Embryology 1 Yen Hsun Chen and Aaron Daluiski

Contents Abstract Introduction ...... 4 Developmental biology has greatly contributed to the understanding of upper limb develop- Molecular Events of the Developing ment. Whereas early understanding of limb Upper Limb ...... 4 development centered on morphological First Phase: Early Development change during organogenesis, current empha- and Limb Identity ...... 7 sis is on discovery of molecular signaling Second Phase: Limb Patterning mechanisms that drive the remarkable transfor- and Initial Growth ...... 8 Proximodistal (PD) ...... 8 mation of single cells into fully functioning Anteroposterior (AP) ...... 10 limbs and the human body. These discoveries Dorsoventral (DV) ...... 13 have laid a foundation for fundamental Coordination Between Axes ...... 15 embryology-based concepts that have Third Phase: Tissue Differentiation ...... 19 reshaped the way congenital limb differences Vascular System ...... 19 are conceptualized, with the ability to trace a Nervous System ...... 19 phenotype back to single genes, and, con- Musculoskeletal System ...... 20 versely, the ability to predict developmental Extrinsic Factors ...... 20 differences from single-gene mutations. Not International Federation of Societies for Surgery only do these discoveries advance understand- of the Hand (IFSSH) Classification System ...... 20 ing of limb development, but clinical benefits Summary ...... 21 are also realized. Clinicians are provided with References ...... 21 the information they need to adequately inform patients and their families about the nature of limb differences, the hereditary implications, and the downstream developmental needs and challenges that the patient may face. Pediatric upper limb surgeons, standing at the interface between clinical care and genetic research, play a unique role in this field. Through recognition of novel human variants, pediatric upper limb surgeons act as gatekeepers by referring patients for appropriate work-up, facilitating Y.H. Chen (*) • A. Daluiski Hospital for Special Surgery, New York, NY, USA research that offers novel insights into human e-mail: [email protected]; [email protected] limb development. The goal of the discussion

# Springer Science+Business Media New York 2015 3 J.M. Abzug et al. (eds.), The Pediatric Upper Extremity, DOI 10.1007/978-1-4614-8515-5_1 4 Y.H. Chen and A. Daluiski

that follows is to provide the pediatric upper stage than the more distal structures due to earlier limb surgeon with the fundamentals of limb onset of formation. embryology that have implications both clini- A host of genes and molecular signaling work cally and academically. harmoniously together to ensure proper development of these structures. Much of these mechanisms and pathways remain to be worked out, but Introduction there is sufficient knowledge to be able to charac- terize defects in the context of basic embryologi- Congenital birth defects affect 3% of all live births cal events that define the complex, coordinated in the United States, with upper limb differences process of limb development. Whereas early occurring at a rate of approximately 1 per every developmental work focused on the morphology 3,000 live births (Parker et al. 2010). Develop- of these embryological events, the emphasis is ment of the upper limb can be described in terms now placed on understanding the molecular of the anatomic changes that occur during basis of these changes, enabling detailed under- embryonic growth or in terms of the molecular standing of genotype-phenotype correlations that cues that cause the developmental processes. The may have substantial clinical implications. basics of both are important for the upper limb In the discussion that follows, the gene name surgeon to understand to properly evaluate nomenclature will be followed in which human congenital limb anomalies that present clinically. genes are designated by having all letters in upper- It is particularly important to recognize genetic case (e.g., SHH for Sonic Hedgehog) and their defects as they may have wider clinical implica- animal counterparts have only the first letter in tions for the patient. Conversely, the treating uppercase (e.g., Shh). While the two counterparts upper limb surgeon may be the first to identify may be considered interchangeable, this distinc- new clinical presentations that may in turn tion serves as a reminder that not all molecular advance the understanding of embryonic limb mechanisms for limb development may be con- development. served between humans and the mouse, chick, or Normal limb development begins with the other model species. appearance of the upper limb bud as early as postconception day 24 and attains all the major structures of an adult by the end of week 8, the end Molecular Events of the Developing of the embryonic period (O’Rahilly and Gardner Upper Limb 1975). An overview of the milestones of human upper limb development is described in Table 1, Molecular signaling pathways control the growth with select milestones for development of the and tissue differentiation, leading to the gross lower limb for comparison. anatomic milestones of limb development. These The upper limb develops proximal to distal, molecular events can be divided into three phases. starting from the trunk, and begins initially as a The first phase is early limb development, which homogenous mass of undifferentiated mesenchy- includes initial establishment of limb identity and mal cells. During limb outgrowth, musculoskele- the initiation of limb bud outgrowth. The second tal elements generally precede development of phase is generally considered “classical” limb other elements such as nerves, vasculature, and development, characterized by basic patterning lymphatics. Three distinct segments are identifi- of the developing limb. During this phase, limb able in both the developing and mature limb: the patterning is commonly subdivided into three spa- stylopod (upper arm), zeugopod (forearm), and tial axes: proximodistal, anteroposterior autopod (hand plate) (Fig. 1). Development of (radioulnar), and dorsoventral. The third phase is the three segments occurs both sequentially and characterized by growth to increase limb size and concurrently. That is, the most proximal structures cellular differentiation to form discrete tissues that tend to be at a slightly more mature developmental make up the individual structures of the limb. 1 Embryology 5

Table 1 Key milestones in the development of the human upper limb (O’Rahilly and Gardner 1975). Select milestones for the lower limb are provided for comparison. AER apical ectodermal ridge Carnegie Week Day stage Upper limb Lower limb 4 24 11 Swelling appears in region of upper limb bud 28 13 Scattered blood vessels Appearance of lower limb bud 5 32 14 Upper limb AER Early marginal vessel Early brachial plexus development 33 15 Hand plate appears Lower limb AER Humerus mesenchymal condensations 6 37 16 Humerus chondrification Lumbosacral plexus Radius and ulna mesenchymal condensations Brachial plexus with radial, median, and ulnar nerves to the elbow Early muscle masses 41 17 Finger rays (webbed) Femur, tibia, fibula, and tarsus Radius, ulna, and metacarpal chondrification mesenchymal condensations 7 44 18 Interdigital apoptosis Femur, tibia, and fibula chondrification Scapula and humeral head chondrification Carpals and proximal phalanges chondrification Trapezius innervated (accessory nerve) Major muscles distinguishable 48 19 Middle phalanges chondrification Shoulder and elbow interzones (joint cavity formation) 8 51 20 Distal phalanges chondrification 52 21 Humerus ossification Radius ossification All muscles distinguishable Wrist and carpal interzones 54 22 Ulna ossification Femur and tibia ossification 9 57 23 Scapula ossification Fibula ossification Intramembranous ossification of distal tip of Tarsus and digits chondrification distal phalanges

These phases are continuous and overlapping. these events produces many of the congenital Early events initiate signaling processes that abnormalities seen clinically. trigger establishment of proper limb patterning. Several key tenets apply: Appropriately patterned groups of cells subsequently undergo expansion and differentiation to 1. Tissues and structures that develop concurrently form speciﬁc limb structures and tissues. While it may be driven by common molecular signaling is more practical to consider each of these pro- pathways, although not always. Defects in com- cesses as distinct steps, these processes overlap in mon pathways may explain constellations of time and space, are dynamic, and are often symptoms that are frequently seen together. interdependent through the cross talk of different 2. Many of the molecular mechanisms identiﬁed signaling pathways. Disruption in any number of are derived from experimental work performed 6 Y.H. Chen and A. Daluiski

Fig. 1 Anatomy of the developing limb. The development of proximal structures precedes the development of more distal structures due to earlier onset of formation (Zeller et al. 2009)

