The document summarizes key aspects of musculoskeletal development in humans: - The musculoskeletal system develops from mesoderm, with bones arising from paraxial mesoderm (somites) and lateral plate mesoderm. - Somites form sequentially through somitogenesis and give rise to vertebrae, ribs, skeletal muscle and tendons. - Specification of axial skeleton identity relies on Hox gene expression in somites. - Muscle development occurs through myogenesis from dermomyotome of somites.

1. Author(s): Deneen Wellik, Ph.D., 2009 License: Unless otherwise noted, this material is made available under the terms of the Creative Commons Attribution – Non-Commercial 3.0 License : http://creativecommons.org/licenses/by-nc/3.0/ We have reviewed this material in accordance with U.S. Copyright Law and have tried to maximize your ability to use, share, and adapt it. The citation key on the following slide provides information about how you may share and adapt this material. Copyright holders of content included in this material should contact open.michigan@umich.edu with any questions, corrections, or clarification regarding the use of content. For more information about how to cite these materials visit http://open.umich.edu/education/about/terms-of-use. Any medical information in this material is intended to inform and educate and is not a tool for self-diagnosis or a replacement for medical evaluation, advice, diagnosis or treatment by a healthcare professional. Please speak to your physician if you have questions about your medical condition. Viewer discretion is advised: Some medical content is graphic and may not be suitable for all viewers.

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3. Musculoskeletal Deneen Wellik M1 Embryology Spring 2009 Day 34 - Human Embryo Reading: Langman’s Medical Embryology, Chapters 9, 10 Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3 rd . Ed.

4. Gastrulation produces the three germ layers: endoderm, ectoderm and mesoderm HN PS Som PS PSM Most visible result of gastrulation - somites! Source Undetermined

8. intermediate mesoderm Somites give rise to: vertebra and ribs dermis skeletal muscles of back, body wall and limbs (important for migration of neural crest and spinal nerves) lateral plate mesoderm Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

9. BMP noggin BMP/noggin: early, critical signal for mesodermal differentiation Noggin-secreting cells ectopically placed in lateral plate mesoderm respecify that mesoderm into somite-forming paraxial mesoderm Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition (Both images)

11. As somitogenesis is a continuous process, various stages of somitogenesis are present simultaneously in the developing embryo. Source Undetermined

12. Overview of somitogenesis/segmentation process: Source Undetermined

13. Raldh2 Fgf8 Molecular model of somitogenesis: Source Undetermined

14. Cycling of Notch Pathway in PSM (pre-somitic mesoderm) Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

15. Disruptions in the Notch pathway result in segmentation defects (irregular and fused elements) Dll3 Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

16. Each somite differentiates into sclerotome, dermatome and myotome; Sclerotome forms the axial skeleton. Source Undetermined Source Undetermined

17. Progression of development after somite formation: Specification and more changes in epithelialization. Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

18. Hoxa11 Sox9 A molecular snapshot of differentiation along the AP axis: D. Wellik

19. Resegmentation: Cells from the caudal half of one somite and cells from the cranial half of the adjacent caudal somite form one vertebral body. Source Undetermined

20. Patterning the axial skeleton - specification of the somite along the anteroposterior (AP) axis: Although the basic cellular differentiation pattern of somites at different axial positions is very similar, unique vertebral structures form along the craniocaudal axis, indicating that somites acquire specific identities according to their axial position. Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed.

21. Morphological identity of axial skeleton determined at pre-somitic mesoderm stages; correlates with maintenance of Hox expression. Hoxa5 Hoxa6 Hoxa7 Hoxa7 Hoxa6 Hoxa5 Source Undetermined

22. In heterotopic transplants, Hox expression is conserved Hoxa9 Hoxa9 Source Undetermined

23. Paralogous Hox genes are redundant in mammalian patterning (paralog groups shown color-coded below) ANT-C Drosophila BX-C lab pb Dfd Scr Antp Ubx AbdA AbdB A1 A3 A4 A5 A6 A7 A9 A10 A11 A13 A2 HoxA B1 B3 B4 B5 B6 B7 B8 B9 B13 B2 HoxB C4 C5 C6 C8 C9 C10 C11 C12 C13 HoxC D1 D3 D4 D8 D9 D10 D11 D12 D13 HoxD Mouse/ Human Evx1 Evx2 D. Wellik

