Development of the Limbs 2024 Jan PDF

Development of the Limbs By: Dr. Siraj Ahmed & Dr. Sujatha Bangalore Department of Anatomy Email: [email protected] & [email protected] Monday, January 15, 2024 Learning Objectives ▪ ▪ ▪ ▪ ▪ ▪ Understand the timing, position and pattern of formation of the limb buds Understand the differentiati...

Development of the Limbs By: Dr. Siraj Ahmed & Dr. Sujatha Bangalore Department of Anatomy Email: [email protected] & [email protected] Monday, January 15, 2024 Learning Objectives ▪ ▪ ▪ ▪ ▪ ▪ Understand the timing, position and pattern of formation of the limb buds Understand the differentiation of the limbs Understand the origin of limb mesenchyme and its differentiation Appreciate that the limbs ‘rotate’ in their orientation Appreciate the factors that influence the pattern of innervation of the limbs. Consider some congenital abnormalities and relevance of understanding limb development. Monday, January 15, 2024 Presentation title Normal Development Why Bother Studying All This? • Aid in understanding adult pattern of anatomy • Essential to understanding anomalies Monday, January 15, 2024 Presentation title Development of the Limbs Timing of developmental events Differentiation of Limbs The upper and lower limbs develop from limb buds Occurs in weeks 5-8 At the end of the fourth week of development, Limb buds become visible as outpocketings from the ventrolateral body wall. The upper bud appears first at C4-T2 level, The lower limb bud appears 1 to 2 days later at L2-S2 level. Each upper limb starts as an elongation with discernible parts separated by bends (future joints) with paddle like digital plate AXIS OF LIMBS Positioning of the limbs along the craniocaudal axis in the flank regions of the embryo is regulated by the HOX genes expressed along this axis. Once positioning along the craniocaudal axis is determined, the limb bud develops (growth) is regulated along three axis: Segmentation Digit formation Development of cartilage and bone Limb Musculature Nerve supply The proximal part of the limb begins to differentiate first followed by the distal part Indentations appear in the hand plate. Bones of the limbs form by endochondral ossification; they are laid down as cartilage models that then ossify. The limb musculature is derived from the myotomes of the somite that migrate into limb. Upper Limb buds receive branches from C5-T1 Nerves and Lower Limb buds from L4-S3 Nerves. The distal end flatten into a paddle like plate to form hand and foot plates At 6th week mesenchymal condensation appears as digital rays. The first sign of cartilage differentiation is condensation of limb mesenchyme. This ridge has an inducing effect on underlying mesenchyme causing cells to proliferation. Limb outgrowth is initiated by lateral plate mesoderm cells It is separated by proximal segment by a circular constriction Bones develop in a proximodistal direction, such that the pubic bones develop first, then the femur, etc. With further development the webs between the fingers regress. Cells condense and then differentiate into chondrocytes that establish a cartilage model of the bone. Tbx5 is the gene initiating the development of forelimbs and Tbx4 is the gene for hind limbs. A second constriction appears and divides the proximal part into two segments The fingers are separated by each other. Main points Osteoblasts then replace the cartilage cells and ossification occurs. 1. Ectodermal covering. What does the limb bud consist of? FGFs (fibroblast growth factors) secreted by the AER create the progress zone in the immediately adjacent mesoderm. 2. Core of mesenchyme derived from: Apical Ectodermal Ridge (AER) is essential for limb bud development in proximal to distal axis, At the tip of each limb bud, the ectoderm is thickened to form the AER. • Somatopleuric lateral plate mesoderm: which forms cartilage, ligaments, tendons, connective tissue and blood vessels. • Paraxial mesoderm: which forms muscles. FGFs keep cells in the progress zone proliferating and prevent their differentiation. 1. Proximodistal: limb grows from humerus to fingers As the limb grows, cells at the back of the progress zone are moved farther away from the influence of FGFs secreted by the AER. Inhibition of BMPs (Bone morphegenetic proteins) results in webbed digits Or ‘Making a chicken into a duck’ As cells are proliferating the limb bud grows distally and form progress zone. The earliest mesenchyme (the oldest) form base and proximal cells differentiate into cartilage. Anteroposterior axis is control by zone of polarizing activity (ZPA). The master gene for this regulation is sonic hedgehog (SHH), secreted by ZAP. The signal from the polarizing region appears to control both digit number and digit pattern. 