Chapter 17 Limb Development 2024 PDF

2024 Chapter 17 Chapter 17 - Limb Development: Appendicular Musculoskeletal Development Key terms and concepts: Lateral plate mesoderm Apical ectodermal ridge (AER) Progress Zone (PZ) Zone of Pola...

2024 Chapter 17 Chapter 17 - Limb Development: Appendicular Musculoskeletal Development Key terms and concepts: Lateral plate mesoderm Apical ectodermal ridge (AER) Progress Zone (PZ) Zone of Polarizing Activity (ZPA) Limb bud Epithelial-mesenchymal interaction Amelia, Polymelia Polydactyly, Oligodactyly, Syndactyly Arthrogryposis Endochondral ossification Apoptosis (programmed cell death) Fibroblast growth factor (FGF) Sonic hedgehog (Shh) Enhancer region Apical ectodermal cap Blastema Critical Periods Learning objectives: By the end if this unit you should be able to: 1. List three structures important in patterning the limb bud and describe the role of each in limb morphogenesis. 2. Describe how “epithelial-mesenchymal” interactions regulate outgrowth of the limb. 3. Understand the role of Sonic Hedgehog signaling in patterning and formation of the digits 4. Understand one role apoptosis plays in limb development. 5. Describe one role movement plays in development of the skeletal system. 6. Explain the embryonic origins of each abnormality listed in “key terms and concepts”. 7. Give several examples of how limb regeneration in salamanders is similar to limb formation in development and how it is different. I. Limb Development A. The first sign of limb formation is a condensation of lateral plate somatic mesoderm beneath the surface ectoderm at four points along the sides of the embryo. The upper limb buds lie opposite the lower cervical and upper thoracic segments and develop first. The lower limb buds lie opposite the lower lumbar and upper sacral segments. 1. The bones, tendons, and other connective tissues of the limb develop from the lateral plate mesoderm. Tendons develop along with the joints, and only later join with migrating muscle cells to form a functional unit. 1 2024 Chapter 17 2. Three interacting tissues are responsible for the organization of the skeletal pattern of the developing limb. a. The apical ectodermal ridge (AER) - a thickening of the ectoderm along the distal margin of the bud. Normal limb development is dependent on the interaction between limb bud mesoderm and the AER. If a limb bud develops, but the AER does not, the limb will not form. The formation of the AER from the ectoderm overlying the region of the future limb-bud is induced by a growth factor of the FGF family (FGF-10) secreted by the mesodermal mesenchyme of the future limb bud. In turn, the AER maintains the underlying mesoderm by secreting FGF growth factors, particularly FGF-8. Thus, the limb bud mesenchyme (of mesodermal origin) and the AER (ectodermal origin) are inter-dependent. b. The progress zone (PZ) – the mesenchyme underlying the AER. Signals from the AER maintain the PZ cells in a proliferative state. Reciprocally, the limb mesenchyme maintains the AER; if limb mesenchyme is experimentally replaced with non-limb mesenchyme, the AER will regress. As the limb grows, limb skeletal structures form in a proximodistal sequence. c. The zone of polarizing activity (ZPA) – a specialized region of mesoderm of the caudal distal margin of the limb bud. The ZPA is mesodermal tissue that governs the cranial-caudal pattern of the limb, i.e. on which side the first digit will form. Sonic hedgehog (Shh) production by the ZPA is responsible for its patterning activity. 3. As the limb bud grows, differentiation starts in the proximal part (to form a humerus), while the cells at its tip remain undifferentiated. The formation of limb structures in appropriate proximal to distal succession depends on the presence of the AER, but also depends on patterning of the limb bud mesoderm. If the ridge is removed during development, the distal structures yet to be laid down (such as digits) are not formed. However, if early development limb mesoderm is placed under a late development AER, the mesoderm will form structures appropriate for its developmental age, not the age of the AER. This indicates that the mesoderm carries the instructions for which part of the proximal/distal axis of the limb to form, but that the AER is required for normal development to occur. The exact mechanism of proximal/distal pattern formation in the limb is currently controversial. 