Original Communications

The Molecular Control of Upper

Extremity Development:
Implications for Congenital
Hand Anomalies
Aaron Daluiski, MD, Soyun E. Yi, PhD, Karen M. Lyons, PhD,
Los Angeles, CA

As the molecular aspects of limb development are being unraveled, more of the congenital
anomalies seen by hand surgeons in the clinical setting will have an identifiable molecular
basis. The majority of the data available regarding the molecular development of the upper
extremity have come from experimental animal studies, specifically the mouse and chicken.
These findings are being discovered by either direct surgical and molecular manipulation of
the developing limb or by production of mice deficient in specific genes. Relatively few
specific human mutations that cause limb abnormalities have been identified. Hand
surgeons should be aware of the basic molecular pathways controlling limb development
because they are in a unique position to be able to identify patients with such deformities.
In turn, detailed clinical descriptions of congenital anomalies affecting the upper extrem-
ity will advance the understanding of the cellular events controlled by the molecular
pathways of limb development. This review describes the general molecular basis of limb
development and correlates it with disease processes affecting the upper extremity. (J
Hand Surg 2001;26A:8 –22. Copyright © 2001 by the American Society for Surgery of the
Hand.)
Key words: Molecular biology, limb development.

Congenital deformities of the upper extremity are
common, occurring in as many as 1 in 626 live
births, with many of these being single gene disor-
From the Departments of Orthopaedic Surgery, Biological Chemis-
try, and Molecular, Cellular, and Developmental Biology, UCLA
School of Medicine, Los Angeles, CA.
Received for publication May 4, 1999; accepted in revised form
April 7, 2000.
No benefits in any form have been received or will be received from
a commercial party related directly or indirectly to the subject of this
article.
Reprint requests: Aaron Daluiski, MD, Hand and Microvascular
Surgery Fellow, Hospital for Special Surgery, 535 E 70th St, New
York, NY 10021.
Copyright © 2001 by the American Society for Surgery of the Hand
0363-5023/01/26A01-0010$35.00/0
doi: 10.1053/jhsu.2001.9419
ders.1 These deformities traditionally have been di-
vided into 7 categories based on the morphologic
characteristics of the deformity (Table 1).2– 4 These 7
categories do not define the etiology of the deformity
but only reference their morphologic appearance.
Mutations of genes controlling limb development
produce many of these deformities. Such molecular
abnormalities have been discovered in 3 of the main
types of proteins: transcription factors, secreted pro-
teins, and receptors. Once the molecular events con-
trolling normal limb development are known, aber-
rations occurring within this developmental cascade
can be identified and described. Understanding these
events will lead to earlier diagnoses and new thera-
peutic approaches. This review will emphasize the
major molecular events of limb development.

8 The Journal of Hand Surgery

 The Journal of Hand Surgery / Vol. 26A No. 1 January 2001 9

Table 1. Classification of Congenital
Hand Deformities2
I Failure of formation of parts
II Failure of differentiation of parts
III Duplication
IV Overgrowth
V Hypoplasia
VI Congenital constriction band syndrome
VII Generalized skeletal abnormalities
Development of the Upper Extremity
The development of the human upper extremity is
first visualized with the appearance of the limb bud at
26 days of life and continues through approximately
47 days when the joints of the hand develop (Fig. 1).5
Initiation of limb bud development can first be seen
when the underlying mesoderm of the appropriate
regions along the axis of the embryo forms an out-
growth into the overlying ectoderm. The mesodermal
tissue derives from 2 sources: the somites and the
lateral plate. Somitic mesoderm eventually forms the
muscles of the limb whereas the lateral plate meso-
derm develops into cartilage and bone (Fig. 2).
Molecular Biology and the
Developing Limb
A variety of techniques have been used to help
decipher the molecular aspects of limb development.
A full explanation of these techniques is beyond the
scope of this review, but a few definitions of terms
will help the reader understand how molecular path-
ways are identified. The protein factors discussed in
this review can be grossly divided into ligands, re-
ceptors, or transcription factors based on their rela-
tionship to a cell. Ligands, such as the bone morpho-
genetic proteins (BMPs), Sonic hedgehog (Shh), and
the fibroblast growth factors (FGFs), are proteins that
are secreted from a cell into the surrounding area.
These proteins signal neighboring cells to differen-
tiate or proliferate (Fig. 3).
Receptor proteins for secreted molecules, such as
the BMP receptors, are tethered to the cell membrane
where they bind ligands extracellularly and thereby
transmit the desired signal intracellularly. Receptors
may be made up of more than one protein. For
example, both BMP and transforming growth fac-
tor-␤ receptors consist of a combination of type I and
type II receptors. Signal transduction, caused by
binding of specific ligands to these receptors, causes
some change in cellular behavior, such as the expres-
sion of a new gene.
The third type of protein is a transcription factor,
which transmits the signal from an activated receptor
to the cell nucleus. These proteins physically bind to
DNA and influence the expression of new gene(s).
Transcription factors act within the nucleus to con-
trol gene expression. The transcription factors re-
quired for BMP or transforming growth factor-␤
signaling belong to the SMAD family of transcrip-
tion factors. In general; activated transcription fac-