2. Anteroposterior (craniocaudal): limb grows from thumb to little finger Digits appear in the proper order, with the thumb on the radial (anterior) side. As the limb grows, the ZPA moves distal ward to remain in proximity to the posterior border of the AER. Misexpression of ZPA or SHH in the anterior margin of a limb results in a mirror image duplication of limb structures. (polydactily) Dorsoventral (back of the hands versus the palms) is regulated by genes expressed in the dorsal and ventral ectoderm. 3. Dorsoventral: differentiation of the extensor and flexor compartments Bone patterning is regulated by a complex overlapping HOX gene expression, bmp and WNT-7 gene. They also control the pattern of muscles, orientation of the joints and eventual characteristic differentiation of the epidermis. Most growth plates close (stop cell production) after the growth spurt during puberty. Joints are formed in the cartilaginous condensations when chondrogenesis is arrested, and a joint interzone is induced. Digits continue growing under the influence of the remaining parts of the AER that cover each one. syndactyly As FGF concentrations decrease around these cells, they begin to form condensing mesenchyme, the first step in differentiating into cartilage. Growth plates remain for a time to produce cartilage cells and continued bone growth. Interdigital tissue is removed by programmed cell death(apoptosis) to create 5 separate digits. Pattern of Skin Innervation Upper Limb buds lie opposite the lower 5 cervical and upper 2 thoracic segments (C5-T1 Nerves). Rotation of limb Rotation occurs during the 7th week along limb’s long axis: Developmental Defects Upper limb rotates 90o laterally, whereas the lower limb rotates 90o medially. Limb innervation is segmental Muscle cells for the limbs are derived from somites at specific segmental levels. For the upper limb, these segments are C5–T2; for the hind limb they are L2–S2. As muscle cells move into the limb, they split into dorsal (extensor) and ventral (flexor) compartments. In polydactyly, patterns of programmed cell death have been disrupted, leaving too many parts of the AER. Lower Limb buds lie opposite the lower 4 lumbar and upper 2 sacral segments (L4- S3 Nerves). Polydactyly and Brachydactyly The ventral muscle mass give rise to flexor and pronator in UL and flexor and adductor in LL. In polydactyly, patterns of programmed cell death have been disrupted, leaving too many parts of the AER. Ultimately, branches from their respective dorsal and ventral divisions unite into large dorsal and ventral nerves. The muscles are arranged into ventral and dorsal muscular mass with respect to axis. The dorsal muscle mass give rise to extensor group of muscles. are innervated by ventral primary rami that initially divide to form dorsal and ventral branches to these compartments. Split foot is caused by excessive programmed cell death in either the AER or the interdigital spaces (or both). Thus, the radial nerve, which supplies the extensor musculature, is formed by a combination of the dorsal segmental branches. Split foot and Syndactyly (fused digits) Syndactyly is an example of insufficient programmed cell death in the interdigital spaces. whereas the ulnar and median nerves, which supply the flexor musculature, are formed by a combination of the ventral branches. Genetic or Environment or mixture of both Cells in this region increase in number and density, and then a joint cavity is formed by cell death. Thalidomide, Warfarin, Phenytoin, Valproic acid TERATOGEN Formation of Joints Studies indicate that the most sensitive period for teratogen-induced limb malformations is the fourth and fifth weeks of development Surrounding cells differentiate into a joint capsule. Causes The drug interrupted function of the AER and proximo distal limb growth. Factors regulating the positioning of joints are not clear, but the secreted molecule WNT14 appears to be the inductive signal Children with thalidomide-induced limb defects Thalidomide For some reason, digits often developed on short limbs (phocomelia), suggesting that function of the AER was disrupted for a time, but then re-established. Mother took the drug around the fourth to sixth weeks at the time of limb development. Timing of developmental events • The upper and lower limbs develop from limb buds • At the end of the fourth week of development, Limb buds become visible as outpocketings from the ventrolateral body wall. • The upper bud appears first at C4-T2 level. • The lower limb bud appears 1 to 2 days later at L2-S2 level. Monday, January 15, 2024 Presentation title Limb buds with their segments of origin indicated. With further development, the segmental pattern disappears; however, an orderly sequence in the dermatome pattern can still be recognized in the adult. A. Upper limb bud at 5 weeks. B. Upper limb bud at 6 weeks. C. Limb buds at 7 weeks Differentiation of Limbs • Occurs in weeks 5-8 • Each upper limb starts as an elongation with discernible parts separated by bends (future joints) with paddle like digital plate • Limb outgrowth is initiated by lateral plate mesoderm cells • Tbx5 is the gene initiating the development of forelimbs and Tbx4 is the gene for hind limbs. Monday, January 15, 2024 Presentation title Q. What does the limb bud consist of? 1. Ectodermal covering. 