Musculoskeletal System - Limb Development

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Introduction

Human embryonic limb development (week 8)
Appendicular skeleton
Limb bud geometry and patterning[1]

The mesoderm forms nearly all the connective tissues of the musculoskeletal system. Each tissue (cartilage, bone, and muscle) goes through many different mechanisms of differentiation.


The musculoskeletal system consists of skeletal muscle, bone, and cartilage and is mainly mesoderm in origin with some neural crest contribution.


The intraembryonic mesoderm can be broken into paraxial, intermediate and lateral mesoderm relative to its midline position. During the 3rd week the paraxial mesoderm forms into "balls" of mesoderm paired either side of the neural groove, called somites.


Somites appear bilaterally as pairs at the same time and form earliest at the cranial (rostral,brain) end of the neural groove and add sequentially at the caudal end. This addition occurs so regularly that embryos are staged according to the number of somites that are present. Different regions of the somite differentiate into dermomyotome (dermal and muscle component) and sclerotome (forms vertebral column). An example of a specialized musculoskeletal structure can be seen in the development of the limbs.


Skeletal muscle forms by fusion of mononucleated myoblasts to form mutinucleated myotubes. Bone is formed through a lengthy process involving ossification of a cartilage formed from mesenchyme. Two main forms of ossification occur in different bones, intramembranous (eg skull) and endochondrial (eg limb long bones) ossification. Ossification continues postnatally, through puberty until mid 20s. Early ossification occurs at the ends of long bones.

Musculoskeletal and limb abnormalities are one of the largest groups of congenital abnormalities.


Musculoskeletal Links: Introduction | Mesoderm | Somitogenesis | Limb | Cartilage | Bone | Bone Timeline | Axial Skeleton | Skull | Joint | Muscle | Muscle Timeline | Tendon | Diaphragm | Lecture - Musculoskeletal Development | Abnormalities | Limb Abnormalities | Cartilage Histology | Bone Histology | Skeletal Muscle Histology | Category:Musculoskeletal
Historic Embryology
1902 - Pubo-femoral Region | Spinal Column and Back | Body Segmentation | Cranium | Body Wall, Ribs, and Sternum | Limbs | 1907 - Muscular System | Skeleton and Limbs | 1910 - Skeleton and Connective Tissues | Muscular System | Coelom and Diaphragm | 1921 - External body form | Connective tissues and skeletal | Muscular | Diaphragm
| Shoulder Development | Pelvis Development
Historic Embryology
1902 Limbs | 1910 Limb Muscles | 1922 Pig Limb Vasculature
Factor Links: BMP | Sonic hedgehog | Homeobox | FGF | Nanog | Notch | Pax | Retinoic acid | Sox | Tbx | TGF-beta | VEGF | Wnt | hCG

