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4 Limb Development Development of the Limbs Competencies By the end of the block you should be able to: 1.Know the origin of the structures found in the upper and lower limb. 2.Identify the factors involved in limb development. 3.Recall the origin and development of skeletal muscle. 4.Understand the development of the nerves of the limbs. Limb bud development • Day 31 – Limb bud appears • Day 33 – Hand paddle appears • Day 36 – Chondrification begins • Day 54 – Fingers separated • Day 54 – Ossifications begins Paraxial Mesoderm ! ! ! Somite ! Paraxial Mesoderm ! gives rise to ! skeletal muscle. Lateral Plate ! Mesoderm Splanchnic mesoderm Gives rise to Smooth muscle Cardiac muscle. Paraxial Mesoderm Somites ! 42 - 44 paired somites 4 occipital 8 cervical 12 thoracic 5 lumbar 5 sacral 8(10) coccygeal ! Myotomes develop into muscles Development of the Limb Bud • At 4 weeks limb buds appear • Core of mesoderm covered by ectoderm • Ectoderm becomes Mesoderm Pseudostratified columnar ectoderm Apical Ectodermal Ridge Limb Development • Three axes: – Proximal/Distal – Anterior/Posterior Anterior – Dorsal/Ventral Proximal Ventral Posterior Distal Dorsal http://www.med.unc.edu/embryo_images/unit-mslimb/mslimb_htms/mslimb017.htm TBX5 (upper limb) and TBX4 (lower limb) are involved in determining forelimb from hindlimb. Somites Upper Limb TBX5 TBX4 Lower Limb 1 2 3 4 5 6 7 8 9 10 11 12 13 HoxA HoxB HoxC HoxD Upper Limb Starts Here Tetra-Amelia Syndrome • WNT is the only gene known to cause this syndrome. 10 Development of the Limb Bud Proximal/Distal Axis • AER influences the underlying limb mesoderm • Zone immediately below AER is the progress zone • Progress zone contains proliferating and undifferentiated cells • As cells leave the zone they begin to differentiate. Development of the Limb Bud • If the AER is removed • The limb bud stops developing • If a bead soaked in FGF is implanted in its place. • Then limb development will continue normally. Development of the Limb Bud • Mesoderm first induces the overlying ectoderm • Mesoderm cells secrete FGF-10 • Ectoderm becomes Apical Ectodermal Ridge • Without the AER the limb will not develop. • If the AER is removed surgically, only the parts that have been determined already develop. • After AER is established FGF-4,8 maintain the progress zone of mesoderm. • Maintains proximal – distal polarity • Without FGF4,8 cell death occurs. • All distal structure do not develop. Mesoderm FGF10 Apical Ectodermal Ridge Dorsal/Ventral Axis • Radical Fringe expressed in dorsal ectoderm • Engrailed-1 expressed in ventral ectoderm and represses Radical Fringe • SER-2 expressed in border area. Dorsal Radical Fringe SER-2 FGF4,8 Engrailed Ventral Dorsal/Ventral Axis • Engrailed1 represses Wnt7a in the ventral ectoderm Dorsal • Wnt7a induces LMX1 in Wnt7a the dorsal mesoderm LMX1 making them dorsal. Engrailed-1 Ventral Limb bud development • The anteroposterior pattern of digit formation is dependent upon the posterior limb bud mesoderm. • This region is the ZPA - zone of polarizing activity. • The ZPA influences the overlying AER. Limb bud development Limb Bud Zone of Polarizing Activity Limb bud Development Normal Limb II IV III Limb Bud Development Limb bud development IV III II II IV III Mirror Image Duplication of Digits Anterior/Posterior polarity • ZPA produces Retinoic