2. Core of mesenchyme derived from: • Somatopleuric lateral plate mesoderm: forms cartilage, ligaments, tendons, connective tissue and blood vessels. • Ventrolateral cells of the somites (Paraxial Mesoderm): forms muscles. Monday, January 15, 2024 Presentation title Monday, January 15, 2024 Presentation title Monday, January 15, 2024 Presentation title Monday, January 15, 2024 Presentation title AXIS OF LIMBS Positioning of the limbs along the craniocaudal axis in the flank regions of the embryo is regulated by the HOX genes expressed along this axis. Once positioning along the craniocaudal axis is determined, the limb bud develops (growth) is regulated along three axis: 1. Proximodistal: limb grows from humerus to fingers 2. Anteroposterior (craniocaudal): limb grows from thumb to little finger 3. Dorsoventral: differentiation of the extensor and flexor compartments Monday, January 15, 2024 Presentation title 1. Proximodistal axis • Apical Ectodermal Ridge (AER) is essential for limb bud development in proximal to distal axis. • At the tip of each limb bud, the ectoderm is thickened to form the AER. • This ridge has an inducing effect on underlying mesenchyme causing cells to proliferation. • As cells are proliferating the limb bud grows distally and form progress zone. • The earliest mesenchyme (the oldest) form base and proximal cells differentiate into cartilage. Monday, January 15, 2024 Presentation title • FGFs (fibroblast growth factors) secreted by the AER create the progress zone in the immediately adjacent mesoderm. • FGFs keep cells in the progress zone proliferating and prevent their differentiation. • As the limb grows, cells at the back of the progress zone are moved farther away from the influence of FGFs secreted by the AER. • As FGF concentrations decrease around these cells, they begin to form condensing mesenchyme, the first step in differentiating into cartilage. Monday, January 15, 2024 Presentation title 2. Anteroposterior axis • Anteroposterior axis is control by zone of polarizing activity (ZPA). • The master gene for this regulation is sonic hedgehog (SHH), secreted by ZAP. • The signal from the polarizing region appears to control both digit number and digit pattern. • Digits appear in the proper order, with the thumb on the radial (anterior) side. As the limb grows, the ZPA moves distal ward to remain in proximity to the posterior border of the AER. • Misexpression of ZPA or SHH in the anterior margin of a limb results in a mirror image duplication of limb structures. (polydactily) Monday, January 15, 2024 Presentation title 3. Dorsoventral axis • Dorsoventral (back of the hands versus the palms) is regulated by genes expressed in the dorsal and ventral ectoderm. • Bone patterning is regulated by a complex pattern of overlapping HOX gene expression, bmp and WNT-7 gene. • They also control the pattern of muscles, orientation of the joints and eventual characteristic differentiation of the epidermis. Monday, January 15, 2024 Presentation title s Segmentation • The proximal part of the limb begins to differentiate first followed by the distal part • The distal end flatten into a paddle like plate to form hand and foot plates • It is separated by proximal segment by a circular constriction • A second constriction appears and divides the proximal part into two segments 15 Digit formation • Indentations appear in the hand plate. • At 6th week mesenchymal condensation appears as digital rays. • With further development the webs between the fingers regress. • The fingers are separated by each other. • Development of the foot is like that of the hand. 16 Digit formation ▪ Interdigital tissue is removed by programmed cell death(apoptosis) to create 5 separate digits. ▪ Digits continue growing under the influence of the remaining parts of the AER that cover each one. ▪ Inhibition of BMPs (Bone morphegenetic proteins) results in webbed digits Or ‘Making a chicken into a duck’ ▪ Ducks, however, do not exhibit cell death between the digits, and the result is webbing (syndactyly). Monday, January 15, 2024 Presentation title Development Of Cartilage And Bone ▪ Bones of the limbs form by endochondral ossification; they are laid down as cartilage models that then ossify. ▪ The first sign of cartilage differentiation is condensation of limb mesenchyme. ▪ Bones develop in a proximodistal direction, such that the pubic bones develop first, then the femur, etc. Main points: ▪ Cells condense and then differentiate into chondrocytes that establish a cartilage model of the bone. ▪ Osteoblasts