Table 2 Terminology for etiology-based description of congenital birth defects Terminology Deﬁnition Sequence A set of defects in which the steps involved in pathogenesis to produce a distinctive phenotype are known, e.g., Potter sequence Syndrome Recurring pattern or constellation of defects, commonly due to genetic defect Association Defects that occur together more commonly than would be expected by chance Disruption/ External forces damages a normal developmental process deformation Malformation Genetic or developmental abnormality Dysplasia Normal genetic programming, but aberrant tissue development

in nonhuman species, especially the chicken from nongenetic insults. This terminology is and the mouse. These findings may or may defined in Table 2. not be conserved in human limb development. 3. Many molecular pathways are critical for For the purposes of this discussion, an exhaus- development of other organ systems. Severe tive review of all the molecular events contributing defects in these pathways may not be encoun- to the developing limb is beyond the scope of this tered clinically due to the failure of develop- chapter or the need of the pediatric upper limb ment of major organs, resulting in a nonviable surgeon. Molecular events presented in this review fetus. Conversely, milder defects may be will be key signals that play a major role in limb encountered clinically, but necessitate screen- development and/or are clinically relevant. Two ing for dysfunction in these other organ things are important for the pediatric upper limb systems. surgeon: (1) the recognition of associated patterns 4. Not all congenital limb differences are directly of deformities and (2) the recognition of novel caused by changes in molecular signaling path- abnormalities or unique variants that are inherit- ways. Amniotic band syndrome, for example, able. The former will help with diagnosis of comor- results from mechanical insults to normally bid conditions that may help our patients, whereas developing tissue in utero. the latter will help our scientist colleagues who 5. Specific terminology is used to distinguish continue deciphering the molecular mechanisms clinical phenotypes due to genetic defects underlying the deformities that afflict our patients. 1 Embryology 7

clearly identifies the limb based on this pattern in First Phase: Early Development both mouse and chick embryos. Further and Limb Identity supporting the specificity of Tbx4, Tbx5, and Pitx1, Fgf-soaked bead-induced ectopic upper Limb buds initiate in the region of the developing limbs express Tbx5 whereas ectopic lower limbs somites and first appear in the region of the upper express Tbx4 and Pitx1. Limb buds with a mixture limb on approximately day 24. This is followed of upper and lower limb characteristics express all 4 days later with the appearance of the lower limb three. In the upper limb, retinoic acid from the buds on day 28. trunk plays a permissive role in limb bud initiation Upper and lower limb bud identities are by permitting induction of Tbx5 expression believed to be predetermined early on during (Cunningham et al. 2013). cranial-caudal patterning by a conserved, sequen- Functionally, Pitx1 is clearly a determinant of tial genetic program encoded by Hox genes (Cohn lower limb morphology whereas the exact contri- et al. 1997). Differential Hox gene expression bution of Tbx4 and Tbx5 to the development of establishes the upper limb- and lower limb- the corresponding limb morphology is less clear. forming regions of the corresponding lateral Loss of Pitx1 results in loss of lower limb charac- plate mesoderm and somites from which the teristics, which can be rescued by Tbx4 since limb buds initiate. Tbx4 is downstream of Pitx1 signaling (Ouimette These limb-forming regions dictate the mor- et al. 2010). Misexpression of Pitx1 in the upper phology of the limb created in this region. Ectopic limb causes a partial forelimb-to-hind limb trans- induction of the lateral plate mesoderm using formation in mice and humans (Liebenberg syn- Fgf-soaked beads in either region results in for- drome, Mendelian Inheritance in Man [MIM] mation of complete upper or lower limbs number 186550) (Spielmann et al. 2012), depending on where the limb was initiated reflecting the role of Pitx1 in directing develop- (Cohn et al. 1995). Determination of whether the ment of lower limb structures. Conversely, ectopic limb develops upper or lower limb structures was expression of Tbx4 in the upper limb does not correlated with proximity of bead placement to produce the same effect. In fact, Tbx4 the limb-forming regions. Beads placed close to misexpression in the upper limb bud can substi- the lower limb-forming region produced ectopic tute for Tbx5 in a conditional knockout mouse lower limbs, whereas beads placed close to the with Tbx5 deleted in the upper limb-forming upper limb-forming region induced formation of region to form an intact upper limb (Minguillon ectopic upper limbs (Cohn et al. 1995, 1997). et al. 2005). The Tbx5 conditional knockout Beads placed in between the two regions pro- mouse also indicated that Tbx5 (or misexpressed duced chimeric limbs with both upper and lower Tbx4) is needed for limb bud initiation, the limb characteristics (Ohuchi et al. 1998). absence of which results in absent upper limbs A triggering signal is required to initiate limb (Rallis et al. 2003). bud outgrowth from the programmed limb bud Mutations to all three genes are associated with forming regions of the embryo. The exact clinical syndromes in humans. Tbx4 mutations sequence of events leading to activation of growth cause small patella syndrome (MIM 147891), remains unclear, but at least three transcription characterized by patellar and hip defects. Tbx5 factors are critical for initiation of the correct mutations can cause Holt-Oram syndrome (MIM upper or lower limb bud development: Tbx4, 142900), characterized by upper limb and cardiac Tbx5, and Pitx1 (Agarwal et al. 2003; Duboc defects. Pitx1 mutations can cause clubfoot (MIM and Logan 2011a; Rallis et al. 2003). Tbx5 is 199800) or Liebenberg syndrome (MIM 186550). specifically expressed in the upper limb bud, A key milestone for limb bud initiation by whereas Pitx1 and Tbx4 (downstream of Pitx1) Tbx5 (upper limb) and Tbx4 (lower limb) is the are specifically expressed in the lower limb bud. induction of Fgf10 expression. Maintained Fgf10 The expression of these genes is quite specific and expression is required for successful completion 8 Y.H. Chen and A. Daluiski of limb bud initiation and subsequent limb development (Duboc and Logan 2011b). In the upper limb, Tbx5 is a direct activator of Fgf10 signaling (Agarwal et al. 2003). In the lower limb, some Fgf10 expression persists despite loss of Tbx4, likely due to other contributors. This overlap in signaling may explain the formed but significantly smaller lower limbs in mice with loss of Tbx4 (Naiche and Papaioannou 2003). In mice, complete loss of Fgf10 resulted in initiated limb buds but no limb outgrowth (Sekine et al. 1999). Once initiated, similar Fgf-dependent signaling mechanisms enable limb outgrowth for both upper and lower limbs.

Second Phase: Limb Patterning and Initial Growth Fig. 2 Whole-mount RNA in situ hybridization visualiza- tion the AER at the tip of a developing mouse limb bud After limb bud initiation, outgrowth of the limb (Soshnikova and Birchmeier 2006) bud begins. All limb buds consist of mesodermal tissue originating from lateral plate mesoderm limb bud outgrowth and elongation, directly (forms bone, cartilage, and tendons) and somitic inducing formation of upper limb structures in a mesoderm (forms muscles, nerves, and vascula- proximal (early) to distal (late) manner. These ture) and are covered by a layer of ectoderm. structures may be viewed as three distinct seg- Anatomically, the limb goes through a progressive ments achieved by patterning along the set of morphological changes starting with a thin proximodistal axis: the stylopod (upper arm), nubbin of tissue along the chest wall of the zeugopod (forearm), and autopod (wrist and embryo through the limb paddle stages. During hand). This PD patterning appears to occur very this process, limb development can be thought of early when the AER is established in the initiating as occurring in three spatially distinct axes. Each limb bud and is dependent on FGF signaling from of these limb growth axes contains a signaling the AER (Mariani et al. 2008; Sun et al. 2002). center, an area or group of cells, that is responsible The importance of the AER is demonstrated by for establishing the corresponding axis. The three limb differences along the PD axis if AER func- axes are proximodistal (PD), anteroposterior tion is impaired. Disruption results in limb trun- (AP) (radioulnar or pre-/postaxial), and dorsoven- cations at a level corresponding with the stage of tral (DV). development when the disruption occurred (Summerbell 1974). The later the removal or disruption of the apical ectodermal ridge in develop- Proximodistal (PD) ment, the more distal the resulting truncation. Structures proximal to the level of truncation At the distal tip of the developing limb buds, at the remain intact. These truncations can be rescued interface between dorsal and ventral ectoderm, a by grafting an AER from a different chick ridge of thickened ectoderm forms in response to embryo, defining the AER as both necessary and signals from the underlying mesoderm. This sufficient to promote outgrowth. thickening develops into the apical ectodermal The molecular cue from the AER was isolated ridge (AER), the signaling center for the PD axis and identified to be one of several growth factors (Fig. 2). Signaling from the AER is critical for from the fibroblast growth factor (Fgf) family. 1 Embryology 9

