paddy is activated in response to polarizing
signals only in early limb development. The limb develops following a proximodistal progression of differentiation (Saunders, 1948). Accordingly, the expression of
paddy in proximal regions of the limb correlates with a
role in girdle/humerus differentiation.
Paddy in Hair Follicle Development
Fig. 4. The 58-RACE corresponds to the cDNA of the trapped gene.
A: Southern blot of DNAs digested with NcoI and hybridized with lacZ
probe. In the transgenic mice (asterisks), only one band of 3 Kb was
detected. B: Reverse transcriptase-polymerase chain (RT-PCR) reaction
of samples derived from E12.5 transgenic animals. RNA were isolated
from the limb (left lane) and from the brain (right lane). PCR was
performed with primers that anneal to the gene-trap vector and the
58-RACE sequence (top) and for GAPDH (bottom).
sion patterns in these late stages (E14.5–E16.5) of limb
development (Neubuser et al., 1995). The function of
Pax-9 in these stages, however, is unknown.
The onset of expression of paddy along the A/P axis
correlates with the timing of this axial polarity determination. The ZPA, which is comprised of a population of
mesenchymal cells in the posterior part of the limb, is
The hair follicles arise in a stepwise process: The
dermal mesenchyme initiates the thickening and ingrowth of the overlying epithelium (Hardy, 1992), the
mesenchymal cells then aggregate and form the dermal
papilla, and the adjacent epithelial cells are stimulated
by the dermal papilla to divide rapidly, forming the
‘‘hair matrix.’’ Cells derived from the hair matrix will
later differentiate into hair cells and inner root sheath
cells. There are only a few molecules that are known to
be involved in these induction processes. The expression of Bmp-4 in the condensing mesenchyme, for
example, could be involved in epithelial invagination
(Bitgood and McMahon, 1995). The fact that paddy has
an expression pattern similar to Bmp-4 suggests that it
could have a similar or related role. Other possible
functions are the aggregation of mesenchymal cells and
the innervation of the hair follicle.
Paddy Expression Suggests Many Roles
During Development
In summary, paddy is a new gene with restricted
expression domains in several tissues. The expression
in the limb suggests that paddy may be involved in the
324
PIRES-DASILVA AND GRUSS
determination of A/P and D/V polarities, probably in a
different signaling pathway than those described thus
far. Its relatively late expression polarization along the
D/V axis suggests that paddy is more likely to act in
late differentiation processes rather than in axis determination.
The absence of a phenotype can be explained in a
number of ways. There is the possibility, for instance,
that alternative splicing is occurring. This would generate the wild type transcript that rescues the phenotype.
Other possibilities include integration in a part of the
gene that does not interfere in its function or the
presence of related genes with overlapping functions.
EXPERIMENTAL PROCEDURES
Generation of Gene-Trapped ES Cell Clones
and Mouse Chimeras
The vector IRESbGeo contains an IRES between the
splice-acceptor site and the bgeo sequence (Chowdhury
et al., 1997). Gene-trapped ES clones were produced by
electroporating 1 3 107 R1 ES cells (Nagy et al., 1993)
with 25 µg of vector linearized at the SacI site. ES cell
colonies resistant for G418 were selected, picked, and
expanded essentially as described by Wurst and Joyner
(1993). Clones positive for lacZ activity were aggregated with morulas to generate chimeric mice (Nagy et
al., 1993). Chimeras with a strong contribution from ES
cells, as judged by coat color, were tested for contribution to the germ line by crossing to NMRI mice.
X-Gal Staining of Mouse Embryos
In all animals, the day on which the vaginal plug was
detected was considered to be day 0.5 of gestation
(E0.5). Embryos were fixed in 1% formaldehyde, 0.2%
glutaraldehyde, and 0.02% NP-40 at 4°C for 30–120
min and then washed twice in phosphate-buffered
saline (PBS) at room temperature. Staining in X-Gal
solution (1 mg/ml X-Gal, 5 mM K3Fe(CN)6, 5 mM
K4Fe(CN)6, 2 mM MgCl2) was performed at 30°C overnight to reveal lacZ activity. Embryos processed for
paraffin sections were postfixed in 4% paraformaldehyde overnight at 4°C and counterstained with neutral
red.
Quantitative Southern Blot
Heterozygous mice for the vector integration were
identified by Southern blot analysis on genomic DNA
digested with BamHI and hybridized with a 32P-labeled
lacZ probe. In the presently described mouse line, there
are about four tandem integrations in one locus (data
not shown). The offspring of heterozygous matings were
genotyped by adding a second probe, Fkh-5, which was
used as an internal control (Wehr, 1996). The homozygous animals had a higher lacZ:Fkh-5 ratio than the
heterozygous animals.
