Limb development

The upper limb bud appears during the fourth week of development as an outgrowth on the sides
of the embryo. Cells of the somatic lateral plate mesoderm form the skeletal framework of the
limb, while mesoderm from the somite migrates in to form the muscular component and
contributes to the limb’s vascular network. Over the next 4 weeks, growth and differentiation
transform the limb bud into an elegant asymmetric organ that is one of the defining features of
the human species. The limb bud growth and differentiation are under the control of signaling
centers, specialized areas within the limb which dictate, in concert, the appropriate sequential
events of development.

The establishment of signaling centers and their subsequent behavior are under genetic control.
The genes and the morphogens (proteins) they encode control the process of limb growth and
differentiation and act as messengers between signaling centers and developing cells.

As knowledge of the process of limb development becomes more sophisticated, an

understanding of the causation of limb anomalies becomes clearer.

Genetic mutations can disrupt the molecular function of a number of proteins orchestrating limb
development, including secreted proteins (ligands), ligand receptors, and transcription factors.
The mutation may be inherited or may arise spontaneously. Environmental factors, including
mechanisms such as thalidomide, which resulted in an epidemic of limb malformations during
the 1960s, radiation, nutritional defects, and infections may affect the molecular pathways of
development or be responsible for a more gross insult resulting in tissue hemorrhage and/or
necrosis. For example, if such direct damage occurs obliterating the apical ectodermal ridge
(AER), an important signaling center that directs limb outgrowth, transverse truncation will
result.

Classification
Congenital anomalies in the hand demand a reproducible and consistent terminology, a universal
language which allows discussion of complex clinical entities, indications for treatment, and
comparisons of results.

Swanson’s classification was adapted as the standard system by which congenital hand
anomalies are described by the IFSSH in 1976.1 It is derived from ideas existing in the 1970s
regarding limb embryology and is largely based on morphological appearance. Knight and Kay2
have presented an extended version, attempting to incorporate a list of all congenital anomalies.
Regrettably, this classification, based as it is on appearance and relying on knowledge available
in the 1960s and 1970s, is intrinsically unsuited to alterations based on causation and etiology at
a molecular level. It is perhaps time to consider alternatives.

Ideally, an overall classification of congenital hand anomalies would be based on etiology, with
such a classification indicating the site in the molecular pathway and/or the anatomical site in the
limb bud and the time at which the aberration occurs.
It would reflect whether the fault is a problem of longitudinal outgrowth, lies within one of the
axes of differentiation, and affects primarily the hand plate alone or the whole upper limb. It is
probable that the causation of some anomalies will confound our attempts to assign an etiology.

Assessment and principles of treatment

It is clear that the terminology applied to our efforts should reflect a language common to, and
understood by, the geneticist, the anatomist, the pathologist, and the surgeon. For this reason,
these authors have chosen to link the terminology of embryology of the limb bud at the
molecular level, the terminology of dysmorphology, and the terminology of classification. This
consistency would appear to provide less controversy in a rapidly changing field of knowledge.
Furthermore, such an approach is of benefit when attempting to explain to parents how the limb
normally forms and how any particular anomaly may occur. This process plays a vital role in the
assessment of any specific anomaly and leads to a sensible plan of management.

Limb development (embryology)