In the developing limb, members of the Fgf family humerus. Cells that exit late end up in distal loca- exhibit varying degrees of functional redundancy. tions as the limb elongates and develop into distal Of the Fgfs, Fgf10 alone is both necessary and structures such as the forearm and hand. The time- sufficient to produce an intact limb (Duboc and dependent mechanism of the progress zone model Logan 2011b) and is normally expressed by the provided a mechanistic explanation for the time- mesenchyme underlying the AER. The AER itself dependent transverse deficit phenotype produced specifically expresses Fgf4, Fgf8, Fgf9, and Fgf17 by removal of the AER. The later the AER exci- (the AER-FGFs). Mice deficient in Fgf4, Fgf9, or sion, the more distal the defects due to loss of Fgf17 retain normal limb development, likely res- progenitors with longer residence in the cued by the functional redundancy of the Fgfs. In progress zone. contrast, Fgf8 is critical as loss of Fgf8 caused Subsequent experiments, however, demon- impaired limb outgrowth and significantly smaller strated that the progress zone model is inaccurate. limbs (Mariani et al. 2008). Lineage tracing in X-irradiation experiments to Fgf10 from the sub-AER mesenchyme and induce phocomelia in chick embryos demon- Fgf8 from the AER are intrinsically related in a strated that proximodistal patterning was unaf- positive feedback loop that forms the core signal- fected despite radiation-induced defects in ing required for growth along the PD axis (Ohuchi proximal structures (Galloway et al. 2009). Simi- et al. 1997). Early expression of Tbx5 during larly, fate mapping in chick limb buds demon- upper limb bud initiation first induces expression strated that proximodistal cell fates were of Fgf10 from the sub-AER mesenchyme established early, with limb truncations occurring (Agarwal et al. 2003). Fgf10 signals to the over- due to apoptosis of these fated cells rather than lying AER to express Wnt3a, which in turn drives defects in PD patterning (Dudley et al. 2002). Fgf8 expression. Fgf8 from the AER then signals Modern attempts to understand PD patterning to the sub-AER mesenchyme to maintain Fgf10 emphasizes the conceptualization of limb pattern- expression from the underlying mesenchyme, ing in the context of dynamic interactions between resulting in an Fgf8-Fgf10 positive feedback molecular events (Tabin and Wolpert 2007). More loop located at the distal end of the developing recently, a “two-signal” model was proposed to limb. This Fgf/Wnt signaling loop is critical for explain proximodistal determination of limb limb development – loss of Wnt3 (tetra-amelia, structures. Specifically, the two opposing signals MIM 273395) (Niemann et al. 2004) or Fgf10 were retinoic acid (RA) for induction of proximal (Sekine et al. 1999) results in amelia, or absence structures, with the FGFs from the AER determin- of limb formation. ing formation of distal structures (Cooper The importance of the distal end of the limb et al. 2011; Roselló-Díez et al. 2011). Supporting bud in limb outgrowth was initially conceptual- this model, ectopic introduction of RA to the ized in a “progress zone” model to explain distal limb bud resulted in proximalization of the proximodistal development. The progress zone distal limb (Mercader et al. 2000). Additionally, model posits that there is a zone of undiffer- the AER-FGFs were demonstrated to establish entiated mesenchymal cells within the sub-AER distal structures by repression of Meis1/2, homeo- mesenchyme with an intrinsic timing mechanism. box genes that establish the proximal limb The cells in this zone are maintained by Wnt3a (Mariani et al. 2008). and Fgf8 from the AER, which maintains a pool The role of retinoic acid appears to be permis- of progenitor cells by stimulating proliferation sive rather than actively establishing PD pattern- and inhibiting differentiation (ten Berge ing (Cunningham et al. 2013). Fgf8 expression by et al. 2008). A timing mechanism would provide the developing heart suppresses limb bud initia- progenitor cells with positional cues based on how tion due to inhibition of Tbx5 expression. long the cells reside in the progress zone. Mesen- Retinoic acid from the trunk functions to block chymal stem cells that exit relatively early cardiac Fgf8, enabling limb bud expression of develop into proximal structures such as the Tbx5 and Meis1/2 and limb bud initiation. For 10 Y.H. Chen and A. Daluiski

Fig. 3 In situ hybridization for SHH, visualizing the SHH-expressing ZPA located in the posterior margin of both the upper and lower limb buds in a mouse embryo (e10.5) (Daluiski et al. 2001)

proximodistal patterning itself, RA is unneces- the radial (or preaxial) from the ulnar sary. Interestingly, Tbx5 itself is needed for (or postaxial) side. This axis was initially discov- cardiomyocyte differentiation (Holt-Oram syn- ered when sections of posterior (ulnar) limb bud drome, MIM 142900), the mutation of which is tissue were excised at varying stages of chicken characterized by upper limb and cardiac defects. limb development resulting in limbs that devel- PD patterning remains incompletely under- oped longitudinally but that did not develop stood and is an evolving ﬁeld. It is also not radioulnar-based digit identities. Excision of known the extent to which these mechanisms are radial tissue did not produce the same effect. It conserved in human limb development. was discovered that a small region of posterior Despite our lack of understanding, the clinical cells, at the junction of the limb paddle and the implications have not changed. Defects along the trunk, was a signaling center for AP development proximodistal axis, such as limb truncations and termed the zone of polarizing activity (ZPA). This longitudinal defects, remain intrinsically related zone of tissue polarized the limb along the AP to the AER and FGF function. Insult to the AER axis. When ZPA tissue was grafted onto the radial during development will lead to clinically side, a mirror image of the limb along the AP observed truncations at variable stages depending plane was produced (Tickle 1981). on the timing of the insult. Frequently, isolated The key signaling molecule of the ZPA is limb cases suspected to be secondary to insults to Sonic Hedgehog (SHH). This ligand was named the AER will predominantly be mechanical and for its molecular resemblance to the drosophila nonheritable due to the extensive role FGFs play molecule Hedgehog, which is important for ﬂy in other biological systems, resulting in severe segmentation, thus leading to its similar name systemic defects that may render limb defects despite different phyla. SHH is a diffusible sig- lower in priority. naling molecule with expression restricted to the posteriorly located ZPA (Fig. 3) and forms a posterior (high) to anterior (low) gradient of SHH Anteroposterior (AP) signaling. Functionally, SHH is critical for regulating AP patterning and growth of the zeugopod Arguably, the most studied axis of limb pattern- (forearm) and autopod (hand). ing, and perhaps the most clinically relevant, is the Restriction of SHH expression to the posterior- AP axis. The anteroposterior axis distinguishes located ZPA is accomplished by Gli3, a 1 Embryology 11