Figure 1. Stages of upper extremity development of the mouse. Representative examples of mouse forelimb development
at embryonic day (A) 11, (B) 13.5, and (C) 15 (corresponding to 33, 48, and 56 days of development in humans,
respectively). Human limb development closely resembles these stages but occurs over longer period of time (see reference
5 for a stage comparison between human and mouse limb development).
10 Daluiski, Yi, and Lyons / Molecular Development of the Limb

Figure 2. Axial view of an embryo cut through the level of the developing limb. The spine, notochord, and gut endoderm
are shown. Two sources of mesoderm migrate from their origins into the limb laterally, causing the outgrowth of the limb
bud. Cells from the somitic mesoderm form putative muscle tissue and those from the lateral plate mesoderm form tendon
and bone.

tors travel into the nucleus and associate with DNA
molecules. Through DNA interactions, transcription
factors activate or modulate the transcription of a
gene or groups of genes. This affects transcription, or
production, of a gene’s mRNA. Once the mRNA is
transcribed. it is processed and shuttled into the cy-
toplasm, where it is eventually translated into the
appropriate protein. Regulation of a gene’s expres-
sion at the level of transcription is only one of the
many regulatory mechanisms that is active in the
development of the limb.
Although we will focus on these 3 types of pro-
teins in this review, it is important to note that other
classes of proteins can play important roles in limb
development. These include proteins that comprise
the extracellular matrix and intracellular cytoplasmic
proteins that interact with and/or modify the activity
of transcription factors.
Although each of these 3 types of proteins has a
different role in controlling cellular function, each of
the messenger RNAs (mRNAs) encoding their gene
products is intracellular. Determining the spatial dis-
tribution of cells that express each gene helps to
determine a gene’s ultimate function in limb devel-
opment. For example, genes expressed within the
interdigital mesenchyme may be involved in the pro-
grammed cell death of the interdigital webbing (Fig.
4). In contrast, genes that are expressed in regions of
putative tendon formation may have some role in
tendon development.
One powerful method of determining the expres-
sion pattern of a specific gene is in situ hybridization.
This technique reveals the spatial and temporal dis-
tribution of the mRNA encoding the protein of in-
terest. In situ hybridization is performed by incubat-
ing a tissue (ie, a section of developing limb tissue)
in solution with an antisense, or complimentary se-
quence, of RNA of the gene of interest (ie, a specific
FGF or BMP gene). The antisense probe is labeled;
therefore, when the probe binds to the RNA on the
tissue of interest, the exact cells that express the gene
mRNA are visually distinguishable (either through a
 The Journal of Hand Surgery / Vol. 26A No. 1 January 2001 11

Figure 3. Diagram of cells producing and secreting a growth factor (secreted ligand), a receptor for such a ligand, and
an intracellular transcription factor. Secreted factors must be processed properly while still intracellular and are then
expelled from the cell diffusing to its target (diamonds). Ligands act extracellularly, attaching to their appropriate receptors,
and cause signals to then be transmitted into the target cells. This activates one or more transcription factors (hatched ovals),
which are entirely intracellular proteins. Activated transcription factors associate with DNA and modulate the transcription
of new genes. It is the expression of new genes that eventually carries out the action of the ligand. Representative examples
of each type of protein are included in the figure. Wnt, wingless-type mouse mammary tumor virus integration site family;
BMPRIB, BMP receptor type IB; BMPRII, BMP receptor type II; FGFR, FGF receptor.