Overview of upper limb morphogenesis
Around day 26 (4 weeks) after fertilization (Carnegie stage 12), the upper limb bud appears as an
oblong ventrolateral bulge on the body wall between somites 9–12 (C5–8) (Fig. 25.1).3 The
emerging limb bud is composed of somatic lateral plate mesoderm covered by ectoderm.
Subsequent limb bud growth and differentiation are described in terms of three coordinate axes:
proximodistal, dorsoventral (dorsovolar), and anterioposterior (or radioulnar) (Fig. 25.2), each
controlled by distinctive regions – signaling centers.
Fig. 25.1 Upper limb and hand development. (A) Human embryo at Carnegie stage 13 (about 28
days of development), showing early limb buds. (B, C) Cross section through an upper limb bud
at 32 days of development (Carnegie stage 14), showing dorsal (Do)-ventral (Ve) limb bud
flattening and the thickened apical ectodermal ridge at the distal tip of the limb bud (that runs
along the dorsal-ventral border from anterior to posterior). (D) The handplate at 41 days of
development (Carnegie stage 17). The scalloped edge conforms to the condensing digital
mesenchyme. (E) Limb at 46 days of development (Carnegie stage 19). Separate fingers are
evident. The proximal limb skeleton is well developed and ossification has begun in the humerus
and shoulder girdle. (F) The embryo at 49 days of development (Carnegie stage 20), showing
ossification centers in the upper limb, lower limb, and face. Note that upper limb development is
more advanced than lower limb development. (G) Upper limbs at week 8 (Carnegie stage 22).
The fingers are completely separate and ossification centers have developed in the forearm, i.e.,
radius and ulna. (H) The upper limb at week 9 (Carnegie stage 23). Metacarpal and distal
phalangeal ossification has begun. (I) The upper limb at week 11. Ossification in proximal and
middle phalanges is now evident in addition to distal phalanges and metacarpals. (J) The hand at
week 12, showing developing nail fields (nails become visible during week 16). (Portions
redrawn from England MA. A colour atlas of life before birth. London: Year Book Medical
Publications, 1983.)

Fig. 25.2 Limb bud coordinate axes and signaling centers: (A) The forelimb (boxed region) of a
Carnegie stage 13 embryo depicting the three coordinate axes – each with its own signaling
center: the apical ectodermal ridge (AER) coordinating proximodistal (Pr-Di) outgrowth and
differentiation; dorsovolar (Do-Vo) asymmetry is regulated by dorsal ectoderm; radioulnar
asymmetry is controlled by the zone of polarizing activity (ZPA). Within the progress zone (PZ)
the fate of mesodermal cells is determined by these signaling centers. The axes and signaling
centers are shown in three different orientations: (B) dorsal view, (C) lateral, end-on view, and
(D) axial cross-section.

(Modified from Oberg KC, Greer LF, Naruse T. 2004. Embryology of the upper limb: the
molecular orchestration of morphogenesis. Handchir Mikrochir Plast Chir 36:98–107.)

The ectoderm overlying the distal edge of the limb bud at the dorsovolar boundary thickens and
forms a distinct ridge of stratified ectoderm, the AER.4 The thickened AER is critical for
proximodistal outgrowth and also adds mechanical rigidity to the distal rim of the limb bud,
flattening it along the dorsovolar axis. Excision of the AER in chicken embryo limb buds
prevents further proximodistal outgrowth and results in limb truncation.5 Underlying the AER,
the distal mesoderm exhibits robust proliferation and has been termed the progress zone (PZ).
Cells in the PZ ultimately differentiate into specific cell types and are directed to specific
positions within the limb.
The dorsal ectoderm is a critical signaling center for directing formation of dorsal and volar
characteristics of the limb. Excision and rotation of dorsal ectoderm cause the formation of
dorsal structures on the limb ventral surface.6 Another collection of mesodermal cells at the distal
ulnar (posterior) margin is called the zone of polarizing activity (ZPA). Although not
morphologically distinct, these cells direct radioulnar patterning and coordinate asymmetric limb
patterning with the other signaling centers during development. Removal of the ZPA in animal
models leads to loss of the ulna and the ulnar digits.7 Conversely, transplantation of these cells to
the anterior (radial) aspect of limb buds of chicken embryos will result in formation of a mirrored
complement of ulnar digits radially.8

Over the next week, the limb bud expands and elongates, particularly along the proximodistal
axis (Table 25.1). By day 33 of development (Carnegie stage 14), differential growth and
programmed cell death transform the distal portion into a paddle-shaped hand plate. There is also
progressive mesodermal condensation along the proximodistal axis, forming a tripartite skeleton
composed of a proximal section, the stylopod (shoulder girdle and humerus), a middle section,
the zeugopod (radius and ulna), and a distal section, the autopod (hand). The joints become fully
evident about day 51 (Carnegie stage 20) when the elbow and wrist joints flex and by day 56
(end of eighth week, Carnegie stage 23), the major morphologic features of the limb are
complete (Fig. 25.1).

