1. Ernest F. Talarico, Jr., Ph.D. Associate Director of Medical Education Associate Professor of Anatomy & Cell Biology Associate Faculty, Radiologic Sciences Indiana University School of Medicine – Northwest Gary, IN Embryology of the Extremities AY14-15 1

2. General Information Development begins with the activation of mesodermal cells in the lateral plate mesoderm – lateral plate mesoderm migrates into the limb bud – condenses along a central axis to form vasculature and skeletal components. Somatic mesoderm (somites) – also migrates into the limb bud – condenses to form muscular components Homeobox gene Hox 3.3 – Hox genes are a group of related genes that determine the basic structure and orientation of an organism – Hox genes are critical for the proper placement of segment structures of animals during early embryonic development (e.g. limbs, ribs, wings, etc.) – regulate limb patterning Apical Ectodermal Ridge (AER) – Formed from a thickening of ectoderm at the distal end of the limb bud (mesodermal signal) – Promotes outgrowth of the limb bud Promotes mitosis Prevents terminal differentiation of mesodermal cells Zone of Proliferative Activity (ZPA) – Zone of mesodermal cells at the proximal base of the limb bud – Directs organization of the limb bud and patterning of the digits – Hox 4 and retinoic acid Digit Formation – Involves selective apoptosis within the AER – 5 separate regions of AER remain (future digits) UPPER EXTREMITY d26-27LOWER EXTREMITY d27-28 2

3. General Information d28 Site of midbrain 1st, 2nd, 3rd, and 4th pharyngeal (branchial) arches Left atrial prominence of heart Upper limb bud Somites Actual size 4.5 mm Mesonephric prominence Site of lens placode Umbilical cord Tail Lower limb bud Site of nasal placode Left ventricular prominence of heart 3

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5. The Organization and Polarity of the Developing Limb Bud The limb bud has a strict pattern and polarity. Development is organized around the A-P, D-V and P-D axes. The tissues of the limb will differentiate in a specific pattern that is defined in part by the existing embryonic regions: the Apical Ectodermal Ridge (AER) = s a transient embryonic structure essential for the induction, patterning and outgrowth of the vertebrate limb. the Zone of Polarizing Activity (ZPA) = is an area of mesenchyme that contains signals which instruct the developing limb bud to form along the anterior/posterior axis. and the Progress Zone (PZ) = is a layer of mesodermal cells immediately beneath the apical ectodermal ridge in the developing limb bud. The fate of the mesodermal cells is thought to be patterned by the length of time spent in the progress zone during limb outgrowth 5

6. The AER acts as the organizing region for the proximodistal axis of the limb. The ZPA organizes the limb along the A-P axis. It does this in part through the expression of Sonic hedgehog resulting in the production of the soluble sonic hedgehog protein. Sonic hedgehog mediates many developmental events. In the limb it not only meditates A-P Axis formation but also the maintenance of the AER. AER 6

7. Signal Transduction & Limb Formation Progress-Zone Model During limb development the limb bud grows away from the body in a proximo- (close) distal (away from) fashion. Developmentally, it is important to realize that as the limb bud lengthens and limb components are specified and start to differentiate, what were once distal regions become proximal as new distal regions form. Example: The humerus as it forms is initially the most distal component but once the radius and ulna and subsequent components form, it becomes proximal to them. This continues until the limb is fully developed and the final relationship of limb components is defined. 7

8. Signal Transduction & Limb Formation Progress-Zone Model The AER secretes FGF that influences the closest cells (those in the progress zone) to develop into distal structures. FGF is a distalizing factor in limb development. Those cells that are not within range of the AERs influence remain proximal in nature. As the AER extends out due to the continued division of cells in the progress zone, it continues to affect the closest cells by causing them to be specified as distal structure cells. Those that fall out of the range of influence of the AER are no longer influenced by the effects of FGF and retain their previously defined status (i.e., are now proximal components not influenced by the distalizing effect of FGF). FGF and T-box genes (tbx-4 and tbx-5) are essential for this process. The T-box gene family encodes DNA-binding transcriptional regulators: tbx-4 (hindlimbs); tbx-5 (forelimbs). 8