color reaction or through radiography) from other
cells (Fig. 4). Using this technique specific groups of
cells, such as those that make up the apical ectoder-
mal ridge (AER) or the interdigital mesenchyme of
the developing limb, can be distinguished based on
the genes they express. For example, Figure 4 shows
patterns of expression of the secreted factors bone
morphogenetic factor 7 (BMP-7) and the growth and
differentiation factor 5 (GDF-5), the transcription
factor Msx-2, and the BMP type II receptor protein.
Each of these genes is expressed at the mRNA level
in a unique pattern. Identifying the temporal and
spatial patterns of expression of the genes that con-
tribute to limb formation provides clues to the actual
functions of these genes throughout development.
Early Limb Formation: The Three Axes of
Limb Development
Limb formation is a continuous process. For ease
of categorization it can be divided into early and late
events. Most of this information has been determined
from experimentation in mouse and chicken limbs,
although the small number of human mutations caus-
ing limb anomalies suggests that a similar sequence
of molecular events occurs in humans as well. Early
events include limb bud outgrowth and dictate the
basic pattern and layout of the limb. These early
events of limb development can be divided into
growth along 1 of the 3 spatial axes of the limb (Fig.
5). These axes are proximodistal (PD), anteroposte-
rior (AP), and dorsoventral (DV). The PD axis de-
fines the outgrowth of the limb. The AP axis occurs
in the radioulnar (preaxial–postaxial) direction. The
third axis is the DV, or dorsopalmar, axis. In lower
vertebrates, the AP (radioulnar) axis is determined
before a limb bud is visible (see review by Tickle6).
After the AP axis is determined, the DV is estab-
lished followed by the PD axis. As discussed below,
the molecular pathways controlling establishment of
these axes intersect at multiple points, which ensures
coordinated development of the limb.
Proximodistal Growth is Controlled by
Fibroblast Growth Factors Secreted by
the Apical Ectodermal Ridge
Where the ventral and dorsal layers of ectoderm of
the developing fetus meet, a ridge of ectoderm, the
AER, is formed in the region where the limb will
12 Daluiski, Yi, and Lyons / Molecular Development of the Limb

Figure 4. In situ hybridization of bone morphogenetic factor 7 (BMP-7), Msx-2, growth and differentiation factor 5
(GDF-5), and type II BMP receptor (BMPR type II) in a developing mouse forelimb. BMP-7 is expressed in the tips of
the forming digits and in the perichondrium of the developing digital rays. The transcription factor Msx-2 is highly
expressed in the interdigital mesenchyme and ectoderm of the tips of the digits. Growth and differentiation factor 5 is
expressed primarily in areas of future joint formation (metacarpophalangeal, interphalangeal, and carpal joints). It also is
expressed highly in developing tendon (not shown). The BMP type II receptor is expressed in the perichondrium of the
developing digits. This information suggests that these genes may be important in the formation of the tissues in which they
are expressed. The exact function of each molecule is not determined through this method, but these data provide
information regarding potential developmental functions.

develop (Fig. 5; Table 2). Several lines of evidence
have shown that this ridge of tissue is the mediator of
PD growth. If the AER is surgically removed, PD
growth ceases.7 The time point at which the ridge is
removed determines at which level limb truncation
occurs; the earlier the ablation of the AER, the more
proximal the limb deficiency. Conversely, if the AER
tissue is implanted ectopically at a remote site, an
extra limb forms at the new site.5 These experiments
demonstrate that the AER produces a diffusable sig-
nal that controls limb growth along the PD axis.
In situ hybridization studies in both mouse and
chick limbs have shown that several members of the
FGF family (FGF-2, -4, and -8) are highly and spe-
cifically expressed by the AER.8,9 Since these mol-
ecules are secreted proteins, they are excellent can-
didates for the signal-stimulating PD growth of the
developing limb.
Expression of a gene in the correct location and the
correct developmental stage is an important criterion
for identifying potential mediators of limb develop-
ment, but it only suggests a mechanistic link. Appli-
cation of these FGF proteins (FGF-2, FGF-4, and
FGF-8) can completely substitute for the AER in
limbs where the ridge has been surgically excised,
restoring normal PD growth of the limb.7,10 Thus,
application of FGFs is sufficient to induce limb
growth, implying that these growth factors are pri-
marily involved in the PD growth of limbs. Another
FGF gene, FGF-10, is expressed in a location con-
sistent with factors responsible for PD growth. Mice
deficient in FGF-10 have complete transverse limb
defects.11 This provides further evidence that genes
in the FGF family are responsible for primary lon-
gitudinal growth of the limb.
Although FGF secreted by the AER is the direct
 The Journal of Hand Surgery / Vol. 26A No. 1 January 2001 13