Fig. 4 SHH and GLI3A:GLI3R ratio in the hand plate (Anderson et al. 2012) transcription factor that pre-patterns the early limb Defects in digit development are closely corre- bud along the AP axis before SHH expression is lated with SHH function because SHH signaling activated (te Welscher et al. 2002). Gli3 exists in specifies the number of digits as well as the iden- two forms: GLI3A (activator) and GLI3R (repres- tity of each digit. To achieve this, SHH is sor). By default, Gli3 is modified to form the Gli3 expressed in a gradient that varies both spatially repressor form. On the posterior (ZPA) side, SHH and temporally (Harfe et al. 2004). Spatially, the inhibits this modification to allow for production concentration gradient exposes the mesoderm to of the GLI3A activator form, producing a gradient varying concentrations of SHH, with the concen- of high GLI3A:GLI3R ratio posteriorly to low trations greatest on the posterior (ulnar) side and GLI3A:GLI3R anteriorly. Manipulation in chick none on the anterior (radial) side. The mesenchy- embryos to produce high GLI3A:GLI3R ratios mal progenitors of the fifth digit are exposed to the throughout results in polydactyly with posterior greatest SHH concentration whereas the thumb digit identities of all extraneous digits (Litingtung develops in the absence of SHH signaling. Tem- et al. 2002), reflecting the role of high SHH signal- porally, the length of exposure to SHH determines ing in specifying posterior structures. Of note, the digit identity. Shorter-term exposure of the mes- stylopod (upper arm) is patterned independently of enchyme is sufficient to specify anterior digits SHH, presumably accomplished by Gli3-mediated (second digit), whereas the fifth digit requires the pre-patterning (Niswander 2002). The elbow repre- longest SHH exposure for correct specification sents the transition from SHH-independent to (Scherz et al. 2007). In fact, the posterior three SHH-dependent limb development, which may digits contain SHH-expressing cells from the ZPA have clinical implications in such clinical pheno- while the SHH contribution to the second digit types as below-elbow truncations. derives from paracrine signaling (Harfe The interactions between SHH and Gli3 result et al. 2004). Furthermore, SHH acts as a mitogen in a gradient of SHH signaling and GLI3A:GLI3R to produce the necessary progenitor pool for ratio along the AP axis that directs specification forming five complete digits (Malik 2014). and development of autopod ulnar-sided to radial- Digit identity is established by the SHH gradi- sided structures (Fig. 4). In humans, defects in the ent from the ZPA, but digits initially develop as function of either SHH or Gli3 may cause limb “webbed” fingers. Extraneous tissue between fin- defects along the AP axis, frequently manifesting gers must undergo apoptosis to form digits with clinically with digit abnormalities such as poly- interdigital separations. Bone morphogenetic pro- dactyly and syndactyly (Anderson et al. 2012). teins (BMPs), widely known for their role in Mutations of GLI3, for example, result in various chondrogenesis and osteogenesis, mediate apo- preaxial and postaxial polydactylies in Greig ptosis of the interdigital mesenchyme to produce cephalopolysyndactyly syndrome (MIM 175700) separated fingers. The interdigital mesenchyme (Hui and Joyner 1993). expresses BMP2, BMP4, and BMP7 which 12 Y.H. Chen and A. Daluiski antagonizes the pro-survival effects of the Fgfs preaxial polydactyly (MIM 174500), isolated from the overlying AER (Pajni-Underwood triphalangeal thumb (MIM 174500), syndromic et al. 2007; Suzuki 2013). In animals with webbed triphalangeal thumb (MIM 174500), syndactyly limbs such as bats, BMP antagonists block BMP (MIM 186200), and acheiropody (bilateral con- signaling to produce persistent interdigital tissue genital amputations, MIM 200500). (Oberg et al. 2010). Enhanced function or signal- Triphalangeal thumbs and preaxial polydactyly ing of Fgfs can also prevent interdigital apoptosis arise secondary to point mutations that result in by overcoming BMP-mediated inhibition, as ectopic SHH expression, producing an ectopic occurs in the syndactyly observed in Apert syn- ZPA at the anterior margin of the limb bud (Lettice drome (MIM 101200). et al. 2008). This ectopic SHH may respecify The factors governing formation of the digits anterior digits into posterior digits (triphalangeal are less well understood. Digit formation is cur- thumb or thumb-to-finger transformation) or rently thought to occur from the combination of induce the formation of a mirror hand (duplication the initial AP patterning established by SHH, of posterior digits in the anterior margin) (Fig. 5). BMP signaling from the interdigital mesenchyme, Syndactyly and polysyndactyly may occur and phalangeal growth mediated by the phalanx- from overexpression of SHH, particularly in the forming region (PFR) found in the sub-AER mes- interdigital mesenchyme, and are associated with enchyme. Cells from the sub-AER mesenchyme mutations that increase the dosage of SHH such as are continuously incorporated into each of the ZRS duplications (Klopocki et al. 2008) or the PFRs and subsequently into the growing digit. adoption of a more widely expressed enhancer Cells incorporated earlier form the proximal by SHH (Anderson et al. 2012). Acheiropodia, digit, undergoing condensation to form the prox- characterized by congenital upper and lower imal phalanges; cells incorporated later form the limb amputations and aplasia of the hands distal phalanges. BMP signaling from the and feet, is associated with mutations to interdigital mesenchyme promotes this process the LMBR1 gene not involving the ZRS via its stimulatory effects on the PFR, in contrast sequence, raising the possibility of additional reg- to the inhibitory effect BMPs have on the ulatory sites in addition to the ZRS (Ianakiev AER-FGFs that produces interdigital apoptosis et al. 2001). (Suzuki et al. 2008). GLI3 regulates SHH signaling via the GLI3A: SHH is critical in various development pro- GLI3R ratio along the AP axis. Mutations to GLI3 cesses, most notably is the development of the are associated with Greig cephalopolysyndactyly notochord. Mutations to the SHH gene proper, syndrome (GCPS, MIM 175700), Pallister-Hall such as large deletions that produce a defective syndrome (PHS, MIM 146510), preaxial polydac- protein, occur but are unlikely to be encountered, tyly (MIM 174200), and postaxial polydactyly due to either developmental lethality or serious (MIM 174700). GCPS and PHS yield interesting neurologic defects that take precedence. More genotype-phenotype correlations. Splitting the relevant, however, are mutations to regulators of GLI3 gene into thirds, GCPS is frequently asso- SHH signaling. Several of these regulators have ciated with mutations to the first and last third of clinical importance and include ZRS mutations, GLI3 whereas PHS is frequently associated with GLI3 mutations, and ciliopathies. mutations to the middle third. Correspondingly, The ZRS (ZPA regulatory sequence) is an GCPS exhibits pre- or postaxial polydactyly enhancer sequence that is necessary and sufficient (or crossed polydactyly) whereas PHS exhibits for regulating the spatiotemporal SHH activity in central polydactyly (Biesecker 2011). PHS muta- the developing limb (Anderson et al. 2012). This tions are frequently gain-of-function mutations sequence is located at a distance (1 Mb upstream) that generate constitutively active GLI3R. This from SHH, in an intron in the LMBR1 gene. GLI3R disrupts the normally high GLI3A: Mutations to this region produces a range of phe- GLI3R ratio in the posterior limb, resulting in notypes involving the upper limb including anteriorization. These disruptions to GLI3A: 1 Embryology 13

Fig. 5 Development of a mirror hand due to a ZRS mutation causing formation of a second, ectopic ZPA on the anterior margin of the hand. A mirror hand develops due to duplication of the posterior digits (Zeller et al. 2009)

GLI3R ratios correlate to the pre- or postaxial syndrome (MIM 146510). In accordance with polydactyly observed in GLI3 mutations. the involvement of SHH and GLI3, upper limb Ciliopathies involve mutations to cilia, which differences seen in these syndromes frequently actively mediate signaling of the Hedgehog fam- include polydactyly. ily of proteins including SHH (Hildebrandt et al. 2011). GLI3 has also been shown to be localized to the tips of cilia. Consequently, in Dorsoventral (DV) addition to disrupting SHH signaling, cilia defects may also disrupt GLI3 function or GLI3 Dorsoventral patterning is less well understood processing into GLI3A and/or GLI3R. Such and deformities in this plane are infrequently defects result in the pre- and postaxial polydactyly encountered. Similar experiments to those done seen in the preceding discussion on SHH regula- for the PD and AP axes initially established that tory and GLI3 mutations. Examples of syndromes the dorsal ectoderm provides the signal for DV associated with ciliopathies that affect the limbs development. However, unlike AP development, include Bardet-Biedl syndrome (MIM 209900), the entire dorsal ectoderm provides the source of Joubert syndrome (MIM 213300), Meckel syn- signaling rather than a single section or zone. drome (MIM 249000), and Pallister-Hall When the dorsal ectoderm was excised, the limb 14 Y.H. Chen and A. Daluiski