Table 25.1 Timing of hand formation

Time after fertilization Hand development

27 days Development of arm bud
28–30 days Further development of arm bud
34–36 days Elongation of arm bud
34–38 days Formation of hand paddle
38–40 days Early separation of digits
44–46 days Digits separated
Week 9–10 Formation of fingernails begins

(Reproduced from: Gupta A, Kay SPJ, Scheker LR, editors. The growing hand: diagnosis and
management of the upper extremity in children. London: CV Mosby; 2000, p. 25.)

The molecular control of limb outgrowth and patterning

During early embryonic development, Hox transcription factors set up a segmental body plan
along the cranial–caudal axis.9 By the fourth week of development the presumptive upper limb
fields are established, triggering the expression of Tbx5, Wnt3, and Fgf10 which initiate limb
formation.10 Tbx5 determines forelimb identity, while Wnt3 and Fgf10 induce mesodermal
proliferation and limb outgrowth. Mesodermal Fgf10, in conjunction with ectodermal radical
fringe (R-fng) at the apical dorsovolar boundary, induces ectodermal thickening to form the
AER.11–13 Formation of the AER initiates the expression of AER-related Wnt and Fgf proteins
(including Fgf2/4/8/9/17)14,15 which, in turn, act on the mesoderm just underneath the AER to
sustain Wnt3 and Fgf10 expression in the PZ. This reciprocal loop of ectodermal and
mesodermal Fgf/Wnt proteins promotes progressive proximodistal outgrowth16,17 (Fig. 25.3). In
the absence of Wnt3 or Fgf10 limbs fail to develop and tetramelia results.18,19 Application of Fgfs
to the distal chick limb bud after AER removal will return proximodistal limb outgrowth. 15
Within the PZ, mesodermal cells persist in an undifferentiated or receptive state, allowing the
signaling centers to direct their fates.

Fig. 25.3 Morphologic impact of signaling centers. Upper panel: role of apical ectodermal ridge
(AER)-related Fgfs (orange) on skeletal outgrowth (humerus, blue; radius and ulna, green; hand,
magenta). Middle panel: role of Shh secretion from the zone of polarizing activity (ZPA):
influence on the forearm and digits (depicted in purple). Lower left panel: role of Wnt7a
(medium green) and Lmx1b (light green), from the dorsal ectoderm and mesoderm respectively,
in tendon and ligament formation in a digit. Do, dorsal; Vo, volar; Pr, proximal; Di, distal; PZ,
progress zone. Lower right panel: illustration depicting communication between signaling
centers – reciprocal Shh-Fgf loop (white bidirectional arrow). Wnt7a influence on Shh
expression (white unidirectional arrow). Rad, radial; Uln, ulnar.

The ZPA in the posterior (ulnar) limb mesoderm secretes a potent morphogen, sonic hedgehog
(Shh), that regulates radioulnar patterning20 (Fig. 25.3). Shh induces posterior (ulnar)
proliferation of the limb bud expanding its width.21,22 Moreover, Shh posteriorizes (ulnarizes) the
developing forearm and defines the identity of the ulnar four digits. In a naturally occurring
chicken mutant, limb-specific Shh expression is lacking and the upper limbs develop without
ulnas and without digits.23 Application of exogenous Shh to the posterior (ulnar) aspect of the
limb bud can fully recover normal limb morphology. Furthermore, application of Shh to the
anterior margin produces mirrored duplication of ulnar digits radially.20