Figure 5. Early limb bud development. (Left) The developing limb in the frontal plane. The limb is paddle shaped at this
stage of development. The thin ridge of tissue at the distal-most end of the limb is called the apical ectodermal ridge (AER).
The mesoderm is all the nonectodermal soft tissue. The posterior (ulnar)-most region of mesoderm is a specialized tissue
called the zone of polarizing activity (ZPA). (Right) The same limb in cross-section along the dotted line shown in the left
panel. The dorsal and ventral ectoderm, key tissues in dorsoventral patterning, are shown. The ridges seen at the anterior
and posterior ends are the cut ends of the AER.

stimulus for PD growth of the limb, the underlying dioulnar (AP) axis. This links the formation of lon-
mesoderm supplies a signal that causes the AER to gitudinal limb outgrowth with radioulnar growth,
produce FGF.6 Thus, a complex interaction between ensuring proportional growth of the limb.
the AER and the underlying mesoderm is necessary
for normal PD growth. This portion of the underlying
Anteroposterior Growth Is Caused by the
mesoderm also controls the establishment of the ra-
Secretion of Sonic Hedgehog From the
Zone of Polarizing Activity
The second axis described is the AP, or radio-
Table 2. Primary Axis Formation and Key Growth ulnar, axis (the preaxial–postaxial axis). The po-
Factors Involved sitional information for this axis is determined
very early within the lateral plate mesoderm be-
Primary
Axis Anatomic Location Growth Factor fore limb bud formation. Experiments have shown
PD AER FGF
that after the limb bud has formed and the AP axis
DV Dorsal ectoderm Wnt-7a has been established, a group of cells from the
AP (radioulnar) ZPA Shh posterior (ulnar) tissue of the limb secretes a pro-
Wnt-7a, wingless-type mouse mammary tumor virus integra- tein, or morphogen, that acts to pattern the AP
tion site family member 7a; ZPA, zone of polarizing activity. structures. This region of posterior mesenchyme
14 Daluiski, Yi, and Lyons / Molecular Development of the Limb

polarizes the limb into a radioulnar axis and is
called the zone of polarizing activity (ZPA).12
Transplantation of the ZPA mesenchyme from the
posterior (ulnar) to the anterior (radial) region of
the limb causes a mirror duplication of the ulnar
part of the hand. The number and morphology of
the formed neodigits is a function of the number of
ZPA cells transplanted.13 This finding suggests
that this axis is dependent on the amount of the
morphogen secreted from these mesodermal cells.
Sonic hedgehog, a secreted protein, is expressed
by the cells that make up the ZPA (Fig. 6).14 This
finding provided initial evidence that Shh may be
the molecular signal for AP axis formation. Sonic
hedgehog protein ectopically implanted into the
anterior side of developing chick limbs causes a
mirror image duplication of the limb.12 This also
results when ZPA tissue is implanted into the
anterior side.12 Thus, Shh protein alone is suffi-
cient to replace the function of the ZPA in the
formation of the AP axis. Naturally occurring
polydactylous mouse mutants exist that have inap-
propriate expression of Shh anteriorly.15 The poly-
dactylous digits have the appearance of ulnar dig-
its, which is also consistent with this molecular
model.15
Although the primary function of Shh is to act as
a stimulus for AP growth, it is also necessary for the
maintenance of PD growth. Deletion of the ZPA
without transplantation elsewhere in the limb causes
PD (transverse) truncation of the limb.16 Mice defi-
cient in Shh have complete transverse limb trunca-
tions, implicating Shh as a key factor in both PD and
AP axis formation.17
Sonic hedgehog induces FGF-4 in the AER and is
part of a positive feedback loop involving the ZPA
and AER.18,19 Thus, the PD and AP axes are not
independent of each other in limb formation. This
connection between the ZPA controlling the AP axis
and the AER controlling the PD growth regulates the
proportional growth of the limb in each of these
directions.

