19 Development of the Tetrapod Limb CONSIDER YOUR LIMB. It has fingers or toes at one end, a humerus or femur at the other. You won’t find anyone with fingers in the middle of their arm. Also consider the subtle but obvious differences between your hands and your feet. If your fingers were replaced by toes, you would certainly know it. Despite these differences, the bones of your feet are similar to the bones of your hand. It’s easy to see that they share a common pat- tern. And finally, consider that both your hands are remarkably similar in size, as are both your feet. These commonplace phenomena present fascinating questions to the develop- mental biologist. How is it that vertebrates have four limbs and not six or eight? How is it that the little finger develops at one edge of the limb and the thumb at the other? How does the forelimb grow differently than the hindlimb? How can limb size be so precisely regulated? Is there a conserved set of developmental mechanisms that can explain why our hands have five digits, a chick’s wing three digits, and the horse’s hoof one? Limb Anatomy As the name denotes, tetrapods are four-limbed vertebrates (amphibians, reptiles, birds, How many !ngers and mammals). The bones of any tetrapod limb—be it arm or leg, wing or flipper—consist am I holding up? The Punchline Designed for locomotion on land, tetrapod limbs have joints between the bones and a set of digits at their distal ends. The limb begins as a “bud” of tissue on the sides of the embryo when cells from the developing myotome and lateral plate mesoderm migrate to and proliferate within the presumptive limb !eld, the limb mesenchyme. This proliferative “progress zone” is covered by ectoderm, with a thickening at the distal tip called the apical ectodermal ridge. Fgf8 signaling from this ridge antagonizes "ank-derived retinoic acid, initiating a positive feedback loop with mesenchyme signaling (Fgf10 and Wnt) to promote limb bud outgrowth. Fgf8 speci!es the posterior mesenchyme into the “zone of polarizing activity,” which secretes Shh to set up the anterior-posterior (thumb-pinkie) axis of the limb. Wnt signaling determines the dorsal- ventral axis (knuckle-palm). Skeletogenesis is controlled by a “Turing-type” model of self-organization through morphogen interactions. In certain animals the early webbed tissue between the digits remains; in others, it dies via BMP-mediated apoptosis. Each limb signaling system in"uences the differential expression of Hox genes along each axis, modi!cation of which supports the evolution of !ns to !ngers. 19_Gilbert_DevBio.11e.indd 613 6/6/16 12:02 PM 614 Chapter 19 (A) (B) (C) Dorsal Stylopod Zeugopod Autopod Pharyngeal Posterior Anterior Humerus Ulna Radius Carpals Digits mesoderm Anterior 1 Ventral 2 Anterior 3 4 Distal lateral plate Proximal Dorsal 5 mesoderm Posterior Anterior Posterior (heart) AER Forelimb Ventral "eld 2 Posterior Anterior lateral plate mesoderm (somitic) 4 3 Progress zone Distal Proximal 3 AER ZPA FIGURE 19.1 Limb anatomy. (A) Illus- Posterior tration of a chick embryo just prior to limb growth showing three important mesodermal cell types as well as the of a proximal stylopod (humerus/femur) adjacent to the body wall, a zeugopod (radius- emergence of the limb field. (B) Axis ulna/tibia-fibula) in the middle region, and a distal autopod (carpals-fingers/tarsals- orientation and anatomy of the limb bud. 1 Apical ectodermal ridge (AER); zone of toes) (FIGURE 19.1). Fingers and toes can be referred to as phalanges or, more gener- polarizing activity (ZPA). (C) Skeletal pat- ally, digits. The positional information needed to construct a limb has to function in a tern of the human arm, chick wing, and three-dimensional2 coordinate system: horse forelimb. (According to convention, The first dimension is the proximal-distal axis (“close-far”; that is, shoulder-to- the digits of the chick wing are numbered finger or hip-to-toe). The bones of the limb are formed by endochondral ossi- 2, 3, and 4. The cartilage condensations fication. They are initially cartilaginous, but eventually most of the cartilage forming the digits appear similar to those forming digits 2, 3, and 4 of mice and is replaced by bone. Somehow the limb cells develop differently at early stages humans; however, new evidence sug- of limb morphogenesis (when they make the stylopod) than at later stages gests that the correct designation may be (when they make the autopod). 