Fig. 6 Dorsal dimelia, with a palmar nail on the fifth digit. Dorsalized skin is apparent on the palm (Al-Qattan 2013) lost dorsal structures (nail plates, extensor ten- ventrally (Loomis et al. 1996). This dorsal dupli- dons) and assumed a more ventral appearance cation can also be reproduced by ectopic (with palmar-like flexion creases, sweat glands, overexpression of Wnt7a on the ventral surface. and lack of hair follicles) (Loomis et al. 1996). Defects in either Wnt7a or En-1 signaling are While it is unclear what initially establishes DV implicated in clinical presentations of ventral polarity, it does appear that this mechanism may (Wnt7a-deficient) or dorsal (En-1-deficient) be distinct from that of the AP and PD axis. Fgf10 duplication phenotypes of varying severity. In null mice are characterized by initiated limb buds humans, dorsalization may manifest as a circum- without any subsequent limb growth. In these ferential or palmar nail, frequently afflicting the limb buds, neither the ZPA nor the AER formed, fifth digit (Fig. 6) (Al-Qattan 2013; Rider 1992). but expression of molecules that establish DV Conversely, defects in Wnt7a or its mediator patterning remained normal (i.e., PD and AP Lmx1b result in defects in dorsal structures, fre- axes disrupted, but DV axis intact) (Sekine quently presenting with fingernail hypoplasia et al. 1999). (Fig. 7). Greater degrees of palmar duplication, The causative agent for establishing DV polar- for instance, involving the entire hand, may also ity is the secreted factor Wnt7a. Wnt7a from the occur in humans (Al-Qattan 2013). Some patients dorsal ectoderm induces expression of Lmbx1b, a do not have identifiable abnormalities in the cod- LIM homeobox transcription factor necessary and ing regions of the Wnt7a gene, reflecting our sufficient for the development of dorsal limb incomplete understanding of the molecular mech- structures (Riddle et al. 1995). Excision of the anisms of DV patterning. dorsal ectoderm results in loss of dorsalization Clinically, DV defects are very rarely seen in associated with deficiency of Wnt7a, which can isolation. Mutations to Wnt7a that causes be rescued by application of a Wnt7a-soaked bead Fuhrmann syndrome (MIM 228930) or (Yang and Niswander 1995). Conversely, the ven- Al-Awadi-Raas-Rothschild/Schinzel phocomelia tral ectoderm expresses Engrailed-1 (En-1). (AARS) syndrome (MIM 276820) have a broad Engrailed-1 inhibits Wnt7a, restricting expression range of defects that include deficits along the AP of Wnt7a to the dorsum, and allows for the devel- and PD axis (Woods et al. 2006). Defects isolated opment of ventral limb structures. In mice, loss of to a single developmental axis should be consid- Engrailed-1 results in dorsalization of the ventral ered the exception rather than the rule as the three surface due to uninhibited expression of Wnt7a axes develop in combination to form the upper 1 Embryology 15

Fig. 7 Example of defects in dorsalization. (a) Nail dysplasia in nail-patella syndrome (LMX1B mutation). (b) Palmar duplication with hypoplastic nails. Note the palmar creases localized on the dorsum of the hand (Reproduced with permission of A Daluiski)

limb. Selected clinical phenotypes are provided in SHH-GREM1-FGF ectodermal-mesenchymal Table 3, demonstrating the frequent involvement feedback signaling loop (Zeller et al. 2009) that of multiple axes as well as other organ systems. has been proposed to explain each of the phases of limb development (initiation, propagation, and termination) (Bénazet et al. 2009). The default Coordination Between Axes loop involves SHH inducing expression of GREM1 from the sub-AER mesenchyme. Mesen- While each of the three axes (AP, PD, and DV) chymal GREM1 antagonizes BMP signaling to was discussed separately in the preceding section, disinhibit expression of ectodermal AER-FGFs these axes are highly coordinated through com- (recall that BMP signaling inhibits the plex interrelated pathways. Integration of the AER-FGFs). AER-FGFs, in turn, signal back to molecular events provides a more complete pic- the ZPA to maintain SHH expression. ture of events of limb development, despite the At the time of limb bud initiation, high BMP signiﬁcant gaps in knowledge that still remain. signaling initially induces formation of the AER. This integrated approach will assist in the recog- Feedback upregulation of GREM1 quickly fol- nition of associated developmental defects that lows, blocking BMP signaling to permit may provide clues as to the nature of the underly- FGF-mediated limb bud outgrowth during the ing genetic defect, as it places distinctive pheno- propagation phase (Ahn et al. 2001; Bénazet types in the context of underlying developmental et al. 2009). As limb outgrowth nears completion, biology. An overview of the interdependency of the SHH-GREM1-FGF feedback loop is capable the major players of each spatial axis is shown in of self-termination to restrict limb size Fig. 8. (Verheyden and Sun 2008). Termination occurs once the sub-AER extends sufﬁciently far away Anteroposterior and Proximodistal from the SHH/ZPA in the growing hand plate such Arguably, the AP and PD axes are the most that SHH is no longer able to maintain GREM1 intricately related. From the early establishment expression from the sub-AER mesenchyme. Loss of the two axes during limb bud initiation, the of GREM1 inhibition allows BMP signaling to AER and ZPA mutually induce the other in a redirect undifferentiated cells from proliferation positive feedback loop to sustain normal limb to tissue differentiation (digits) or apoptosis development; loss of one results in loss of the (interdigital mesenchyme). other (Niswander et al. 1994). Molecularly, this The interdependence between the AP and PD positive feedback loop has been referred to as the axes through the SHH-GREM1-FGF feedback 16

Table 3 Selected genes involved in limb identity or patterning along the three axes (anteroposterior, proximodistal, and dorsoventral). Limb findings as well as findings in other organ systems are provided to illustrate the frequently pleiotropic manifestations of mutations to single genes critical for upper limb development Limb ﬁndings Gene Phenotypes Inheritance Gene function (bold ¼ characteristic) Other ﬁndings Limb TBX4 Small patella syndrome AD Lower limb Pelvic and lower limb defects – identity (147891) development Aplasia/hypoplasia of patellae PITX1 Clubfoot (119800) Multifactorial Lower limb Clubfoot – development Various lower limb malformations Liebenberg syndrome AD with Lower limb Upper limb malformations – (186550) variable development (dysplastic elbow joints, penetrance carpal fusion, radial deviation) Homeotic arm-to-leg transformation TBX5 Holt-Oram syndrome AD Upper limb Thumb anomaly (various) Cardiac defects (142900) development Atrial septal defect

Cardiomyocyte Various upper limb anomalies Daluiski A. and Chen Y.H. differentiation (aplasia/hypoplasia) PD axis FGF10 LADD Tissue growth Clinodactyly (5th digit) Puncta aplasia/hypoplasia, lacrimal duct (FGFR3, (lacrimoauriculodentodigital) and Thumb anomalies obstruction FGFR2) syndrome (149730) differentiation Mild syndactyly Hearing deficits Wnt3 Tetra-amelia (273395) AR Tissue growth Amelia Pulmonary hypoplasia and Urogenital defects differentiation Craniofacial defects Embryology 1 AP axis GLI3 Greig cephalopolysyndactyly AD Digit Pre-/postaxial polydactyly Cranial (frontal bossing, scaphocephaly, (GLI3 inactivating mutation) development Variable syndactyly hypertelorism) (175700) Craniosynostosis Pallister-Hall syndrome AD Digit Central polydactyly Pituitary dysfunction (GLI3 truncation) development Postaxial polydactyly Visceral malformations Syndactyly/polysyndactyly Hypothalamic hamartomas Brachydactyly Postaxial polydactyly, types AD Digit Postaxial polydactyly (often – A1 and B (GLI3 point development functional) mutation) (174200) Preaxial polydactyly, type IV AD Digit Preaxial polydactyly +/À – (GLI3 point mutation) development postaxial polydactyly (174700) DV axis WNT7A Fuhrmann syndrome AR Dorsalization Posterior (ulnar) aplasia/ Primary (major axes), secondary (heart or (228930) hypoplasia limb primordial), and local (tibial-fibular Digit abnormalities differentiation) developmental fields Pelvic abnormalities Femur bowing Absence of ulna and fibula AR Dorsalization Absent ulnae and fibulae (276820) Femoral hypoplasia Pelvic hypoplasia Abnormal genitalia LMX1B Nail-patella syndrome AD Dorsalization Nail dysplasia Distal neuropathy (161200) Patellar aplasia/hypoplasia Nephropathy, renal disorders Iliac horns 17 18 Y.H. Chen and A. Daluiski