9. Signal Transduction & Limb Formation Progress-Zone Model Fibroblast Growth Factor (FGF): a family of factors FGF is linked to the initiation of bud formation, maintaining bud outgrowth, and the induction of a regeneration FGFs are secreted primarily by AER Tyrosine kinase FGF receptor is expressed on the surface mesenchyme cells Events of Signal Transduction & Limb Formation FGF Released by AER binds to FGF Receptor (a receptor tyosine kinase or RTK) and activates it RTK then phosphorylates critical proteins This causes – the mesenchyme cells to release retinoic acid (RA) – activation of sonic hedgehog (Shh) genes RA induces Hox Gene Expression in target cells Shh secretions (i.e., morphogens) may control the patterning of the limb along the anterior-posterior axis 9

10. Signal Transduction & Limb Formation Progress-Zone Model Sonic Hedgehog (Shh) SHH is the ZPA signal – SHH is a secreted cholesterol linked-protein – a Morphogen SHH receptor is PATCHED (Ptc) – a transmembrane lipoprotein SHH signaling pathway is responsible for some types of cancers Other Hedgehog related genes – Indian Hedgehog (proliferation & differentiation of chondrocytes; bone formation) – Summer Hedgehog (?) 10

11. Signal Transduction & Limb Formation Progress-Zone Model Other involved Factors: Wnt7 from the dorsal epidermis of the limb bud – Helps to specify the dorsal- ventral axis – Along with Shh, helps to maintain AER via induction Engrailed-1 (EN-1) from the ventral aspect of the limb bud – En-1 Knockout Circumferential nails Ectopic ventral digits Absence of ventral tendon Dorsal flexion Absent Sesamoid bone 11

12. CLINICAL CORRELATION Understanding Thalidomide and Limb Development Thalidomide leads to an inhibition of the growth of the limb bud mesenchyme. The end product is a limb with distal structures (hand/fingers) attached directly to the shoulders (i.e., the long bones are lost) resembling the flippers of a seal. By the progress-zone model one interpretation is that the lack of growth of the limb mesenchyme means that all of those cells are continually influenced by FGF released by the AER (Tabin, 1996. Nature 396: 322-323). Thus these cells never fall out of the sphere of influence of the AER to become proximal cells. Instead they only receive the distalizing signals ultimately resulting in their differentiation only into distal components. Thalidomide was introduced as a sedative drug in the late 1950s that was typically used to cure morning sickness. There are a WIDE range of limb shortening (phocomelia) syndromes reflected in thalidomide syndromes but this model begins to elucidate the possible underlying mechanisms and may lead to a better understanding of normal and defective limb development. A proposed mechanism of thalidomide teratogenesis at the molecular level has been recently reviewed (Stephens et al, 2000. Biochemical Pharmacology 59: 1489-1499). 12

13. Limb Bud (lateral plate mesoderm) 13

14. Digits By the end of week 6, mesenchymal tissue in hand plates condenses to form digital rays (week 7 in lower limbs -> footplates) Notches between digital rays are formed of loose mesenchyme Separate digits are formed by the end of the 8 th week via apoptosis and may be mediated by bone morphogenic proteins. Week 5 35 days Hand plate formed Week 6 44 days Digital rays appear in hand plate Digital ray Notch between digital rays 14