58-RACE, RT-PCR, and Sequence Analysis
Total RNA was prepared from E12.5 limbs by using
TRIzol reagent (GIBCO-BRL, Gaithersburg, MD). Ge-
nomic DNA was removed by DNAseI (BoehringerMannheim, Mannheim, Germany) digestion. 58-RACE
was performed by using the GIBCO kit (catalog no.
18374–025; GIBCO-BRL) following the manufacturer’s
instructions. The primer PB1 (58-AGGGAGAGGGGCGGATT-38), which hybridizes to the IRES sequence, was
used for the RT reaction. Nested PCRs were performed
with primers PB2 (58-CGATGATCTTCCGGGTACCGAGCT-38) and PB3 (58-TACCGAGCTCCTGTGCCAGACTCT-38) under the following conditions: 94°C
for 45 sec, 57°C for 25 sec, and 72°C for 2 min using 1
unit of Taq DNA polymerase in Taq buffer for 30 cycles.
PCR products were cloned into pGEM-T vector (Promega, Madison, WI) and sequenced by dideoxy chain
termination using T7 DNA polymerase (Pharmacia,
Uppsala, Sweden). The sequences of seven clones were
analyzed by using the software from Genetics Computer Group (Madison, WI) and were compared with
GenBank/EMBL sequence data links. All clones presented the same sequence.
To confirm that the 58-RACE sequence corresponded
to the trapped gene, an RT-PCR was performed using
RNA isolated from the limbs and brains of E12.5
transgenic embryos, as identified previously by Southern blot. RT was performed with the first-strand cDNA
synthesis kit (Pharmacia) by using random hexamer
primers. The primers for the PCR amplification of the
derived cDNAs were as follows: AS31 (58-TGCGGTTGAGGCTCACTT-38) derived from the 58-RACE sequence positions 18–29; AS32 (58-ATCTTCCGGGTACCGAGC-38) derived from the gene-trap vector sequence;
GAPDH, a typical ‘‘housekeeping gene,’’ was used as an
internal control; GAPDH 58 primer (58-ACCACAGTCCATGCCATCAC-38; positions 566–585); and GAPDH
38 primer (58-TCCACCACCCTGTTGCTGTA-38; positions 998–1,017). The DNA fragments produced after
25 or 30 amplification cycles with an annealing temperature of 60°C were 420 bp and 451 bp, respectively.
ACKNOWLEDGMENTS
We thank R. Scholz for ES cell aggregation; F.
Cecconi for helping with the 58-RACE; and C. Tickle, K.
Ewan, and G. Alvarez-Bolado for critically reading the
paper. This research was funded by AmGen Inc. and
the Max Planck Society.
REFERENCES
Bitgood MJ, McMahon AP. Hedgehog and Bmp genes are coexpressed
at many diverse sites of cell-cell interaction in the mouse embryo.
Dev. Biol. 1995;172:126–138.
Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal H,
Beachy PA. Cyclopia and defective axial patterning in mice lacking
sonic hedgehog gene function. Nature 1996;383:407–413.
Chowdhury K, Bonaldo P, Torres M, Stoykova A, Gruss P. Evidence for
the stochastic integration of gene trap vectors into the mouse
germline. Nucleic Acids Res. 1997;25:1531–1536.
Dealey CN, Roth A, Ferrari D, Brown AMC, Kosher RA. Wnt-5a and
Wnt-7a are expressed in the developing chick limb bud in a manner
suggesting roles in pattern formation along the proximo-distal and
dorsoventral axes. Mech. Dev. 1993;43:175–186.
Paddy EXPRESSION IN THE LIMB
Dolle P, Dierich A, LeMeur M, Shimmang T, Schuhbaur B, Chambon P,
Duboule D. Disruption of the Hoxd-13 gene induces localised
heterochrony leading to mice with neotenic limbs. Cell 1993;75:
431–441.
Fallon JF, Lopez A, Ros MA, Savage MP, Olwin BB, Simandl K. FGF2:
Apical ectodermal ridge outgrowth signal for chick limb development. Science 1994;264:104–107.
Frohman MA, Dush MK, Martin GR. Rapid amplification of cDNA
ends (RACE): A new method for obtaining full-length cDNA clones.
Proc. Natl. Acad. Sci. USA 198885:8998–9002.
Gaunt SJ. Chick limbs, fly wings and homology at the fringe. Nature
1997;386:324–325.