The AER and the ZPA are also closely linked by a reciprocal feedback loop that maintains Shh
expression at the distal posterior (ulnar) border of the limb bud adjacent to the AER during
progressive outgrowth.24–26 Removal of the AER causes regression of Shh expression and
ablation of the ZPA induces a loss of Fgf signaling.25 Secretion of Wnt7a from the dorsal
ectoderm induces the homeodomain transcription factor Lmx1b in the underlying mesoderm and
asymmetrically dorsalizes the developing limb13,27,28 (Fig. 25.3). Wnt7a also contributes to the
maintenance of Shh secretion from the ZPA29 linking the dorsovolar and radioulnar axes.
Removal of dorsal ectoderm reduces Shh expression and disrupts posterior (ulnar) patterning.30
Thus, Shh plays a pivotal role during limb development linking dorsovolar, proximodistal, and
radioulnar axes during outgrowth.30

Around the end of the fifth week of development the hand plate becomes visible. Interplay
between Hox transcription factors (particularly Hoxd9-13 and Hoxa9-13) and Shh establishes
digit number and identity31–34 (Fig. 25.4). Shh also induces an ulnar to radial (posterior to
anterior) gradient that appears to involve Bmps in at least two roles in the formation and
differentiation of digits. First, Bmps induce programmed cell death or apoptosis via discrete
interdigital signaling centers, in part by repressing Fgf expression in the overlying AER.35–37 In
addition, Bmps play a role in completing digital identity via the phalanx-forming region, a region
overlying the distal digital anlagen that regulates Sox9 expression and chondrogenesis.38
Applying a Bmp-laden bead or transplanting Bmp expressing interdigital tissue from the third
interdigital space to the second transforms the second digit into a third digit.35 The phalanx-
forming region also maintains digit-associated Fgf expression in the overlying AER for
continued digital outgrowth.38 However, it is still unclear how the various members of the Bmp
family (Bmp2/4/5/6/7 and Gdf5/6) and their receptors (BmpR1a, BmpR1b) that are expressed in
the digital and interdigital mesenchyme establish periodic thresholds that alternate between
chondrogenesis and apoptosis. With regression of the phalanx-forming region and loss of
overlying Fgf, the terminal phalanges form at the distal tip of each digit, invoking a unique
poorly characterized mechanism that includes membranous ossification in addition to anlagen
formation.39,40
Fig. 25.4 Hand plate development. Progressive stages of hand development. ID, interdigital
region. The phalanx-forming region is shown in pink capping the digital anlagen. Apical
ectodermal ridge, orange; black speckling of interdigital regions, apoptosis.

The development/differentiation of specific tissues

Limb bud formation and the progressive morphologic changes evident externally are
accompanied by differentiation of various tissues internally that are regulated by the coordinate
signaling centers described above. Differentiation of these tissues is coincident, but will be
discussed separately to add clarity and to relate timing of formation to tissue-specific
malformations.

Limb vasculature

As the limb bud grows, nutrients and oxygen are needed to maintain rapid cell proliferation and
the secretory activity of the signaling centers. Induction of a primitive vascular system in the
limb begins with transformation of mesoderm into angioblasts, cells that express the basic helix-
loop-helix transcription factor Tal-1 and the vascular endothelial growth factor (VEGF) receptor
Flk1.41 A dense meshwork of primitive vascular channels forms de novo from angioblasts in the
limb mesoderm.42,43 Angioblasts formed in nearby somites also migrate into the limb bud and
contribute to the formation of new limb vessels through continued vasculogenesis.44 As
angioblasts aggregate, differentiate into endothelium, and form primitive vascular tubes, new
vascular markers emerge. Flk1 persists to allow further remodeling by VEGF, Tal-1 diminishes
and VE-cadherin, a cell–cell adhesion molecule, is up-regulated.

The primitive vascular network undergoes significant remodeling as the limb develops. By stage
13, the vascular channels coalesce proximally to form a central artery (subclavian) that connects
to the dorsal aorta via the seventh intersegmental artery45 and two peripheral veins form that
drain into the posterior cardinal system.43 In addition to vasculogenesis, angiogenesis, i.e., vessel
sprouting from a preformed vessel, also contributes to the definitive architecture of the limb
vasculature.