then replace the cartilage cells and ossification occurs. ▪ Growth plates remain for a time to produce cartilage cells and continued bone growth. ▪ Most growth plates close (stop cell production) after the growth spurt during puberty. Monday, January 15, 2024 Presentation title Bone Development Monday, January 15, 2024 Presentation title Formation of Joints • Joints are formed in the cartilaginous condensations when chondrogenesis is arrested, and a joint interzone is induced. • Cells in this region increase in number and density, and then a joint cavity is formed by cell death. • Surrounding cells differentiate into a joint capsule. • Factors regulating the positioning of joints are not clear, but the secreted molecule WNT14 appears to be the inductive signal Monday, January 15, 2024 Presentation title Limb Musculature • The limb musculature is derived from the myotomes of the somite that migrate into limb. • Muscle cells for the limbs are derived from somites at specific segmental levels. • For the upper limb, these segments are C5–T2; for the hind limb they are L2–S2. • The muscles are arranged into ventral and dorsal muscular mass with respect to axis. • The ventral muscle mass give rise to flexor and pronator in UL and flexor and adductor in LL. • The dorsal muscle mass give rise to extensor group of muscles. Monday, January 15, 2024 Presentation title Nerve Supply • Upper Limb buds receive branches from C5-T1 Nerves and Lower Limb buds from L4-S3 Nerves. • As muscle cells move into the limb, they split into dorsal (extensor) and ventral (flexor) compartments. • Muscles are innervated by ventral primary rami that initially divide to form dorsal and ventral branches to these compartments. • Ultimately, branches from their respective dorsal and ventral divisions unite into large dorsal and ventral nerves. • Thus, the radial nerve, which supplies the extensor musculature, is formed by a combination of the dorsal segmental branches. • whereas the ulnar and median nerves, which supply the flexor musculature, are formed by a combination of the ventral branches. Monday, January 15, 2024 Presentation title Monday, January 15, 2024 Presentation title Pattern of Skin Innervation Limb innervation is segmental • Upper Limb buds lie opposite the lower 5 cervical and upper 2 thoracic segments (C5-T1 Nerves). • Lower Limb buds lie opposite the lower 4 lumbar and upper 2 sacral segments (L4S3 Nerves). Monday, January 15, 2024 Presentation title Rotation of limb • Rotation occurs during the 7th week along limb’s long axis: • Upper limb rotates 90o laterally, whereas the lower limb rotates 90o medially. Monday, January 15, 2024 Presentation title Developmental Defects • Period of vulnerability weeks 5-8 • Rate 12.9/100,000 population (Rosano et al 2000) Monday, January 15, 2024 Presentation title Terms for Limb Abnormalities Abnormality Description Amelia Absence of limb Meromelia Absence of a part of a limb Phocomelia Short or ill-formed limbs (flippers) Hemimelia Stunting of distal segments Polydactyly Presence of extra digits or parts of digits Syndactyly Fusion of digits Adactyly Absence of all digits on a limb Monday, January 15, 2024 Presentation title Monday, January 15, 2024 Presentation title Monday, January 15, 2024 Presentation title Polydactyly and Brachydactyly ▪ In polydactyly, patterns of programmed cell death have been disrupted, leaving too many parts of the AER. ▪ In brachydactyly, something has caused the AER to stop working or the progress zone to stop responding. Split foot and Syndactyly (fused digits) ▪ Split foot is caused by excessive programmed cell death in either the AER or the interdigital spaces (or both). ▪ Syndactyly is an example of insufficient programmed cell death in the interdigital spaces. Monday, January 15, 2024 Presentation title Causes: • May be Genetic or Environment or mixture of both • TERATOGEN ? • Thalidomide, Warfarin, Phenytoin, Valproic acid • Studies indicate that the most sensitive period for teratogen-induced limb malformations is the fourth and fifth weeks of development Monday, January 15, 2024 Presentation title Children with thalidomide-induced limb defects ▪ Thalidomide was used in the 1960s as a sedative and antinauseant. ▪ The drug interrupted function of the AER and proximo distal limb growth. ▪ For some reason, digits often developed on short limbs (phocomelia), suggesting that function of the AER was disrupted for a time, but then re-established. ▪ Mother took the drug around the fourth to sixth weeks at the time of limb development. Monday, January 15, 2024 Presentation title Readings Main: • Sadler "Langman's Medical Embryology" chapter 12 (13th edition) page no. :151-161 For more information: • Gary's Anatomy, 41 edition, chapter 15, Development of limb, page: 218220. Monday, January 15, 2024 Presentation title THANK YOU Monday, January 15, 2024 Presentation title