4. The presence of the AER simply maintains the growth and development of the PZ ("Keep doing what you're meant to do!"); it will not tell the mesenchyme what to become. Transplantation experiments have shown that mesenchyme of the PZ determines what type of limb will form; hind-limb PZ tissue will form hind-limb if transplanted beneath a fore-limb AER. The AER, however, is needed to keep the PZ in a proliferative state. 2 2024 Chapter 17 CONCEPT: Epithelial-mesenchymal interactions We see here the influence of limb bud epithelium and mesoderm on each other. In other areas of the body there are many examples of induction involving the interaction of epithelia (from any of the germ layers) and adjacent mesenchyme (usually from mesoderm). The mesenchyme often plays an instructive role, altering the expression of genes by the epithelium. 5. The dorsal/ventral (palmar or plantar) axis of the limb appears to be determined by its ectoderm. B. As the limb bud begins to form, blood vessels differentiate from mesoderm and form a dense network. Vascularization is necessary for successful differentiation and development of bone and cartilage. One possible explanation (there are others) for the lack of a single bone, such as the radius (radial hemimelia), is a lack of vascularization at the appropriate time. 1. Amelia - complete absence of a limb; can result from a defect in the AER, and/or in the underlying mesoderm. This is normal in snakes, for example; limb buds form initially, but the AER never forms and so the limb buds degenerate rapidly. 2. Polymelia - extra limbs - This is one of the defects recently reported to be widespread in frogs in the midwest. Other limb deformities such as Amelia are also reported in the same frogs. For a discussion of possible mechanisms, see Meteyer et al (2000) Teratology. 2000 Sep;62(3):151-71 C. While the major features of a bone (large processes) are genetically determined, the forces exerted by attached muscles and bearing of weight will cause other bony features. 3 2024 Chapter 17 D. Strange as it seems, the formation of tendons and the differentiation of muscles happens independently at first. The connective tissue of the limb bud, including tendons, creates compartments into which myogenic cells from the hypomere will migrate. These compartments control the organization of muscle cells into different muscle groups. Ventral branches of spinal nerves in the region of the limb buds follow the migrating myogenic cells, apparently attracted by chemicals produced by the muscle cells. Each limb muscle is derived from several segments and so each is innervated by portions of several spinal nerves. 1. Example: The biceps brachii is innervated by the musculocutaneous nerve, which has fibers from C 6, 7 and 8. Muscle cells in the biceps probably originated from myotomes C 6, 7 and 8 II. Development of the Digits A. Above, we discussed the mechanisms that control proximal-distal patterning. But the digits are not all the same. How does the limb “know” on which side to place the first digit? The zone of polarizing activity (ZPA), at the caudal base of each limb bud (near where the fifth digit will develop) controls this aspect of patterning through its expression of Sonic hedgehog (Shh). 1. Polydactyly - extra digits. Ectopic expression of Sonic Hedgehog (Shh) in the cranial aspect of the limb bud can cause postaxial polydactyly. 2. Oligodactyly - abnormal reduction in digit number. Inhibition of the Hedgehog signaling pathway is one possible cause of oligodactyly. In fact, the Shh knockout mouse develops a limb without discernable digits. B. The process of limb formation is similar in all domestic mammals. The most striking differences occur in the formation of the digits. The standard pattern to which we compare all animals is the five digit arrangement seen in the human, cat and dog. The distal portion of maturing limb buds in these animals exhibits five areas of concentrated mesenchyme called digital blastemas. Animals such as the horse and ox experience either loss or fusion of some of these blastema, resulting in an adult that has fewer than five digits. 1. The order of loss tends to be the first, fifth, second, then fourth. Therefore, the horse, which has only a single full digit, has retained the one comparable to the third. C. Even in a five-toed animal, how do individual digits develop from the clumsy looking, paddle- shaped limb bud? Obviously, some cells have got to go! Certain groups of cells undergo apoptosis helping to sculpt the final shape of the limb and digits. 