Fig. 8 A simpliﬁed view of signaling between the major signaling molecules of the three (AP, PD, and DV) spatial axes (Duboc and Logan 2009)

loop accounts for clinical phenotypes that involve AER-FGF signaling due to impaired SHH both axes simultaneously. expression. In humans, disruption of Wnt7a results in dor- Dorsoventral and Anteroposterior soventral patterning defects (MIM 276820), as Excision experiments indicated that the dorsal well as impaired ulnarization of tissue resembling non-AER ectoderm (expressing Wnt7a) plays a SHH deficiencies (MIM 228930). role in maintaining SHH expression from the ZPA. Removal of dorsal non-AER ectoderm Dorsoventral and Proximodistal resulted in deficient expression of SHH from the Disruption of either Wnt7a or En1 results in DV ZPA. Conversely, removal of the ventral defects, but without any impairment to PD non-AER ectoderm had only minor effects on growth, suggesting that Wnt7a or En1 does not SHH expression (Yang and Niswander 1995). regulate AER function. However, simultaneous Furthermore, loss of the dorsal ectoderm induced DVand PD defects can exist due to their common ulnar defects (AP axis) as well as defects in limb dependence on BMP signaling for initial induc- outgrowth (PD axis). tion of the patterning signals. In both mice and The differential effects produced by excision chicks, BMP signaling is necessary and sufficient of either the dorsal or ventral ectoderm can be for establishing the DVaxis and induction of AER explained by the dorsal ectoderm’s expression of formation (Ahn et al. 2001). Wnt7a. Confirmatory experiments in mice dem- Due to the close relationship between the AP onstrated that Wnt7a, in addition to its role in the and PD axes, defects of DV patterning genes such DV axis, also induced and maintained expression as Wnt7a cannot only result in defects in the AP of SHH. Loss of Wnt7a in mice resulted in dorsal- axis, but secondarily affect the PD axis. In accor- to-ventral transformation, as well as loss of pos- dance with its role in dorsalization, relatively mild terior digits that require SHH for formation (Parr Wnt7a mutations produce dorsal defects such as and McMahon 1995). The observed defects in fingernail hypoplasia. Mutations resulting in mod- limb outgrowth are likely the result of defective erate loss of Wnt7a function result in impaired 1 Embryology 19

SHH expression, leading to ulnar ray deficiencies elongates, the marginal sinus is lost whereas the due to deficient ulnarizing SHH signals. Severe vascular plexus maintains continued angiogenesis loss of Wnt7a function produces severe deficiency to supply blastemas of muscle progenitors that of SHH, resulting in phenotypes such as limb develop along the periphery of the forming limb. truncations that resemble defects occurring from The core of the limb receives comparatively fewer AER dysfunction or acheiropodia (LMBR1 gene penetrating vessels, allowing for a lower oxygen defect that affects SHH regulation). The severe tension region that facilitates cartilage develop- loss of SHH likely results in loss of ment and subsequent formation of the skeletal SHH-GREM1-FGF signaling, resulting in limb system via endochondral ossification. truncations due to failure of AER-mediated Initial arterial supply of the rudimentary capil- proximodistal growth. lary system is provided by the subclavian artery originating from the right dorsal aorta. Formation of the brachial artery occurs early as the limb bud Third Phase: Tissue Differentiation elongates. The brachial artery then branches into the median and interosseous arteries. Formation In concert with the establishment of upper limb of the ulnar artery then follows with the radial patterning, progenitor pools must appropriately artery forming last. migrate, expand (for growth), and differentiate (for the development of specific structures). The lateral plate mesoderm, somitic mesoderm, and Nervous System neural crest provide all the necessary progenitors for the formation of a complete limb. From the Two sets of neural cells grow out from the devel- initial proliferating mass of undifferentiated mes- oping spinal cord toward the limb buds to form the enchyme that characterizes the early limb bud, brachial plexus: ventral rami (motor) and dorsal limb structures develop at a rapid pace with rami (sensory). The two sets of proliferating neu- remarkable coordination and organization. Pro- rons coalesce during week 4 to form the brachial gression of development is a fluid process with plexus. From then on, nerves grow into the devel- many events occurring simultaneously. Rather oping limb innervating structures proximal to dis- than presenting individual events chronologically, tal. Motor neurons have cell bodies in the spinal it is useful to consider limb development by cord, yet send out a single axon that extends over system. great distances to innervate a single target. This is accomplished by the enlarged tip at the ends of the growing axon known as the growth cone. The Vascular System growth cone contains numerous highly motile filopodia that interact extensively with local cues One of the earliest systems to appear in the early within the developing limb that progressively limb bud is the vascular system. Progenitors from guide the axon to its final target. Inability of the the somitic mesoderm migrate into the limb bud axon to reach its target muscle results in neuronal and undergo angiogenesis to form a rudimentary cell death. For each individual muscle, multiple capillary system. The AER is instrumental in axons compete to establish innervation with apo- guiding the longitudinal growth of the vascular ptosis of the neurons unable to establish a connec- system as the limb bud elongates and matures. tion. The number of neurons that establishes a Initially, a vascular plexus forms in the connection and survives correlates with the size subectodermal mesenchyme of the early limb of the muscle, with one neuron eventually becom- bud, which coalesces along the peripheral border ing the dominant one. to form a marginal sinus. Formation of the venous The remarkable process of selective axonal system follows the capillary network, with lym- targeting is not completely understood. Experi- phatics differentiating last. As the limb bud mental studies suggest that migrating neural cells 20 Y.H. Chen and A. Daluiski provide the guiding cues. However, it appears that The major constituent on this category is once an axon reaches the brachial plexus, the known as the amniotic band syndrome (ABS) axons are able to find their target muscle regard- (MIM 217100), also known as ADAM (amniotic less of duplication, rotation, or amputation of the deformity, adhesion, and mutilation) sequence limb (Beatty 2000). (Cignini et al. 2012). ABS produces a variety of limb differences attributed to in utero mechanical compression or amputation of normally develop- Musculoskeletal System ing tissue. Theories for pathogenesis include rup- ture of the amniotic sac resulting in either the In the developing limb bud, the muscular blas- formation of fibrous amniotic bands that act as tema and the chondrogenic blastema form the tourniquets or the extrusion of fetal parts through muscles and bones, respectively. The muscular the amniotic sac defect with subsequent constric- blastema is located in the periphery, where oxygen tion. These compressive forces may result in con- tension is higher due to the vascular plexus. The striction bands with or without distal hypoplasia chondrogenic blastema is located centrally, where secondary to impaired vascular supply, classically the oxygen tension is comparatively lower. Mus- seen in the digits. More severe strangulation can cle development occurs sequentially, with proxi- result in outright amputation. mal muscles separating from the muscular Ischemia due to either spontaneous or induced blastema and differentiating before distal muscles vascular insufficiency may also produce similar and superficial muscles differentiating before phenotypes due to necrotic loss of developing deep muscles. The skeletal system in the upper tissue. limb forms through endochondral ossification. The chondrogenic blastema forms cartilage in the central region of the limb bud in a proximal International Federation of Societies to distal manner. The cartilage later undergoes for Surgery of the Hand (IFSSH) ossification to form bones. Classification System Joints form in regions called the “interzone” at the junction between the ends of two blastemas. A The IFSSH classification currently in use was joint capsule forms early on at the interzone. Sub- adopted in 1976 (Swanson 1976). Originally pro- sequently, cavitation occurs within the center of posed by Alfred B. Swanson in 1964 as a modifi- the interzone to produce a joint space with pro- cation of Frantz and O’Rahilly’s proposed system duction of joint fluid. At either end of the (Frantz and O’Rahilly 1961), the classification interzone, articular cartilage forms to cap the two system was intended to be a practical, efficient ends of bone. The formation of a functioning joint method to facilitate identification and diagnosis requires joint motion. In the absence of motion, of upper limb differences. Conception of the sys- the joint space becomes infiltrated by fibrous tis- tem was made with comparatively limited under- sue, resulting in an immobile joint. standing of limb development, resulting in a classification system that was primarily based on morphology (Table 4). Extrinsic Factors Over the years, several modifications have been proposed to change the IFSSH classification Limb differences secondary to extrinsic factors system to better reflect the updated view of human are deformations with a normal genetic develop- upper limb development. While new classification mental program. These differences are not related systems for upper limb differences will undoubt- to dysfunction in molecular signaling or tissue edly emerge, the Swanson IFSSH classification differentiation. Had embryo development been system remains the most universally accepted allowed to proceed unhindered, no differences system as of the writing of this text. Furthermore, would have otherwise been observed. it is a logical, easy-to-use clinical tool that 1 Embryology 21