1, 2, and 3.) (A after Tanaka 2013; B after The second dimension is the anterior-posterior axis (thumb-to-pinkie). Our Logan 2003.) little fingers or toes mark the posterior end and our thumbs or big toes are at the anterior end. In humans, it is obvious that each hand develops as a mirror image of the other. One can imagine other arrangements—such as the thumb developing on the left side of both hands—but these patterns do not occur. Finally, limbs have a dorsal-ventral axis: our palms (ventral) are readily distin- guishable from our knuckles (dorsal). The Limb Bud The first visible sign of limb development is the formation of bilateral bulges called limb buds at the presumptive forelimb and hindlimb locations (FIGURE 19.2A). Fate map- ping studies on salamanders, pioneered by Ross Granville Harrison’s laboratory (see Harrison 1918, 1969), showed that the center of this disc of cells in the somatic region of the lateral plate mesoderm normally gives rise to the limb itself. Adjacent to it are the cells that will form the peribrachial (around the limb) flank tissue and the shoulder 1 These terms can be difficult to remember, but knowing their word origins can help. Stylo = like a pillar; zeugo = joining; auto = self; pod = foot. 2 Actually, it is a four-dimensional system, in which time is the fourth axis. Developmental biologists get used to seeing nature in four dimensions. Developmental Biology 11e Fig. 19.01 Dragon!y Media Group 05/10/16 19_Gilbert_DevBio.11e.indd 614 6/6/16 12:02 PM Development of the Tetrapod Limb 615 (A) (B) Epaxial Central Somites Pronephric myotome bud dermatome kidney Myotome Hypaxial Gills Spinal cord myotome bud Sclerotome Limb muscle Notochord precursors Pronephron Limb bud Limb skeletal Peribrachial Free Shoulder Endoderm precursors !ank tissue limb girdle Lateral plate mesoderm (C) (D) FIGURE 19.2 The limb bud. (A) Prospective forelimb field of the salamander Ambystoma maculatum. The central area contains cells destined to form the limb per se (the free limb). The cells surrounding the free limb girdle. However, if all these cells are extirpated from the embryo, a limb give rise to the peribrachial flank tissue and the shoulder will still form (albeit somewhat later) from an additional ring of cells that Gilbert girdle. The ring of cells outside these regions usually is Developmentalsurrounds Biology this 11e area, Sinauer but would Associates not normally form a limb. If this surround- not included in the limb, but can form a limb if the more DevBio11e_19.02ing ring of cells Dateis included 05-10-16 in the extirpated tissue, no limb will develop. central tissues are extirpated. (B) Emergence of the limb This larger region, representing all the cells in the area capable of forming bud. Proliferation of mesenchymal cells (arrows) from a limb on their own, is the limb field. the somatic region of the lateral plate mesoderm causes The cells that make up the limb bud are derived from the posterior lat- the limb bud in the amphibian embryo to bulge outward. These cells generate the skeletal elements of the limb. eral plate mesoderm, adjacent somites, and the bud’s overlying ectoderm. Contributions of myoblasts from the lateral myotome Lateral plate mesenchyme cells migrate within the limb fields to form the provide the limb’s musculature. (C) Entry of myoblasts limb skeletal precursor cells, while mesenchymal cells from the somites at (purple) into the limb bud. This computer stereogram the same level migrate in to establish the limb muscle precursor cells (FIG- was created from sections of an in situ hybridization to URE 19.2B,C). This accumulating heterogeneous population of mesenchy- the myf5 mRNA found in developing muscle cells. If you mal cells proliferates under the ectodermal tissue, creating the limb bud. can cross your eyes (or try focusing “past” the page, Even the early limb bud possesses its own organization such that looking through it to your toes), the three-dimensionality of the stereogram will become apparent. (D) Scanning the main direction of growth occurs along the proximal-to-distal axis electron micrograph of an early chick forelimb bud, with (somites-to-ectoderm), with lesser growth occurring along the dorsal-to- the apical ectodermal ridge in the foreground. (A after ventral and anterior-to-posterior axes (see Figure 19.1B). The limb bud is Stocum and Fallon 1982; C courtesy of J. Streicher and further regionalized into three functionally distinct domains: G. Müller; D courtesy of K. W. Tosney.) 19_Gilbert_DevBio.11e.indd 615 6/6/16 12:02 PM 616 Chapter 19 1. The highly proliferative mesenchyme that fuels limb bud growth is known as the FIGURE 19.3 Deletion of limb bone progress zone (PZ) mesenchyme (a.k.a. the undifferentiated zone). elements by the deletion of paralogous 2. The cells found within the most posterior region of the progress zone constitute Hox genes. (A) 5 Hox gene patterning the zone of polarizing activity (ZPA), since it patterns cell fates along the anterior- of the forelimb. Hox9 and Hox10 para- posterior axis. logues specify the humerus (stylopod). Hox10 paralogues are expressed to a 3.