Some Recent Findings

  • Notch regulation of myogenic versus endothelial fates of cells that migrate from the somite to the limb[2] "Multipotent Pax3-positive (Pax3(+)) cells in the somites give rise to skeletal muscle and to cells of the vasculature. We had previously proposed that this cell-fate choice depends on the equilibrium between Pax3 and Foxc2 expression. In this study, we report that the Notch pathway promotes vascular versus skeletal muscle cell fates. ...We now demonstrate that in addition to the inhibitory role of Notch signaling on skeletal muscle cell differentiation, the Notch pathway affects the Pax3:Foxc2 balance and promotes the endothelial versus myogenic cell fate, before migration to the limb, in multipotent Pax3(+) cells in the somite of the mouse embryo." Muscle Development | Notch
  • GATA6 Is a Crucial Regulator of Shh in the Limb Bud[3] "In the limb bud, patterning along the anterior-posterior (A-P) axis is controlled by Sonic Hedgehog (Shh), a signaling molecule secreted by the "Zone of Polarizing Activity", an organizer tissue located in the posterior margin of the limb bud. We have found that the transcription factors GATA4 and GATA6, which are key regulators of cell identity, are expressed in an anterior to posterior gradient in the early limb bud, raising the possibility that GATA transcription factors may play an additional role in patterning this tissue. While both GATA4 and GATA6 are expressed in an A-P gradient in the forelimb buds, the hindlimb buds principally express GATA6 in an A-P gradient." Sonic hedgehog
  • Transient downregulation of Bmp signalling induces extra limbs in vertebrates[4] "Bone morphogenetic protein (Bmp) signalling has been implicated in setting up dorsoventral patterning of the vertebrate limb and in its outgrowth. Here, we present evidence that Bmp signalling or, more precisely, its inhibition also plays a role in limb and fin bud initiation. Temporary inhibition of Bmp signalling either by overexpression of noggin or using a synthetic Bmp inhibitor is sufficient to induce extra limbs in the Xenopus tadpole or exogenous fins in the Danio rerio embryo, respectively. We further show that Bmp signalling acts in parallel with retinoic acid signalling, possibly by inhibiting the known limb-inducing gene wnt2ba."
  • Global gene expression analysis of murine limb development[5] "Here we describe the global gene expression dynamics during early murine limb development, when cartilage, tendons, muscle, joints, vasculature and nerves are specified and the musculoskeletal system of limbs is established. We used whole-genome microarrays to identify genes with differential expression at 5 stages of limb development (E9.5 to 13.5), during fore- and hind-limb patterning."
  • Developmental Dynamics - Special Issue: Special Issue on Limb Development May 2011 Volume 240, Issue 5
  • Spatially Controlled Cell Proliferation in Limb Bud Morphogenesis[6]"Our data run contrary to the proliferation gradient hypothesis, indicating instead that oriented cell behaviours are important for driving elongation."
  • Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development[7] "One such event is antero-posterior (AP) polarization of early limb buds and activation of morphogenetic Sonic Hedgehog (SHH) signaling in the posterior mesenchyme, which in turn promotes outgrowth and specifies the pentadactylous autopod. Inactivation of the Hand2 transcriptional regulator from the onset of mouse forelimb bud development disrupts establishment of posterior identity and Shh expression, which results in a skeletal phenotype identical to Shh deficient limb buds. ... Our study uncovers essential components of the transcriptional machinery and key interactions that set-up limb bud asymmetry upstream of establishing the SHH signaling limb bud organizer."
  • The apical ectodermal ridge (AER) can be re-induced by wounding[8] "First, we assessed the sequence of events following limb amputation in chick embryos and compared the features of limb development and regeneration in amphibians and chicks. Based on our findings, we attempted to re-induce the AER. When wnt-2b/fgf-10-expressing cells were inserted concurrently with wounding, successful re-induction of the AER occurred."
More recent papers
Mark Hill.jpg
This table shows an automated computer PubMed search using the listed sub-heading term.
  • Therefore the list of references do not reflect any editorial selection of material based on content or relevance.
  • References appear in this list based upon the date of the actual page viewing.

References listed on the rest of the content page and the associated discussion page (listed under the publication year sub-headings) do include some editorial selection based upon both relevance and availability.

Links: References | Discussion Page | Pubmed Most Recent


Search term: Limb Embryology

Farnoosh Saraee, Mohsen Sagha, Homa Mohseni Kouchesfehani, Alireza Abdanipour, Masoud Maleki, Mahin Nikougoftar Biological parameters influencing the human umbilical cord-derived mesenchymal stem cells' response to retinoic acid. Biofactors: 2014; PMID: 25408532 Maryna Masyuk, Beate Brand-Saberi Recruitment of Skeletal Muscle Progenitors to Secondary Sites: A Role for CXCR4/SDF-1 Signalling in Skeletal Muscle Development. Results Probl Cell Differ: 2015, 56;1-23 PMID: 25344664 Xin Cheng, Jian-Long Chen, Zheng-Lai Ma, Zhao-Long Zhang, Shun Lv, Dong-Mei Mai, Jia-Jia Liu, Manli Chuai, Kenneth Ka Ho Lee, Chao Wan, Xuesong Yang Biphasic influence of dexamethasone exposure on embryonic vertebrate skeleton development. Toxicol. Appl. Pharmacol.: 2014; PMID: 25286332 Dana Horakova, Petra Cela, Pavel Krejci, Lukas Balek, Simona Moravcova Balkova, Eva Matalova, Marcela Buchtova Effect of FGFR inhibitors on chicken limb development. Dev. Growth Differ.: 2014, 56(8);555-72 PMID: 25280231 G Manivannan, Premal Das, G Karthikeyan, Annamma S John Reconstructive surgery in children to correct ulnar claw hand deformity due to leprosy. Lepr Rev: 2014, 85(2);74-80 PMID: 25255610