15. UPPER EXTREMITY LOWER EXTREMITY Lateral plate mesoderm (LPM) – scapula, clavicle, humerus, ulna and radius; carpals, metacarpals and phalanges Endochondral ossification – all bones of the upper extremity – clavicle = intramembranous and endochondral ossification Sequence of Events – Wk5 LPM condenses within the limb bud chondrification centers appear – Wk6 condensed mesoderm chondrifies forming a hyaline cartilage model – Wk7 primary ossification centers are seen in the clavicle (1 st ), humerus, radius and ulna present in all bones by Wk12 rotation takes place as the limb extends ventrally – lateral 90 ° rotation on the longitudinal axis – olecranon process becomes dorsal; extensor muscles posterior and lateral – Wk9 to birth primary ossification centers appear in the scapula, metacarpals and phalanges – Childhood secondary ossification centers form in epiphyses ossification of carpal bones Skeletal Elements Lateral plate mesoderm (LPM) – Ilium, ischium and pubis; femur, tibia, fibula, tarsals, metatarsals and phalanges (all undergo endochondral ossification) Sequence of Events – Wk5 (late) – Wk6 (late) – Wk7 (late) primary ossification centers are seen in the femur and tibia rotation takes place as the limb extends ventrally – medial 90 ° rotation on the longitudinal axis – Future knee becomes ventral; extensor muscles anterior – Wk9 to birth primary ossification centers appear in the ilium, ischiium and pubis; fibula, calcaneus, talus, metatarsals and phalanges Calcaneus (at wk16-10) – Childhood secondary ossification centers form in epiphyses ossification of tarsal bones Important factors: BMPs Indian Hedgehog (IHH) Growth/differentiation factor-5 (Gdf-5) 15

16. UPPER EXTREMITY Somites – C4-C8, T1-T2 – at the limb bud site Sequence of Events (wk5) – migration – mesoderm from somites (myotomes) migrates into the limb bud forming posterior and anterior condensations – condensation – mesoderm condenses and differentiates into myoblasts; condensations split into recognizable muscles Muscular Elements Posterior Condensation – extensor and supinator musculature Anterior Condensation – flexor and pronator musculature 16

17. LOWER EXTREMITY Somites – L1-L5, S1-S2 – at the limb bud site Sequence of Events (wk5) – migration – mesoderm from somites (myotomes) migrates into the limb bud forming posterior and anterior condensations – condensation – mesoderm condenses and differentiates into myoblasts; condensations split into recognizable muscles Muscular Elements Posterior Condensation – extensor and abductor musculature Anterior Condensation – flexor and adductor musculature 17

18. UPPER EXTREMITY LOWER EXTREMITY Vasculature 4 th aortic arch – proximal right subclavian a. 7 th intersegmental artery – distal right subclavian a. – left subclavian a. Subclavian a. – Axis a. radial and ulnar aa. anterior and posterior (1 st ) interosseous aa.; median artery (reduces) becomes the axillary, brachical and anterior interosseous aa.; deep palmar arch – Terminal plexus deep and superficial palmar arches Umbilical a. – Axis a. anterior tibial, dorsalis pedis, posterior tibial, medial and lateral plantar aa. mostly regresses (but for inferior gluteal, sciatic, proximal popliteal and distal peroneal aa.) – Terminal plexus deep plantar arch – External Iliac a. femoral artery – profunda femoris a. UPPER EXTREMITY LOWER EXTREMITY 18

19. UPPER EXTREMITYLOWER EXTREMITY General (Upper & Lower): – Motor Axon from the Spinal cord innervate limb tissues – Local cues guide axons – Sensory axons use motor axons for guidance Brachial Plexus - Ventral Primary Rami (C 5 -C 8, T 1 ) Trunks (upper, middle, lower) Divisions (anterior and posterior) – anterior (flexors) – posterior (extensors) Innervation Lumbosacral Plexus - Ventral Primary Rami (L 2 -L 5, S 1 -S 3 ) Divisions (anterior and posterior) – anterior (flexors) – posterior (extensors) 19

20. Meromelia - Absence of part of the limb Amelia, Ectromelia - Absence of 1 or more limbs Phocomelia - Short, ill-formed limb (flipper limb) Hemimelia - Stunted distal limb Acrodolichomelia - Enlarged autopod (hand,foot) Adactyly - Absence of all digits Ectrodactyly - Absence of digits (one or more) Polydactyly - Extra digits Syndactyly - Fusion of digits Clinical Terms 20

21. 21 http://www.youtube.com/watch?v=VpbdqGJ9LWk