24. Control T12 L3 S2 C5 * * * * Sacral } Lumbar { T13 T13 T13 } { * * * * * * * * } { T12 L3 S2 C5 T12 L3 S2 C5 10aaccdd 11aaccdd ‘ Segment transformation’ Sacral --> Lumbar ‘ Segment transformation’ Lumbar --> Thoracic Loss of paralogous function results in complete loss of regional identity in axial column Wellik and Capecchi, Science, 2003

25. Ribs and Sternum: Unlike the rest of the axial skeleton (vertebrae and vertebral ribs, the sternum derives from lateral plate mesoderm intermediate mesoderm lateral plate mesoderm somites/ paraxial mesoderm Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

26. Two sternal bands condense bilaterally in the lateral plate mesoderm in the ventral body wall, migrate around the developing embryo and fuse at the midline to become the manubrium, sternebrae and xiphoid process Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed.

29. Newborn Skull: At birth, flat bones are separated by sutures and fontanelles; allows molding at birth. Cranial capacity increases very little after 5-7 years of age. Some sutures remain open until adulthood. Palpitation of anterior fontanelle provides valuable information on proper ossification of the skull. Sadler. Langman’s Medical Embryology. Lippincott 2004. 9 th ed.

30. Craniofacial Defects: Cranioschisis: abnormal cranial vault formation due to failure of cranial neuropore closure anencephaly meningocele Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed. Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed.

31. Craniofacial Defects: Craniosynostosis: premature closure of one or more sutures (1:2500 births and more than 100 syndromes) Scaphocephaly: early closure of sagittal suture Brachycephaly: early closure of coronal/lambdoidal sutures Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed.

32. Craniofacial Defects: Thanatophoric dwarfism: Dwarfism with or without cloverleaf skull (abnormal growth of skull base with premature closures of all cranial sutures); neonatal lethal; autosomal dominant. Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed.

33. Craniofacial Defects: Microcephaly: generalized failure of brain growth; results in severe mental retardation Acromegaly: congenital hyperpituitarism which results in excessive production of growth hormone; usually disproportianal enlargement of face, hands and feet (sometimes results in symmetrical growth)

35. Muscular system (Muscle is of mesodermal origin): Skeletal muscle derives from somites Smooth muscle derive from splanchnic lateral plate mesoderm Cardiac muscle derives splanchnic lateral plate mesoderm of the heart tube Voluntary facial muscles derive from anterior somitic mesoderm

36. Skeletal muscles form from somites (paraxial mesoderm) Each somite differentiates into sclerotome, dermatome and myotome; Myotome forms muscles. Sclerotome becomes axial skeleton and ribs Myotome forms most of the body and limb musculature Dermatome gives rise to the dermis of the skin Source Undetermined Source Undetermined

38. By the end of the fifth week, prospective muscles are found divided into two parts: Epimere - back muscles (from dorsomedial lip of myotome) Hypomere - body wall and limb muscles (from ventro medial lip of myotome) Sadler. Langman’s Medical Embryology. Lippincott 2004. 9 th ed.

39. Mature limb has no segments, but dermomyotomal pattern can still be recognized in the adult. 5 weeks 6 weeks 7 weeks Sadler. Langman’s Medical Embryology. Lippincott 2004. 10 th ed.

41. Wnt, Shh, Myf5, MyoD Conversion of myotome to muscles cells require multiple steps: All cells fated to become muscle, express MyoD Developmental Biology, Sinauer and Associates. Eighth Edition

42. Committed myoblasts in culture divide and proliferate (without differentiating) in the presence of growth factors (primarily FGFs), but show no muscle-specific protein expression. When the growth factors are used up, the cells cease to divide, align and fuse into myotubules. When the myoblasts align (but prior to fusion), they cease to divide (shown here by lack of incorporation of radiolabeled thymidine). Myotubes form when myoblasts align and fuse. The cell membranes between the multinucleate, aligned cells dissolve (requires Myogenin ). If adhesion in these cells are experimentally blocked, differentiation does not proceed. Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

43. Myogenesis occurs in two phases, the first results in the formation of primary myotubes - these arise prior to innervation by motor neurons. Secondary myotubes, which are smaller, form adjacent to primary tubules, arise after motoneuron innervation and depend upon it. They are electrically coupled. Satellite cells : Lineage is unclear, but persist after development and are capable of proliferating in response to muscle fiber damage. Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed.