Figure 6. Expression of Shh in a developing mouse embryo. The entire mouse embryo is shown on the left. Note the
paddle-like extremities indicating an early stage of limb development (approximately mouse embryonic day 10.5). The dark
signal at the posterior (ulnar) region of the limbs represents the cells of the ZPA expressing the Shh gene. Two of the axes,
PD and AP, are labeled above. The AER is the thin rim of tissue at the edge of the limb. The AER tissue expresses the
FGF genes (not shown) and is necessary for PD growth of the limb.
 The Journal of Hand Surgery / Vol. 26A No. 1 January 2001 15
The Dorsoventral Axis Is Formed in
Part by Wingless-Type Mouse Mammary
Tumor Virus Integration Site Family
Member 7a Secretion From
the Dorsal Ectoderm
Dorsoventral patterning is somewhat less well un-
derstood than the other two primary axes. Before
limb bud outgrowth, the signaling center for the
instruction of DV patterning is transferred from the
underlying mesoderm to the ectoderm or primitive
skin. In situ hybridization studies show that the dor-
sal ectoderm expresses a secreted protein called
wingless-type mouse mammary tumor virus integra-
tion site family member 7a (Wnt-7a). Mice lacking
Wnt-7a have biventral limbs20,21 with duplicated
palms. This finding suggests that Wnt-7a is neces-
sary for formation of dorsal limb elements during
limb development. These mutant animals also have
shortened digits, which have more anterior (or radial)
characteristics. The transcription factor Lmx1 is in-
duced by Wnt-7a in the dorsal mesoderm. Overex-
pression of this transcription factor in ventral meso-
derm results in ventral cells adopting a dorsal
appearance and produces a mirror image dorsal du-
plication. It is interesting that the loss of Lmx1
causes nail-patella syndrome in humans and a similar
phenotype in mice.22–24
In a similar but reciprocal fashion, the transcrip-
tion factor engrailed-1 is expressed in the ventral
ectoderm. Mice lacking this transcription factor have
bidorsal limbs.25,26 Although the exact nature of the
molecular interaction is unknown, engrailed-1 re-
presses Wnt-7a expression in the ventral portion of
the limb.
As discussed above, these 3 axes are not indepen-
dent of each other. In addition to defining the DV
axis, Wnt-7a also had been shown to induce Shh,
which ties DV patterning to the other 2 axes.27 Thus,
the shortening and lack of posterior (ulnar) digits of
the Wnt-7a mutants can be explained in part by a
concomitant decrease in Shh affecting both the PD
(shortened) and AP (lack of posterior digits) axes in
addition to the role of Wnt-7a in elaboration of the
DV axis.21 It is therefore easy to understand how a
defect in a single gene (Shh) can affect multiple axes
and lead to complex and variable limb deformities.
There are no known human mutations involving the
Shh gene.
A summary of the anatomic location of expression
and primary axis affected for each of the 3 axes is
given in Table 2. Each of these factors and the
general mechanism of interaction is shown in Fig-
ure 7.
Hox Family of Transcription Factors Is
Important in Limb Development
Primary axis formation is only one facet of limb
development. A family of transcription factors,
called Hox (Homeobox) genes, is also important in
limb development. It is thought that the expression of
these transcription factors is induced in part by ex-
tracellular factors acting through a receptor (Fig. 3).
It is unclear which secreted factors cause expression
of Hox genes, although some of the factors discussed
previously (including Shh28) probably play roles in
their induction.
There are 4 clusters or groups of Hox genes:
HoxA, HoxB, HoxC, and HoxD. Each cluster resides
on a different chromosome. Taken together these 4
clusters contain a total of 38 hox genes. In general,
expression of certain hox genes is localized to spe-
cific regions of the developing limb.29 –32 The HoxD
complex, for example, is important in AP differen-
tiation. The different genes of the HoxD group have
expression patterns that correlate with each of the 5
digital anlagen.33 Thus, the HoxD group genes are
excellent candidates for loci causing hand malforma-
tions. Mutations in the HoxD group locus have been
shown to be the cause of several types of human
synpolydactyly (Table 3).34,35 The precise signaling
pathways through which the Hox genes arc activated
are not known.
Specific Abnormalities in Limb Formation
After primary axis formation of the limb occurs,
the cells within the limb bud continue to differentiate
into the specialized tissues of the extremity. The
complexity of cellular processes underlying these
later events (including the formation of joints; the
proper size, shape, and position of the bones of the
wrist and digits; the formation of dorsal and ventral
tendons; and neurovascular differentiation) is im-
mense. Since each of these complex events is gov-
erned and regulated by molecular signals within the
developing limb, the task of dissecting these regula-
tory pathways is daunting.
Knowledge of the expression patterns of specific
genes (ligands, receptors, and transcription factors)
and correlation with specific temporal and spatial
developmental landmarks is one way of determining
the potential importance of a gene in limb develop-
ment. Mutational analyses in mice and ectopic ex-
16 Daluiski, Yi, and Lyons / Molecular Development of the Limb