Table 4 The IFSSH classification for characterizing of the apical ectodermal ridge and dorsal-ventral pat- human congenital differences of the upper limb terning of the limb. Development. 2001;128 (22):4449–61. I. Failure of formation Al-Qattan MM. Classification of dorsal and ventral dimelia II. Failure of differentiation in humans. J Hand Surg Eur Vol. 2013;38(9):928–33. III. Duplication Anderson E, Peluso S, Lettice LA, Hill RE. Human limb IV. Overgrowth (gigantism) abnormalities caused by disruption of hedgehog signal- – V. Undergrowth (hypoplasia) ing. Trends Genet. 2012;28(8):364 73. Beatty E. Tissue differentiation of the upper extremity. In: VI. Congenital constriction band syndrome Gupta A, Kay SPJ, Scheker LR, editors. Grow hand VII. Generalized skeletal abnormalities diagnosis manag up extrem child. 1st ed. London: Mosby; 2000. p. 33–8. Bénazet J-D, Bischofberger M, Tiecke E, Gonçalves A, provides clinicians with a framework with which Martin JF, Zuniga A, et al. A self-regulatory system to begin evaluating patients with upper limb of interlinked signaling feedback loops controls mouse limb patterning. Science. 2009;323 differences. (5917):1050–3. More recently, moves toward overhauling the Biesecker LG. Polydactyly: how many disorders and how IFSSH classification in favor of a more many genes? 2010 update. Dev Dyn. 2011;240 – embryology-based classification system have (5):931 42. Cignini P, Giorlandino C, Padula F, Dugo N, Cafà EV, been made. In particular, Tonkin and colleagues Spata A. Epidemiology and risk factors of amniotic have proposed a system that integrates our current band syndrome, or ADAM sequence. J Prenat Med. understanding of how the upper limb develops in 2012;6(4):59–63. humans to aid in the diagnosis, communication, Cohn MJ, Izpisúa-Belmonte JC, Abud H, Heath JK, Tickle C. Fibroblast growth factors induce additional limb and understanding of congenital limb differences development from the flank of chick embryos. Cell. (Tonkin et al. 2013). Underscoring this move 1995;80(5):739–46. toward defining limb developmental differences Cohn MJ, Patel K, Krumlauf R, Wilkinson DG, Clarke JD, fi on a molecular level, the ability of pediatric upper Tickle C. Hox9 genes and vertebrate limb speci cation. Nature. 1997;387(6628):97–101. limb surgeons to recognize, define, and character- Cooper KL, Hu JK-H, ten Berge D, Fernandez-Teran M, ize limb difference phenotypes becomes all the Ros MA, Tabin CJ. Initiation of proximal-distal pat- more important. terning in the vertebrate limb by signals and growth. Science. 2011;332(6033):1083–6. Cunningham TJ, Zhao X, Sandell LL, Evans SM, Trainor PA, Duester G. Antagonism between retinoic acid and Summary fibroblast growth factor signaling during limb development. Cell Rep. 2013;3(5):1503–11. Many of the “key” molecular players in this phase Daluiski A, Yi SE, Lyons KM. The molecular control of upper extremity development: implications for congen- may not have great clinical significance as we have ital hand anomalies. J Hand Surg Am. 2001;26 not yet identified human mutations in these genes (1):8–22. that produce limb phenotypes presenting for clini- Duboc V, Logan MP. Building limb morphology through cal treatment. Thus, though these may be of vital integration of signalling modules. Curr Opin Genet Dev. 2009;19(5):497–503. importance to the developing limb, they may be of Duboc V, Logan MPO. Pitx1 is necessary for normal little importance to the treatment of our patients. initiation of hindlimb outgrowth through regulation of Tbx4 expression and shapes hindlimb morphologies via targeted growth control. Development. 2011a;138 (24):5301–9. References Duboc V, Logan MPO. Regulation of limb bud initiation and limb-type morphology. Dev Dyn. 2011b;240 Agarwal P, Wylie JN, Galceran J, Arkhitko O, Li C, (5):1017–27. Deng C, et al. Tbx5 is essential for forelimb bud initi- Dudley AT, Ros MA, Tabin CJ. A re-examination of ation following patterning of the limb field in the mouse proximodistal patterning during vertebrate limb devel- embryo. Development. 2003;130(3):623–33. opment. Nature. 2002;418(6897):539–44. Ahn K, Mishina Y, Hanks MC, Behringer RR, Crenshaw Frantz C, O’Rahilly R. Congenital skeletal limb deficien- EB. BMPR-IA signaling is required for the formation cies. J Bone Joint Surg. 1961;43(8):1202–24. 22 Y.H. Chen and A. Daluiski