Geduspan JS, MacCabe JA. Ectodermal control of mesodermal patterns of differentiation in the developing chick wing. Dev. Biol.
1987;124:398–408.
Geduspan JS, MacCabe JA. Transfer of dorsoventral information from
mesoderm to ectoderm at the onset of limb development. Anat. Rec.
1989;224:79–87.
Gossler A, Joyner AL, Rossant J, Skarnes W. Mouse embryonic stem
cells and reporter constructs to detect developmentally regulated
genes. Science 1989;244:463–465.
Hardy MH. The secret of the hair follicle. TIGS 1992;8:55–61.
Laufer E, Nelson CE, Johnson RL, Morgan BA, Tabin C. Sonic
hedgehog and Fgf-4 act through a signalling cascade and feedback
loop to integrate growth and patterning of the developing limb bud.
Cell 1994;79:993–1003.
Loomis CA, Harris E, Michaud J, Wurst W, Hanks M, Joyner AL. The
mouse Engrailed-1 gene and ventral limb patterning. Nature 1996;
382:360–363.
MacCabe JA, Errick J, Saunders JW. Ectodermal control of the
dorsoventral axis in the leg bud of the chick embryo. Dev. Biol.
1974;39:69–82.
Meneely PM, Herman RK. Lethals, steriles and deficiencies in a region
of the X chromosome of Caenorhabditis elegans. Genetics 1979;92:
99–115.
Nagy A, Rossant J. Production of completely ES cell-derived fetuses.
In: Joyner AJ, ed. Gene Targeting: A Practical Approach. Oxford:
Oxford University Press, 1993:147–178.
Neubuser A, Koseki H, Balling R. Characterization and developmental
expression of Pax9, a paired-box-containing gene related to Pax1.
Dev. Biol. 1995;170:701–716.
325
Niswander L, Tickle C, Vogel A, Booth I, Martin G. FGF-4 replaces the
apical ectodermal ridge and directs outgrowth and patterning of the
limb. Cell 1993;75:579–587.
Noji S, Nohno T, Koyama E, Muto K, Ohyama K, Aoki Y, Tamura K,
Ohsugi K, Ide H, Taniguchi S, Saito T. Retinoic acid induces
polarizing activity but is unlikely to be a morphogen in the chick
limb bud. Nature 1991;350:83–86.
Nusslein-Volhard C, Wieschaus E. Mutations affecting segment number and polarity in Drosophila. Nature 1980;287:795–801.
Parr BA, McMahon AP. Dorsalizing signal Wnt-7a required for normal
polarity of D-V and A-P axes of mouse limb. Nature 1995;374:
350–353.
Parr BA, Shea MJ, Vassileva G, McMahon AP. Mouse Wnt genes
exhibit discrete domains of expression in the early embryonic CNS
and limb buds. Development 1993;119:247–261.
Riddle RD, Johnson RL, Laufer E, Tabin C. Sonic hedgehog mediates
polarizing activity of the ZPA. Cell 1993;75:1401–1416.
Riddle RD, Ensini M, Nelson C, Tsuchida T, Jessell TM, Tabin C.
Induction of the LIM homeobox gene Lmx1 by WNT7a establishes
dorsoventral pattern in the vertebrate limb. Cell 1995;83:631–640.
Saunders JW, Jr. The proximo-distal sequence of origin of the parts of
the chick wing and the role of the ectoderm. J. Exp. Zool. 1948;108:
363–403.
Saunders JW, Gasseling MT. Ectoderm mesenchymal interactions in
the origin of wing symmetry. In: Fleischmajer R, Billingham RE,
eds. Epithelial-Mesenchymal Interactions. Baltimore: Williams and
Wilkins, 1968:78–97.
Tickle C. Vertebrate limb development. Annu. Rev. Cell Biol. 1994;10:
121–152.
Vogel A, Rodriguez C, Warnken W, Izpisua-Belmonte JC. Dorsal cell
fate specified by chick Lmx1 during vertebrate limb development.
Nature 1995;378:716–720.
Wehr R. Expressions und Funktionsanalyse des Fkh5 Gens der Maus
[dissertation]. Göttingen, Germany: University of Göttengen, 1996.
Wolpert L. Positional information and the spatial pattern of cellular
differentiation. J. Theor. Biol. 1969;25:1–47.
Wurst W, Joyner AL. Production of targeted embryonic stem cells. In:
Joyner AJ, ed. Gene Targeting: A Practical Approach. Oxford: Oxford
University Press, 1993:33–61.