Acid • This initiates the production of Sonic Hedgehog (SHH) Anterior • This regulates the AP axis Posterior Anterior/Posterior polarity • Shh upregulates Gremlin 1 • Gremlin 1 inhibits BMP and Grem1 maintains FGF RA BMP • FGF maintains FGF SHH. SHH ZPA Anterior/Posterior polarity • ZPA produces Retinoic Acid • This initiates the production of Sonic Hedgehog (SHH) Anterior • This regulates the AP axis Posterior Anterior/Posterior polarity • Shh upregulates Gremlin 1 • Gremlin 1 inhibits BMP and Grem1 maintains FGF RA BMP • FGF maintains FGF SHH. SHH ZPA • HOX expression regulate the types and shapes of bones. • Mostly HOXD and HOXA clusters 1 2 3 4 5 6 7 8 9 10 11 12 13 HoxD HoxD HoxD HoxD HoxD h"p://www.sciencemag.org/content/338/6113/1476.full Fig.%1(A)#Expression#of#Sox9#in#E12.5#limbs#of#the#Hoxa13;Gli3#allelic#series.#Note#the#delayed#differen=a=on#in#the# anterior#mesoderm#in#the#absence#of#Gli3.#The#curved#white#and#yellow#lines#show#the#AP#profiles#used#for#the#analysis# of#Sox9.#The#red#arrowhead#points#to#a#digit#bifurca=on.#WT,#wild#type.#(B)#Sox9#staining#intensity#along#the#yellow# profile#indicated#by#the#curved#arrow.#AP#length#and#the#period#of#each#digit#(from#minimum#to#minimum)#are# measured#and#shown#for#Hoxa13+/–;Gli3XtJ/XtJ.#(C)#Chart#showing#the#average#digit#periods#versus#AP#lengths#for#each# profile#and#limb.#A#linear#rela=on#is#observed#in#controls#and#in#the#Gli3XtJ/XtJ#background#for#either#the#normal#or# heterozygous#dose#of#Hoxa13,#whereas#a#flaMer#rela=on#that#correlates#with#bifurca=ons#(red#arrowhead)#is#observed# in#the#Hoxa13–/–;Gli3XtJ/XtJ#limbs#(red#line).#The#curved#arrow#marks#the#yellow#point#corresponding#to#the#profile#in#(B).# (D#and#E)#Two#simula=ons#of#the#reac=onOdiffusion#model#inside#an#E12.5#Gli3#mutant#limb#shape.#(D)#The#ac=vator# concentra=on#obtained#in#the#simula=on#with#a#uniform#modula=on#of#wavelength#ω#(shown#in#the#graph)#shows#digit# bifurca=on#(red#arrowhead)#similar#to#theHoxa13–/–Gli3XtJ/XtJ#mutants.#(E)#The#simula=on#result#when#wavelength#is# modulated#according#to#a#suitable#PD#gradient#(in#this#case,#a#2D#gradient#of#simulated#FGF#signaling#ac=vity)#avoids# bifurca=ons,#because#the#wavelength#increases#with#increasing#AP#length.#Limbs#shown#in#all#figures#are#forelimbs#with# distal#to#the#right#and#anterior#to#the#top.# 26 # Fig.%2Representa)ve+skeletal+phenotypes+of+newborns+of+ the+Hoxa13;Hoxd11)13;Gli3+allelic+series.+Digit+number+(indicated+for+ the+Gli3XtJ/XtJcondi)on)+increases+as+distal+Hox+dose+is+reduced.+When+only+one+ func)onal+copy+of+Hoxa13+remains+(right+column),+the+)p+of+the+digits+is+ connected+by+a+con)nuous+band+of+ossified+(red)+and+car)laginous+(blue)+ )ssue+rimming+the+distal+border+of+the+limb+and+becoming+more+conspicuous+ as+Gli3+copies+are+removed.+ h"p://www.sciencemag.org/content/338/6113/1476.full+ 27 Fig.%2Representa)ve+skeletal+phenotypes+of+newborns+of+ the+Hoxa13;Hoxd11)13;Gli3+allelic+series.+Digit+number+(indicated+for+ the+Gli3XtJ/XtJcondi)on)+increases+as+distal+Hox+dose+is+reduced.+When+only+one+ func)onal+copy+of+Hoxa13+remains+(right+column),+the+)p+of+the+digits+is+ connected+by+a+con)nuous+band+of+ossified+(red)+and+car)laginous+(blue)+ )ssue+rimming+the+distal+border+of+the+limb+and+becoming+more+conspicuous+ as+Gli3+copies+are+removed.+ + 28 5th Week • The end of the limb is initially a paddle. • As development proceeds cell death occurs along the apical ectodermal ridge. 