Figure 7. Summary of the molecular regulation of limb development. Posterior (ulnar) to anterior (radial) view of the
developing limb showing the AER, ZPA (shaded), and dorsal and ventral ectoderm. Sonic hedgehog from the ZPA is
involved in a feedback loop with FGF from the AER. This ties together the AP and PD axes. Engrailed-1 (En1) is expressed
in the ventral ectoderm and represses the expression of Wnt-7a in ventral tissues. These interactions contribute to DV
patterning. Wnt-7a induces the transcription factor Lmx lb, through which some of the aspects of dorsalization occurs.

pression of potential signaling genes may provide
more insight into their roles. The ultimate goal, how-
ever, is to determine specific mutations in humans
causing congenital hand malformations. Table 3
identifies the genes that are important in limb devel-
opment. Aberrations in these genes cause a variety of
limb malformations in vertebrates. These malforma-
tions and what is known of the molecular causes in
the developing vertebrate limb are listed. The major-
ity of these mutations involve the later events of limb
formation as mutations in factors that disturb early
limb formation often create nonviable embryos.
Polydactyly
Many members of the BMP family, including
BMP-2,- 4, and -7, are expressed in the developing
limb. This suggests that they are important in medi-
ating at least some of the later events of hand devel-
opment. BMP-7, for example, is expressed in the
perichondrium of the developing phalanges and the
finger tip ectoderm (Fig. 4).36 Mice deficient in
BMP-7 have a variable preaxial polydactyly, sug-
gesting that in addition to its role in bone formation,
this growth factor has a primary suppressive role in
digit formation in the vertebrate limb.36,37 Because
BMP-7 is expressed at high levels in the interdigital
mesenchyme, which normally undergoes pro-
grammed cell death, loss of BMP-7 likely allows for
survival of these cells. These surviving cells can then
give rise to an extra digit.
Other factors cause the development of polydac-
tyly. Gli-3 is a transcription factor important in Shh
signaling and is strongly expressed in developing
limbs.38,39 Lack of Gli-3 in both mice and humans
causes polydactyly, indicating that like BMP-7, the
normal function of Gli-3 is to mediate the suppres-
sion of polydactyly. Gli-3 has been shown to be
induced by BMPs, suggesting that Gli-3 and BMPs
are involved in a common pathway controlling digit
formation.40 – 44
Syndactyly
Syndactyly is one of the most common congenital
deformities of the hand. The limb paddle is sculpted
 The Journal of Hand Surgery / Vol. 26A No. 1 January 2001 17