Construction of the vascular pattern is under the direction of the coordinate signaling centers and
involves the regulation of specific VEGF family members and VEGFR3 receptors.45–47 The final
vascular pattern progresses from proximal to distal (Fig. 25.5) – with formation of the axillary
artery by stage 17 and the brachial and major forelimb branches by stage 19. The median artery
is prominent in the forearm.48 Distally the capillary plexus persists. Ulnar differentiation
precedes radial differentiation and is evident distally by the radial artery merging with an
extensive primitive capillary plexus radially, while the ulnar side of the axis is already forming
the palmar arch which communicates with the ulnar associated capillary plexus. 49 The median
and interosseous arteries decrease in size. The median artery degenerates, providing blood supply
to the median nerve only.48

Fig. 25.5 Vasculogenesis of the developing upper limb. Progressive vascular remodeling from
Carnegie stage 13 through 21, showing: subclavian artery (S), axillary (A), brachial (B), median
(M), radial (R), ulnar (U), interosseous (i), palmar arches (PA) and digital (D) arteries (red). Note
the primitive vascular plexuses persist distally on the radial aspect at stage 19. The veins have
also remodeled during this period, from a distal sinus and anterior (radial) and posterior (ulnar)
marginal veins, into the distinctive veins of the limb (illustrated in blue).

The vascular network must include arteries, capillaries, and veins. The diameter of each is
dictated by blood flow, blood pressure, shear stress, and the accumulation of vascular smooth
muscle around the vessel.42 Formation of vascular smooth muscle around limb arteries lags
behind vessel formation by about 2 days. It is unclear whether these muscle cells are derived
from the endothelial cells or are condensations of the surrounding mesoderm. Furthermore, the
mechanism underlying differential smooth-muscle accumulation in arteries and veins is
unknown. Arteries are also differentiated from veins by the expression of Ephrin B2, a
membrane-bound ligand, while the Eph-B4 receptor highlights veins. Presumptive capillaries
will be negative to both Ephrin B2 and the Eph-B4 receptor.

By stage 21 the major vessel architecture is complete. The majority of vascular anomalies arise
between stage 17 and 21, i.e., between days 41 and 52 or largely within the seventh and early
eighth week of development. Lymphatics follow a similar course, albeit less well delineated, and
are also composed of angioblasts that migrate into the limb from somitic mesoderm.44 A defining
molecular difference in lymphatic vessels is the coexpression of PROX-1 and LYVF-1.50

Skeletogenesis

Under the influence of the limb signaling centers, Sox9, a high-mobility group transcription
factor, is up-regulated in a targeted population of limb mesoderm.51 Sox9 transforms these cells
into chondrogenic precursors and induces condensation, the first step in limb skeletogenesis.52,53
Chondrogenesis occurs in a proximal to distal progression (Fig. 25.6). The expression of Sox5
and Sox6 is required for further differentiation of chondrogenic precursors into chondrocytes to
form cartilage anlagen.54

Fig. 25.6 Skeletogenesis of the developing upper limb. By Carnegie stage 17, there is partial
rotation of the humerus (Hu, blue) at the shoulder and the elbow joint is starting to form. Radius
(Rad, green); Ulna (Uln, green); Hand, magenta.

At precise locations within the forming skeletal anlagen, synovial joints form. Hoxa transcription
factors (Hoxa9-13) exhibit expression patterns along the proximodistal axis that correlate with
skeletal segmentation.54 In addition, several molecules are known to participate in the process of
joint formation, including Wnt14 and Gdf5. The first morphologic evidence of joint formation is
the compact cellular condensation called the interzone that expresses Wnt14.55 In addition, Gdf5
is induced in the proximal portion of the interzone, covering the distal end of the proximal
anlagen.56,57 The central region of the interzone begins to expand, accumulates hyaluronan, and
becomes hypocellular in a process termed cavitation.58,59 The two cellular regions of the
interzone begin to differentiate into the opposing articular cartilage surfaces. Patterning signals
and movement work in concert to shape the joint into its definitive morphology57 (Fig. 25.7).
Mesoderm surrounding the developing joint condenses to form the joint capsule.60

Fig. 25.7 Joint formation.