44. Cardiac Muscle: Develops from splanchnic lateral plate mesoderm surrounding heart tube. Myoblasts adhere, but do not fuse to one another and, later in development, form intercalated discs. Later still a few specialized bundles of muscle cells, the Purkinje fibers, form the conducting system. Smooth Muscle: Mostly derived from splanchnic lateral plate mesoderm, but part of aorta and coronary arteries are neural crest derived. Only the sphincter, the dilator muscle of the pupil, mammary and sweat gland muscles are derived from ectoderm.

45. Syndetome 4th Somitic Compartment Defined by expression of scleraxis . This population arises between myotome (after involution under dermomyotome) and sclerotome. From sclerotomal compartment. Gives rise to the tendons. Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

46. Places tendons in correct position in the axial skeleton… C. Taubman

47. Limb scleraxis expression; in limb tendons also direct muscle attachment Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

48. Limb bones and connective tissue derive from lateral plate mesoderm Limb Growth and Development Source Undetermined

49. EARLY LIMB PATTERNING : Limb formation initiates during the fourth week of development (E9.0 in mouse) as the primary axis (AP) is still elongating. First the forelimb and then the hindlimb begin as protrusions from the lateral plate mesoderm at the sides of the embryo. Limb buds consist of a core of mesenchyme and an outer covering of ectoderm. 1 in 200 live human births display limb defects. Forelimb bud Hindlimb bud Source Undetermined

50. Limb skeletal elements: Chicken Mouse Stylopod: The proximal element of a limb that will give rise to the humerus in the forelimb and femur in the hindlimb Zeugopod: The intermediate element of a limb that will give rise to the radius and ulna in the forelimb and the tibia and fibula in the hindlimb Autopod: The distal elements of a limb that will give rise to the wrist and the fingers in the forelimb and the ankle and toes in the hindlimb Niswander. Pattern Formation: Old models out on a limb. Nature.com. February 2003, volume 4.

51. Proximal Distal Posterior Anterior Dorsal : top of hand/paw Ventral : palm Limb axes: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

52. Human Limb Development 5 weeks 6 weeks 8 weeks Sadler. Langman’s Medical Embryology. Lippincott 2004. 9 th ed.

53. Limbs rotate inward Day 33: hand plate, forearm, shoulder Day 37: Carpal region, digital plate Day 38: Finger rays, necrotic zones Day 44: toe rays Day 47: horizontal flexion Day 52: tactile pads A-P (fingers); D-V (palm); P-D (length) 5 weeks 6 weeks 8 weeks Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed.

54. At 6 weeks, the terminal portion of the limb buds flatten to form hand- and footplates. Fingers and toes (digits) are formed when cell death in the AER separates the plate into five parts. Digits continue to grow and cell death in intervening mesenchymal tissue delineates the digits. 41 days 51 days 56 days Bmp signaling Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed.

55. While external shape is being established, the mesenchyme begins condensing and chondrocyte differentiation ensues. The first cartilage models are formed in the sixth week of development. Joints form at regions of arrested chondrogenesis by cell death. early 6 weeks early 8 weeks late 6 weeks Sadler. Langman’s Medical Embryology. Lippincott 2004. 9 th ed.

57. Molecular Regulation of Limb Development: Known molecular interactions coordinate limb growth and patterning along three axes: 1) Dorsal to ventral (Lmx1b, Wnt7A, BMP/En1) 2) Proximal to distal (Fgf4/8) 3) Anterior to posterior (Shh/Gli3) GLI3 R/A University of the Basque Country Press, 1990.

58. LIMB BUD OUTGROWTH: Fgf8 is expressed shortly after limb bud outgrowth and quickly becomes localized to the Apical Ectodermal Ridge (AER) AER Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

60. Juxtaposition of Wnt7a and En1 promotes formation of the AER 1 = AER 6 = dorsal ( Wnt7a ) 5 = ventral ( En1 ) 2 = mesenchyme Junction = AER Source Undetermined

61. At the tip of the limb bud a signaling structure, the apical ectodermal ridge (AER) forms. It signals mesenchyme to proliferate and the limb grows. Removal of the AER inhibits outgrowth of the limb Transplantation of a second AER duplicates the limb; diplopodia. The signals responsible for this are fibroblast growth factors. Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

62. Proximodistal (PD) growth - regulated by AER (Fgfs): Embryological manipulation in the developing chick limb established that the apical ectodermal ridge (AER) is necessary for maintenance of PD outgrowth in the limb bud. Removal of the AER at early stages causes severe truncations of the limb skeletal elements. Progressively later removal of the AER causes progressively more distal truncations of limb elements. Removal of AER in chick at progressively later HH stages of development Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

63. Removal of AER: arrests limb development at stage at which it was removed Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed.