Table 3. Genes and the Limb Abnormalities Associated With Loss or Modification of the Gene Product
Primary Condition
Affecting the
Gene Extremity Species Notes
Ligands (secreted factors)
Bmp-7 Preaxial polydactyly M Primarily affects hind foot36,37
CDMP/GDF-5 Brachydactyly H, M Grebes chondrodysplasia, Hunter-Thompson syndrome54–56
Fgf-10 Transverse limb M Ref 11
deficiency
Ihh Preaxial polydactyly M Ectopic IHH causes 6 digits per limb70
Noggin Symbrachydactyly H, M Lack of joint development in fore and hind feet71
Shh Transverse defect M Ref 17
Wnt-1 Transverse defect M Ref 72
and syndactyly
Wnt-7a Palmar duplication M Ref 21
Receptors
BMP receptor IB Brachydactyly M Ref 57
FGF receptor 1-3 Brachydactyly H, M Aperts, Jackson-Weiss, Crouzon, Pfeiffer syndromes47,49,50,73,74
Patched Preaxial polydactyly M Ref 75
Transcription factors
Alx-4 Preaxial polydactyly M Ectopic ZPA with more anterior (radial) expression of Shh,
HOXD13, and FGF-476
CBP Broad thumbs H Rubinstein-Taybi syndrome77
Gli-3 Preaxial, central, or H, M Pallister-Hall syndrome, Grebes cephalopolysyndactyly40,41,43
postaxial
polysyndactyly
HoxA-13 Preaxial digit loss; H, M HoxA13/HoxD13 double mutant shows transverse deficiency;
brachydactyly functional overlap; HoxA13 loss in human causes hand-foot-
genital syndrome29,78
HoxB-8 Mirror duplication of M ZPA duplication in forelimb79
hands
HoxD-12 Loss of thumb, M Overexpression produces limbs with more ulnar digits and loss of
polydactyly thumb80
HoxD-13 Synpolydactyly H Synpolydactyly34,81,82
HoxD-11-13 Synpolydactyly M Synpolydactyly with loss of all 3 hox genes35
Lbx1h Loss of dorsal M Loss of upper extremity extensors and all lower-extremity
muscles muscles83
Lmx1b Nail changes H, M Nail-patella syndrome22–24
Msx-2 Brachydactyly, M Dominant mutation in the gene not loss of function; associated
simple syndactyly craniosynostosis84
NF-␬␤ Limb truncation C AER malformation85
p63 Truncated limbs M Failure of AER differentiation86,87
Pax-9 Preaxial polydactyly M Cleft palate and absent teeth88
Pitx 1 Lower extremity has M Refs 64,89
features of upper
extremity
Prx-2 Postaxial polydactyly M PRX1/PRX2 double mutant90
Sall1 Polydactyly H Townes-Brocks syndrome91
SHOX Brachydactyly, H Leri-Weill dyschondrosteosis92
meromelia
Sox-9 Campomelic H Bowed long bones93
dysplasia
Tbx-3 Ulnar ray deficiency H Occasional volar nail formation94
Tbx-5 Radial ray deficiency H Holt-Oram syndrome; radial ray limb deficiency with thumb
abnormalities65,66
Twist Brachysyndactyly, H Saethre-Chotzen syndrome95–97
clinodactyly
Other
Connexin-43 Transverse truncation C Split limbs98
CRABP-II Postaxial polydactyly M Variable phenotype depending on genetic background99
Link protein Shortened limbs M Generalized chodrodysplasia100
The limb abnormalities listed here are not all due to functional loss of the involved protein.
H, human limb; M, mouse limb; C, chicken limb.
18 Daluiski, Yi, and Lyons / Molecular Development of the Limb
by cells in the interdigital mesenchyme undergoing
programmed death.45 This cell death leaves behind
tissue that will eventually become each individual
digit. Several genetic causes of syndactylies have
been determined (Table 3). Of particular interest is
Apert’s syndrome, which is characterized by a com-
plex synpolydactyly. This syndrome, and several
others, is caused by mutations in FGF receptors.46 –50
Other molecular causes of syndactyly exist but are
unidentified. Msx-2 is a transcription factor that is
highly expressed in the interdigital mesenchyme of
the hand (Fig. 4). This expression pattern suggests a
potential role of this transcription factor in mediating
interdigital cell death. Tissue culture and chick limb
experiments have implicated Msx-2 in BMP-induced
apoptosis, which suggests a role for this transcription
factor in programmed cell death of the interdigital
mesenchyme.51,52
Brachydactyly
Deficiency in cartilage-derived morphogenetic
protein (known as GDF-5 in mice), a member of the
BMP family, can cause brachydactyly. Growth and
differentiation factor 5 is expressed in the hand in
putative regions of joint and tendon formation53 (Fig.
4). A naturally occurring mouse lacking GDF-5 ex-
ists.54 The brachydactylous forefeet and hindfeet ex-
hibit rhizomelic shortening of the entire limb. Cer-
tain human brachydactylous conditions, including
Grebe and Hunter-Thompson chondrodysplasias,
have been shown to be caused by deficiencies in the
human cognate GDF-5 gene (cartilage-derived mor-
phogenetic protein-1).55,56
Mice deficient in the BMP receptor type IB have a
similar brachydactylous condition. These animals
have absent proximal and middle phalanges, a phe-