4 2024 Chapter 17 CONCEPT: Programmed Cell Death (Apoptosis) Cell death, at very precise stages of development and in well-defined regions of the embryo, is a normal process which is necessary for development of the appropriate shape of an area. This is not a matter of cells becoming malnourished or ill, but of cells being genetically predetermined to die at a particular time or under certain conditions such as receiving a signal from surrounding cells. A great deal of research is currently being done to learn more about apoptosis, since its control may be important in preventing tumor growth 1. Differing degrees of programmed cell death are responsible for the variable extent to which digits are separated from each other in various animals. 2. syndactyly - abnormal webbing or fusion of the digits III. Development of Joints A. At first, long bones of the limbs appear as continuous condensations of mesenchyme at the center of the limb axis. As we’ve said, the first to appear will become the proximally- located humerus or femur. As this “rod” of mesenchyme extends, it starts to branch, forming what will ultimately become the radius and ulna, or tibia and fibula. The distal ends of these primordia then branch further, laying down the more distal structure that are characteristic of that species. B. Within this continuous branching mesenchyme, the positions of the future joints are evident as areas of higher cell density. Cells in these regions change their characteristics, and some undergo apoptosis, leaving a joint cavity and forming the tissues typical of a joint. Movement is necessary for the proper development of joints. C. Arthrogryposis - a limitation in the range of motion of one or more joints at birth. Usually this is not genetic, but can be caused by a wide range of teratogens and conditions. Arthrogryposis can be caused by any condition that prevents normal joint movement before birth: a. improper development of muscles, or the nerves that innervate them b. insufficient room in the uterus for normal prenatal movement, i.e., caused by abnormal uterine shape, decreased amniotic fluid c abnormal formation of tendons, bones, joints or synovial membranes V. An example of modified limb formation: limb regeneration in Salamanders 5 2024 Chapter 17 A. Salamanders can regenerate limbs following amputation at any level. B. Salamander limb regeneration differs from development in that regeneration is dependent on nerve supply to the limb. However, other elements of regeneration are reminiscent of development. C. The tip of the amputated limb develops a mass of proliferative mesenchymal cells termed the “blastema”. Cells of the blastema will regenerate the missing elements of the limb. D. The cellular source of the blastema cells has been controversial. Recent evidence suggests that the blastema cells are at least partially restricted; blastema cells derived from tissues of lateral plate mesoderm origin only give rise to new tissues that also arise from lateral plate mesoderm (eg dermis can arise only from dermal derived blastema, cartilage can arise from cartilage or dermis derived blastema), while muscle arises from muscle derived blastema. E. The blastema is covered by a simple epithelium. Together, the blastema and the epithelium resemble the embryonic limb bud. F. At first, the epithelial covering of the regenerating limb is termed the “wound epidermis”. The distal tip of the epidermis is called the “apical epidermal cap (AEC) and has unique morphologic and molecular features that are similar to those of the apical ectodermal ridge (AER) of the developing limb bud. G. Blastema cells re-express genes similar to those expressed by embryonic limb bud mesenchyme. The mechanism of this restoration of an embryonic pattern of gene expression is currently unknown. H. Nerve supply to the regenerating limb is important for several reasons, including the possibility that the nerves contribute to the patterning of the regenerating limb. Pattern formation in the regenerating limb is not well understood, and is likely to depend on numerous factors, including nerve supply and expression of many genes important in embryonic limb bud patterning. Sonic hedgehog is expressed in the regenerating limb in a similar pattern as in the embryonic limb bud. I. Similar to the embryonic apical ectodermal ridge (AER), the apical epidermal cap (AEC) of the regenerating limb stimulates growth of the blastema through FGF secretion. However, in the regenerating limb, nerves are required for this effect of the AEC. The nerves are believed to additionally stimulate blastema proliferation independently. 6