64. Fgf4/Fgf8 double mutants (Sun, et al. , 2002): No limb outgrowth, BUT early patterning markers are unperturbed. Thus, Fgf’s only permissive for outgrowth, apparently not important for early patterning. Sun, et al. , Nature , 2002 Source Undetermined

65. Molecular control of proximodistal (PD) growth: Identical results have been achieved in mouse genetic models. Genetic removal of Fgf8 and Fgf4 in the AER of developing mouse limbs at progressively earlier time points cause more severe (more proximal) truncations, proving that Fgf4 and Fgf8 represent the AER activity required for PD outgrowth. Important Note: With complete removal of all AER FGF expression, NO limb skeletal elements are formed BUT the limb bud is established at the normal time and location and early markers are expressed normally. Therefore, FGFs are essential for limb bud outgrowth but DO NOT appear to be important for limb patterning. Niswander. Pattern Formation: Old models out on a limb. Nature.com. February 2003, volume 4.

66. Anterior-posterior (AP) patterning: Early embryological experimentation in chick established that an area at the posterior, distal margin of the emerging limb bud, when grafted to an anterior location, was capable of causing mirror-image duplications of the digits. This region was termed Zone of Polarizing Activity (ZPA). Source Undetermined

67. Sonic hedgehog ( Shh ) expression was shown to be coincident with the ZPA region and Shh soaked beads could reproduce the digit phenotype. Shh Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

68. Such disturbances are not uncommon in humans: Sadler. Langman’s Medical Embryology. Lippincott 2004. 10 th ed. Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed.

69. Any cells capable of expressing Shh were able to reproduce this phenotype. Cells contributing to the phenotype were from host and donor cells - example of a cell non-autonomous defect Source Undetermined

70. Shh induces the conversion of Gli3R to Gli3A; results in a gradient of R/A across the AP axis of the limb bud Conc. Gli3 Act. Rep. Source Undetermined Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

71. Loss of Shh results in single stylopod, single zeugopod, single digit - consistent with idea that Shh is critical AP patterning molecule… However, biochemical work was showing that Gli3 was the transcriptional modulator of Shh function. Gli3 loss-of-function resulted in polydactyly. Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity. Nature.com. August, 2002, Volume 418.

72. Loss of Shh results in single stylopod, single zeugopod, single digit - consistent with idea that Shh is critical AP patterning molecule… However, biochemical work was showing that Gli3 was the transcriptional modulator of Shh function. Gli3 loss-of-function resulted in polydactyly. Shockingly, Shh/Gli3 double mutants look identical to Gli3 nulls!! Shh only modulates inherent polydactylous limb ‘ground state.’ Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity. Nature.com. August, 2002, Volume 418.

74. Ectoderm (AER) promotes outgrowth signals, but mesoderm controls limb identity. Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

75. Fgf beads are capable of inducing ectopic limbs. The identity of the ectopic limb is dependent on position within the flank. Very rarely a chimeric ectopic limb is formed. Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

76. Forelimb/Hindlimb Identity : Fgf beads are capable of inducing ectopic limbs. The identity of the ectopic limb is dependent on position within the flank. Very rarely a chimeric ectopic limb is formed. Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition

77. Expression of Tbx4 defines lower limb. Expression of Tbx5 defines upper limb. Post-specification: What molecular signals distinguish forelimb from hindlimb are poorly defined Source Undetermined

78. Experimentally, it has been determined that the flank region between the fore- and hindlimb, but not anterior or posterior to it, can be induced to form limbs (Fgfs, Tbx4 and Tbx5). Tbx5 Tbx4 Whether the ectopic limb becomes forelimb or hindlimb depends on WHERE in the flank it is induced. Identity of ectopic limb is correlated with expression of Tbx. Source Undetermined

79. The correlation of Tbx5/4 expression with some overexpression results in chick led to the hypothesis that Tbx5/4 control the differential identity of forelimbs and hindlimbs. Elegant genetic work has shown this is NOT TRUE!!! Developmental Cell, Vol. 8, 75-84, January, 2005