notype remarkably similar to, but less severe than,
the GDF-5 mutant.57,58 These genes are expressed in
similar locations and times throughout the develop-
ment of the limb, suggesting that this BMP receptor
is necessary for normal signal transduction of he
GDF-5 growth factor.59,60
All Limbs Are Not Created Equal
Until recently little distinction had been made be-
tween the development of the upper and lower ex-
tremities. Although the basic molecular events de-
scribed above are conserved between upper and
lower extremity development, the obvious differ-
ences in morphologic appearance dictate that there
must be differences at the molecular level as well.
The T-box family of transcription factor has 2 mem-
bers (Tbx4 and Tbx5) that are specifically expressed
in the hindlimb and forelimb, respectively. Overex-
pression of upper extremity transcription factor Tbx5
in the lower extremity causes this limb to develop
with features of an upper extremity.61,62 Another
limb-specific gene, the transcription factor Pitx1, is
expressed only in lower extremities. Mice that lack
this transcription factor have a reduction in lower
extremity expression of Tbx4. The lower extremities
of these mice have upper extremity morphologic
features.63,64 These experiments show that at least 3
factors are important in differentiating the develop-
ment of upper and lower extremities in these lower
vertebrates. Interestingly, human mutations in Tbx5
cause Holt-Oram syndrome, which affects only the
upper extremities.65,66
Molecular Development of the Limb:
Filling in the Blanks
One of the easiest ways to decide whether a gene
is potentially involved in the development of the
limb is to determine whether its mRNA is expressed
in the extremity. A large number of genes are ex-
pressed in the developing hand, making them inter-
esting candidates for potential involvement in the
development of the upper extremity. Tissue-specific
expression of genes in tendon (eg, Eya, Six), bone,
and cartilage (eg, GDF, BMPs, BMP receptors) has
provided clues to potential functions of these growth
factors, receptors, and transcription factors in tissue
specification important in human limb development
(Fig. 4).
The basic molecular events of hand formation are
being discovered and characterized in lower verte-
brates at a remarkable rate. An important goal is to
apply this information to human hand and upper
extremity development: Hand and pediatric orthope-
dic surgeons are in unique positions to identify pa-
tients with congenital limb anomalies that may have
a genetic or molecular basis. The contribution from
the surgeon is crucial to carry our understanding of
limb development from the laboratory to the clinical
setting. Conversely, the surgeon must be aware of
basic molecular aspects of limb development to be
able to determine which patients may hold keys to
linking basic science to clinical entities. An under-
standing of the molecular events provides the sur-
geon with information necessary to help identify
 The Journal of Hand Surgery / Vol. 26A No. 1 January 2001 19
which part of the normal developmental cascade may
be defective.
Studies of the developing limb will undoubtedly
lead to secondary and potentially more important
therapeutic uses of these same molecules necessary
for limb formation. For example, BMPs have been
shown to be potent inducers of bone formation and
are currently undergoing clinical trials for use in
fracture treatment.67– 69 GDF-5, another member of
the BMP family, is expressed in tendon tissue as well
as cartilage. This gene has been shown to produce
fibrous tissue resembling tendon and has been used
to augment tendon defects in rodents.60
As the specific mutations that cause certain hand
defects are found, the resolution at which we under-
stand limb deformities will increase. Unraveling
these molecular events will allow us to more accu-
rately diagnose and more effectively treat these de-
formities. The “molecular anatomy” of the human
genome and its influence on musculoskeletal anat-
omy and pathology is having a greater impact on
clinical medicine. The contribution of the molecular
and developmental biologist in isolating these new
molecules is quite evident. The role of the surgeon in
helping to determine the actions of these molecules
in human development and in developing novel ways
of delivering these molecules in the clinical setting is
in many ways more important, further emphasizing
the need for surgeons to understand the molecular
aspects of the developing limb.
The authors thank Dr Roy Meals for useful discussions and critical
review of the manuscript.
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