(Modified from Pacifici M, Koyama E, Iwamoto M. Mechanisms of synovial joint and articular
cartilage formation: recent advances, but many lingering mysteries. Birth Defects Res C Embryo
Today 2005;75:237–248.)

Endochondral ossification converts the cartilage anlagen into the skeletal framework of the
growing limb. This process is under precise regulation and involves Runx2, Twist1, Fgfs, Indian
hedgehog (Ihh), and vascular endothelial growth factors (Vegfs).53 Chondrocytes are induced to
proliferate, undergo hypertrophy, and then die, leaving an extracellular cartilage matrix. This
matrix is subsequently invaded by blood vessels, osteoclasts, and differentiating osteoblasts.
Osteoblast differentiation is also under the control of Runx2 and Osterix (Osx), a bone-specific
transcription factor.61 Ossification begins within the diaphysis of anlagen at the primary
ossification center during early fetal development. Subsequently, vascular invasion of the
proximal epiphysis occurs, followed by the distal epiphysis forming secondary ossification
centers later in development. Each metacarpal and digital phalanx has two ossification centers, a
primary center within the diaphysis and a single secondary center that develops postnatally.

Bony malformations are most likely to involve the disruption of Sox9 and the progressive
proximal to distal anlagen formation, i.e., radiohumeral synostosis would occur earlier in
development than polydactyly or syndactyly.

Myogenesis
Muscular development of the upper limb is a coordinated effort between segment-specific tendon
primordium, migrating myocytes, and migrating motor neurons.62,63 There are three phases to
myogenesis.64 Embryonic myogenesis establishes the primary myotubes and the basic muscle
layout. Later, a second wave of myogenesis occurs with secondary myofibers surrounding
primary myofibers and contributing to the bulk of the muscle mass present at birth. Finally,
satellite cells which take up residence in the basal lamina surrounding myofibers will contribute
to postnatal growth and muscle regeneration.65

During early limb bud formation, limb mesoderm condenses to form the proximal tendon
primordium (PTP), establishing a target and an initial scaffold for migration of myocytes. 62
Myocyte precursors of the limb arise from the dorsolateral aspect of associated somites (the
dermomyotome subdivision) and express the Pax3 transcription factor. Muscle precursors for the
limb and body wall express c-Met, a surface receptor that is modulated by scatter factor initially
emanating from the lateral plate mesoderm and then later from other sites, including the PZ.
Scatter factor acts as a chemoattractant to promote myocyte precursor migration. A population of
the myocyte precursors further differentiates into limb-specific precursors, demarcated by Lbx1
expression66 (Fig. 25.8).

Fig. 25.8 Early neuromuscular development in the upper limb. (A) Dorsovolar view of
presumptive myocyte migration into the limb from the lateral aspect of the dermomyotome (DM)
– molecular markers listed in gray box. (B) Motor and sensory projection of processes into the
developing limb. SK, sclerotome; SF, scatter factor; Do, dorsal; Vo, volar; Pr, proximal; Di,
distal; Cut, cutaneous; Proprio, proprioception.

During embryonic myogenesis, limb-specific myocytes migrate into the proximal limb bud,
initially as dorsal and ventral masses (Fig. 25.8). Continued migration, however, is not
haphazard; rather myocyte precursors are directed into muscle anlagen by the tendon
primordium, e.g., the ventral mass migrates into the biceps and brachialis under the direction of
the PTP (Fig. 25.9, Carnegie stage 15). With continued proliferation and differentiation, the
myocytes up-regulate MyoD and Myogenin, declaring their commitment as myocytes. These
myocytes will then coalesce to form fibers and begin to produce myosin filaments. Satellite cells,
important for later growth and muscle regeneration, take up residence just under the basal lamina
of the developing myofibers.65 Concurrently, the tendon primordia further define the shape of
specific muscles with discrete tendinous attachments. For example, expression of Lmx1b within
the dorsal tendons directs the unique pattern of extensor attachments67 and it is likely that each
upper limb muscle is shaped by a unique combination of patterning factors.68