Galloway JL, Delgado I, Ros MA, Tabin CJ. A Niswander L, Jeffrey S, Martin GR, Tickle C. A positive reevaluation of X-irradiation-induced phocomelia and feedback loop coordinates growth and patterning in the proximodistal limb patterning. Nature. 2009;460 vertebrate limb. Nature. 1994;371(6498):609–12. (7253):400–4. O’Rahilly R, Gardner E. The timing and sequence of Harfe BD, Scherz PJ, Nissim S, Tian H, McMahon AP, events in the development of the limbs in the human Tabin CJ. Evidence for an expansion-based temporal embryo. Anat Embryol (Berl). 1975;148(1):1–23. Shh gradient in specifying vertebrate digit identities. Oberg KC, Feenstra JM, Manske PR, Tonkin Cell. 2004;118(4):517–28. MA. Developmental biology and classification of con- Hildebrandt F, Benzing T, Katsanis N. Ciliopathies. N Engl genital anomalies of the hand and upper extremity. J J Med. 2011;364(16):1533–43. Hand Surg Am. 2010;35(12):2066–76. Hui CC, Joyner AL. A mouse model of greig cephalopo- Ohuchi H, Nakagawa T, Yamamoto A, Araga A, Ohata T, lysyndactyly syndrome: the extra-toesJ mutation con- Ishimaru Y, et al. The mesenchymal factor, FGF10, tains an intragenic deletion of the Gli3 gene. Nat Genet. initiates and maintains the outgrowth of the chick limb 1993;3(3):241–6. bud through interaction with FGF8, an apical ectodermal Ianakiev P, van Baren MJ, Daly MJ, Toledo SP, Cavalcanti factor. Development. 1997;124(11):2235–44. MG, Neto JC, et al. Acheiropodia is caused by a geno- Ohuchi H, Takeuchi J, Yoshioka H, Ishimaru Y, Ogura K, mic deletion in C7orf2, the human orthologue of the Takahashi N, et al. Correlation of wing-leg identity in Lmbr1 gene. Am J Hum Genet. 2001;68(1):38–45. ectopic FGF-induced chimeric limbs with the differen- Klopocki E, Ott C-E, Benatar N, Ullmann R, Mundlos S, tial expression of chick Tbx5 and Tbx4. Development. Lehmann K. A microduplication of the long range SHH 1998;125(1):51–60. limb regulator (ZRS) is associated with triphalangeal Ouimette J-F, Jolin ML, L’honoré A, Gifuni A, Drouin thumb-polysyndactyly syndrome. J Med Genet. J. Divergent transcriptional activities determine limb 2008;45(6):370–5. identity. Nat Commun. 2010;1:35. Lettice LA, Hill AE, Devenney PS, Hill RE. Point muta- Pajni-Underwood S, Wilson CP, Elder C, Mishina Y, tions in a distant sonic hedgehog cis-regulator generate Lewandoski M. BMP signals control limb bud a variable regulatory output responsible for preaxial interdigital programmed cell death by regulating FGF polydactyly. Hum Mol Genet. 2008;17(7):978–85. signaling. Development. 2007;134(12):2359–68. Litingtung Y, Dahn RD, Li Y, Fallon JF, Chiang C. Shh and Parker SE, Mai CT, Canfield MA, Rickard R, Wang Y, Gli3 are dispensable for limb skeleton formation but Meyer RE, et al. Updated National Birth Prevalence regulate digit number and identity. Nature. 2002;418 estimates for selected birth defects in the United States, (6901):979–83. 2004–2006. Birth Defects Res A Clin Mol Teratol. Loomis CA, Harris E, Michaud J, Wurst W, Hanks M, 2010;88(12):1008–16. Joyner AL. The mouse Engrailed-1 gene and ventral Parr BA, McMahon AP. Dorsalizing signal Wnt-7a limb patterning. Nature. 1996;382(6589):360–3. required for normal polarity of D-V and A-P axes of Malik S. Polydactyly: phenotypes, genetics and classifica- mouse limb. Nature. 1995;374(6520):350–3. tion. Clin Genet. 2014;85(3):203–12 Rallis C, Bruneau BG, Del Buono J, Seidman CE, Seidman Mariani FV, Ahn CP, Martin GR. Genetic evidence that JG, Nissim S, et al. Tbx5 is required for forelimb bud FGFs have an instructive role in limb proximal-distal formation and continued outgrowth. Development. patterning. Nature. 2008;453(7193):401–5. 2003;130(12):2741–51. Mercader N, Leonardo E, Piedra ME, Martínez AC, Ros Riddle RD, Ensini M, Nelson C, Tsuchida T, Jessell TM, MA, Torres M. Opposing RA and FGF signals control Tabin C. Induction of the LIM homeobox gene Lmx1 proximodistal vertebrate limb development through by WNT7a establishes dorsoventral pattern in the ver- regulation of Meis genes. Development. 2000;127 tebrate limb. Cell. 1995;83(4):631–40. (18):3961–70. Rider MA. Congenital palmar nail syndrome. J Hand Surg Minguillon C, Del Buono J, Logan MP. Tbx5 and Tbx4 are Br. 1992;17(3):371–2. not sufficient to determine limb-specific morphologies Roselló-Díez A, Ros MA, Torres M. Diffusible signals, not but have common roles in initiating limb outgrowth. autonomous mechanisms, determine the main Dev Cell. 2005;8(1):75–84. proximodistal limb subdivision. Science. 2011;332 Naiche LA, Papaioannou VE. Loss of Tbx4 blocks (6033):1086–8. hindlimb development and affects vascularization and Scherz PJ, McGlinn E, Nissim S, Tabin CJ. Extended fusion of the allantois. Development. 2003;130 exposure to Sonic hedgehog is required for patterning (12):2681–93. the posterior digits of the vertebrate limb. Dev Biol. Niemann S, Zhao C, Pascu F, Stahl U, Aulepp U, 2007;308(2):343–54. Niswander L, et al. Homozygous WNT3 mutation Sekine K, Ohuchi H, Fujiwara M, Yamasaki M, causes tetra-amelia in a large consanguineous family. Yoshizawa T, Sato T, et al. Fgf10 is essential for limb Am J Hum Genet. 2004;74(3):558–63. and lung formation. Nat Genet. 1999;21(1):138–41. Niswander L. Interplay between the molecular signals that Soshnikova N, Birchmeier W (2006) Limb development. control vertebrate limb development. Int J Dev Biol. In: Encyclopedic reference of genomics and proteo- 2002;46(7):877–81. mics in molecular medicine. Springer. 1 Embryology 23

Spielmann M, Brancati F, Krawitz PM, Robinson PN, specification during limb development. Development. Ibrahim DM, Franke M, et al. Homeotic arm-to-leg 2008;135(19):3247–57. transformation associated with genomic Tickle C. The number of polarizing region cells required to rearrangements at the PITX1 locus. Am J Hum Genet. specify additional digits in the developing chick wing. 2012;91(4):629–35. Nature. 1981;289(5795):295–8. Summerbell D. A quantitative analysis of the effect of Tonkin MA, Tolerton SK, Quick TJ, Harvey I, Lawson RD, excision of the AER from the chick limb-bud. J Smith NC, et al. Classification of congenital anomalies Embryol Exp Morphol. 1974;32(3):651–60. of the hand and upper limb: development and assess- Sun X, Mariani FV, Martin GR. Functions of FGF signal- ment of a new system. J Hand Surg Am. 2013;38 ling from the apical ectodermal ridge in limb develop- (9):1845–53. ment. Nature. 2002;418(6897):501–8. Verheyden JM, Sun X. An Fgf/Gremlin inhibitory feed- Suzuki T. How is digit identity determined during limb back loop triggers termination of limb bud outgrowth. development? Dev Growth Differ. 2013;55(1):130–8. Nature. 2008;454(7204):638–41. Suzuki T, Hasso SM, Fallon JF. Unique SMAD1/5/8 activ- Woods CG, Stricker S, Seemann P, Stern R, Cox J, ity at the phalanx-forming region determines digit iden- Sherridan E, et al. Mutations in WNT7A cause a tity. Proc Natl Acad Sci U S A. 2008;105(11):4185–90. range of limb malformations, including Fuhrmann syn- Swanson AB. A classification for congenital limb drome and Al-Awadi/Raas-Rothschild/Schinzel malformations. J Hand Surg Am. 1976;1(1):8–22. phocomelia syndrome. Am J Hum Genet. 2006;79 Tabin C, Wolpert L. Rethinking the proximodistal axis of (2):402–8. the vertebrate limb in the molecular era. Genes Dev. Yang Y, Niswander L. Interaction between the signaling 2007;21(12):1433–42. molecules WNT7a and SHH during vertebrate limb Te Welscher P, Fernandez-Teran M, Ros MA, Zeller development: dorsal signals regulate anteroposterior R. Mutual genetic antagonism involving GLI3 and patterning. Cell. 1995;80(6):939–47. dHAND prepatterns the vertebrate limb bud mesenchyme Zeller R, López-Ríos J, Zuniga A. Vertebrate limb prior to SHH signaling. Genes Dev. 2002;16(4):421–6. bud development: moving towards integrative analysis Ten Berge D, Brugmann SA, Helms JA, Nusse R. Wnt and of organogenesis. Nat Rev Genet. 2009;10(12): FGF signals interact to coordinate growth with cell fate 845–58.