7th Week (49 days) • The ridge is divided into five regions • In between there is cell death that separates these ridges. • Interdigital Necrotic Zone: – Apical ectodermal ridge ceases to influence mesoderm. – FGF8 down regulated – BMPs play role in INZ apoptosis INZ – Retinoic Acid INZ influences cell death INZ • Either directly or thru BMPs The Limb Skeleton • At six weeks of development the cartilage models begin to develop • Mesenchymal cells – differentiation into osteoblasts regulated by CBFA1/RUNX2; – differentiation into chondrocytes regulated by SOX9. The Limb Musculature • The limb buds develop opposite – lower 5 cervical – upper 2 thoracic levels. Somites – lower 4 lumbar and Upper Limb – upper 2 sacral levels. Lower Limb Limb Musculature Myotome • Cells from somites migrate into limb bud to develop into muscle • Nerves that innervated that somite followed the muscles • Muscle cells appear to move actively rather that being moved by other cells or the matrix. Paraxial Mesoderm ! ! ! Somite ! Paraxial Mesoderm ! gives rise to ! skeletal muscle. Lateral Plate ! Mesoderm Splanchnic mesoderm Gives rise to Smooth muscle Cardiac muscle. • Somite: – Dermatome – Myotome – Scleretome From Buckingham and Vincent 2009 Distinct and dynamic myogenic populations in the vertebrate embryo. Current Opinion in Genetics and Development. 19:444-453 WNT Pax3/7 FGF Myostatin Myf5 MyoD Limb musculature event Genes involved Delamination PAX3 Migration C-met Proliferation C-met, PAX3 Determination MyoD, Myf5 Differentiation Myogenin, Mef2 From Buckingham and Vincent 2009 Distinct and dynamic myogenic populations in the vertebrate embryo. Current Opinion in Genetics and Development. 19:444-453 Muscle Type Muscle Stem Determination Differentiation cells Eye muscles Pitx2 Myf5, Mrf4 Myogenin Pharyngeal arch Pitx2 Myf5 Myogenin muscles Trunk/limb Pax3/7 Myf5, MyoD Myogenin muscles BMP4 FGF • WNT from dorsal spinal cord induces Myf5 in myotome to aid in the formation of Epaxial muscles. • WNT from body wall ectoderm induces lateral myotome to express myoD for the formation of anterior and limb musculature (Hypaxial Muscles). Development of Muscle Epaxial muscles Hypaxial muscles Limb Musculature Epimere Extensor muscles Flexor Muscles Limb Musculature • As upper limb bud develops, muscle cells from the lower 4 cervical and the first thoracic myotomes migrate to the upper limb bud. • In the lower limb muscle cells from the lower 3 lumbar and upper 2 sacral myotomes migrate into the lower limb bud. • Initially the limb bud is segmental. • Later the muscle cells combine and fuse. The nerve cells that innervate the skeletal muscle cells arise from the neuroepithelial cells of the neural tube. The neuroepithelial cells migrate into the basal plate of the mantle layer to populate the ventral horn. These motor neurons are called alpha motor neurons or ventral horn cells. The ventral horn cells send their axons out of the gray matter. These axons form the ventral root and join the dorsal root to form the spinal nerve. They continue as either dorsal and ventral rami. • For the limbs the ventral rami will then form either the brachial plexus (upper limb) or the lumbosacral plexus (lower limb). 47 Defects in limb development • There are several genes that have been implicated in limb development.
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