Fig. 25.9 Muscular development in the upper limb. Progressive myocyte migration and muscle
formation between Carnegie stages 13 through 21 showing the ventral or volar surface. Note the
resting flexed position of the upper limb in the stage 21 embryo rotates the upper arm so that the
elbows shift from dorsal to caudal and the forearm rotates medially at the elbow. The palmaris
longus and flexor carpi radialis are not shown. PTP, proximal tendon primordium; ITP,
intermediate tendon primordium; DTP, distal tendon primordium; Bi, biceps; Tri, triceps; B,
brachialis; BR, brachioradialis; PL, flexor pollicus longus; FDP, flexor digitorum profundus;
FCU, flexor carpi ulnaris; FDS, flexor digitorum superficialis.

As in other aspects of upper limb development, there is progressive differentiation from proximal
to distal. Thus, as the muscles of the upper arm take shape, migrating myocytes invade the
forearm to associate with the PTP and a new mesodermal condensation forming at the
presumptive wrist, the intermediate tendon primordium. In the forearm, the superficial muscles
differentiate before the deep muscles. By Carnegie stage 17, the distal tendon primordia form
and associate with migrating myocytes destined to be muscles of the hand (Fig. 25.9). The
intrinsic muscles arise from five embryonic muscle layers, which differentiate and fuse in a
complex but logical manner.69,70 Following embryonic myogenesis, a second wave of myocyte
precursors migrate into the limb and coalesce around primary myofibers, forming secondary
myofibers and adding bulk to the muscle masses as the fetus grows. It is during this secondary or
fetal myogenesis that formation of motor endplates occurs and neuromuscular communication
begins, further differentiating the forming muscle with slow and fast fiber types.71,72
Innervation

Outgrowth of nerves into the limb bud lags behind muscle migration (Fig. 25.10) and involves
both motor and sensory neurons.73 Motor neurons are specified early during spinal cord
development by exposure to Shh, initially from the notochord and later from the floor plate of
the developing neural tube.74,75 Motor neurons begin to express a combination of transcription
factors (e.g., Hb9/Mnx1, Lhx3/4) that promote motor neuron migration into discrete columns
within the spinal cord and direct their axons to specific muscle groups.63 Within the limb fields
(specified by Hox6 in the forelimb and Hox10 in the hindlimb), a Hox accessory factor, FoxP1,
is expressed that assists in deciphering the subsequent Hox-specific code within the limb for
appropriate axon targeting.76 As the axons of the motor neurons enter the limb, those expressing
Lim1 (Lhx1) will project into the dorsal Lmx1b-expressing compartment of the limb to target
dorsal extensor muscle groups. The remaining axons express Isl1 and will enter the ventral
compartment to target flexor muscles of the limb.

Fig. 25.10 Nervous development in the upper limb. Progressive innervation of the upper limb
and formation of the brachial plexus. Rt, nerve roots; T, trunks; U, upper; M, middle; L, lower;
C, cords; Lat, lateral; Med, medial; Pst, posterior; A, axillary; Mc, musculocutaneous; R, radial;
Md, median; U, ulnar.

Sensory processes accompany the axons of motor neurons into the limb. The cell bodies of
sensory neurons reside within the dorsal root ganglion (DRG) which is derived from neural crest
cells. Specification of sensory neurons within the DRG is characterized by up-regulation of
neurogenin1 and 2 (Ngn1/2) and brain-specific homeobox/POU domain protein 3A (Brn3a).
Furthermore, cuntaneous sensory neurons also express a runt homeodomain transcription factor 1
(Runx1), while neurons involved in proprioception express Runx2.