80. S Z A S Z A H I N D L I M B S F O R E L I M B S Hoxa13/ Hoxd13 Hoxa13/ Hoxd13 Hoxa11/ Hoxd11 Hox9P Hox10P ( ) Patterning of the limb elements: Hox9 through Hox13 paralogous groups are responsible for establishing morphological pattern Hox10 Hox11 Hox13 S Z A Fromental-Ramain, et al, 1996 Z Hoxa11/ Hoxc11/ Hoxd11 Hoxa10/ Hoxc10/ Hoxd10 hl hl fl fl & hl Wellik and Capecchi, Science, 2003

81. Failure to develop a limb: amelia Loss of AER or FGF signalling Source Undetermined

82. Polydactyly: duplication of digits: inherent in mesoderm Disrupted Shh/Gli3 function Source Undetermined

83. Lack of BMP4, or expression of an inhibitor in the interdigital space, results in fusions, syndactyly or webbing of fingers and toes Source Undetermined Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed.

85. 47 days 11 weeks A myriad of complex genetic interactions result in the formation and proper development of the human limb…. Source Undetermined Source Undetermined

86. Additional Source Information for more information see: http://open.umich.edu/wiki/CitationPolicy Slide 3: Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed. Slide 4: Source Undetermined Slide 5: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 6: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 7: Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed. Slide 8: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 9: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition (Both images) Slide 10: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 11: Source Undetermined Slide 12: Source Undetermined Slide 13: Source Undetermined Slide 14: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 15: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 16: Source Undetermined Slide 17: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 18: Deneen Wellik Slide 19: Source Undetermined Slide 20: Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed.; Source Undetermined Slide 21: Source Undetermined Slide 22: Source Undetermined Slide 23: Deneen Wellik Slide 24: Wellik and Capecchi, Science, 2003 Slide 25: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 26: Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed. Slide 28: Sadler. Langman’s Medical Embryology. Lippincott 2004. 9th ed.; Sadler. Langman’s Medical Embryology. Lippincott 2004. 9th ed. Slide 29: Sadler. Langman’s Medical Embryology. Lippincott 2004. 9th ed. Slide 30: Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed. Slide 31: Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed. Slide 32: Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed. Slide 36: Source Undetermined; Source Undetermined Slide 37: Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed. Slide 38: Sadler. Langman’s Medical Embryology. Lippincott 2004. 9th ed. Slide 39: Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed. Slide 40: Sadler. Langman’s Medical Embryology. Lippincott 2004. 9th ed. Slide 41: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 42: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 43: Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed.

87. Slide 45: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 46: Cliff Taubman Slide 47: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 48: Source Undetermined Slide 49: Source Undetermined Slide 50: Niswander. Pattern Formation: Old models out on a limb. Nature.com. February 2003, volume 4. Slide 51: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 52: Sadler. Langman’s Medical Embryology. Lippincott 2004. 9th ed. Slide 53: Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed. Slide 54: Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed. Slide 55: Sadler. Langman’s Medical Embryology. Lippincott 2004. 9th ed. Slide 56: Sadler. Langman’s Medical Embryology. Lippincott 2004. 9th ed. Slide 57: University of the Basque Country Press, 1990. Slide 58: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 59: Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed. Slide 60: Source Undetermined Slide 61: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 62: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 63: Carlson: Human Embryology and Developmental Biology. Elsevier, 2004. 3rd. Ed. Slide 64: Source Undetermined; Sun, et al. , Nature , 2002 Slide 65: Niswander. Pattern Formation: Old models out on a limb. Nature.com. February 2003, volume 4. Slide 66: Source Undetermined Slide 67: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 68: Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed. Slide 69: Source Undetermined Slide 70: Source Undetermined; Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 71: Litingtung, Dahn, Li, Fallon, Chiang. Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity. Nature.com. August, 2002, Volume 418. Slide 72: Litingtung, Dahn, Li, Fallon, Chiang. Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity. Nature.com. August, 2002, Volume 418. Slide 73: Niswander. Pattern Formation: Old models out on a limb. Nature.com. February 2003, volume 4. Slide 74: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 75: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 76: Gilbert. Developmental Biology, Sinauer and Associates. Eighth Edition Slide 77: Source Undetermined Slide 78: Source Undetermined Slide 79: Developmental Cell, Vol 8, 75-84, January, 2005 Slide 80: Wellik and Capecchi, Science, 2003; Fromental-Ramain, et al, 1996 Slide 81: Source Undetermined Slide 82: Source Undetermined Slide 83: Source Undetermined; Sadler. Langman’s Medical Embryology. Lippincott 2004. 10th ed. Slide 85: Source Undetermined