Search term: Limb Development

Elisa Bettoni, Giorgio Ferriero, Hadeel Bakhsh, Elisabetta Bravini, Giuseppe Massazza, Franco Franchignoni A systematic review of questionnaires to assess patient satisfaction with limb orthoses. Prosthet Orthot Int: 2014; PMID: 25428901 Z Sedaghat, M Kadkhodaee, B Seifi, P Ahghari Hepatoprotective effects of remote perconditioning during renal ischemia. Bratisl Lek Listy: 2014, 115(11);675-679 PMID: 25428534 Zongxi Li, Emmanuel Roussakis, Pieter G L Koolen, Ahmed M S Ibrahim, Kuylhee Kim, Lloyd F Rose, Jesse Wu, Alexander J Nichols, Yunjung Baek, Reginald Birngruber, Gabriela Apiou-Sbirlea, Robina Matyal, Thomas Huang, Rodney Chan, Samuel J Lin, Conor L Evans Non-invasive transdermal two-dimensional mapping of cutaneous oxygenation with a rapid-drying liquid bandage. Biomed Opt Express: 2014, 5(11);3748-3764 PMID: 25426308 J Crawford, K Nygard, B S Gan, D B O'Gorman Periostin induces fibroblast proliferation and myofibroblast persistence in hypertrophic scarring. Exp. Dermatol.: 2014; PMID: 25421393 Agata Paj K, Anna Szczygie, Dorota Paluszy Ska, Barbara Królak-Olejnik Congenital skin aplasia on the lower limb in a premature infant with ELBW ¿ case report. Ital J Pediatr: 2014, 40(1);88 PMID: 25420905

Textbooks

The Developing Human, 9th edn.jpg Keith L. Moore, T.V.N. Persaud, Mark G. Torchia. (2011). The Developing Human: clinically oriented embryology (9th ed.). Philadelphia: Saunders.
Larsen's human embryology 4th edn.jpg Schoenwolf, G.C., Bleyl, S.B., Brauer, P.R. and Francis-West, P.H. (2009). Larsen’s Human Embryology (4th ed.). New York; Edinburgh: Churchill Livingstone Chapter 18 - Development of the Limbs (chapter links only work with a UNSW connection).
  • Essentials of Human Embryology Larson Chapter 11 p207-228

Objectives

Comparison of mammalian limbs
Comparison of mammalian limbs[9]
Mouse limb (E14.5)
  • Identify the components of a somite and the adult derivatives of each component.
  • Give examples of sites of (a) endochondral and (b) intramembranous ossification and to compare these two processes.
  • Identify the general times (a) of formation of primary and (b) of formation of secondary ossification centres, and (c) of fusion of such centres with each other.
  • Briefly summarise the development of the limbs.
  • Describe the developmental abnormalities responsible for the following malformations: selected growth plate disorders; congenital dislocation of the hip; scoliosis; arthrogryposis; and limb reduction deformities.

Development Overview

Below is a very brief overview using simple figures of 3 aspects of early musculoskeletal development. More detailed overviews are shown on other notes pages Mesoderm and Somite, Vertebral Column, Limb in combination with serial sections and Carnegie images.

Mesoderm Development

Mesoderm cartoon 01.jpg Cells migrate through the primitive streak to form mesodermal layer. Extraembryonic mesoderm lies adjacent to the trilaminar embryo totally enclosing the amnion, yolk sac and forming the connecting stalk.
Mesoderm cartoon 02.jpg Paraxial mesoderm accumulates under the neural plate with thinner mesoderm laterally. This forms 2 thickened streaks running the length of the embryonic disc along the rostrocaudal axis. In humans, during the 3rd week, this mesoderm begins to segment. The neural plate folds to form a neural groove and folds.
Mesoderm cartoon 03.jpg Segmentation of the paraxial mesoderm into somites continues caudally at 1 somite/90minutes and a cavity (intraembryonic coelom) forms in the lateral plate mesoderm separating somatic and splanchnic mesoderm.

Note intraembryonic coelomic cavity communicates with extraembryonic coelom through portals (holes) initially on lateral margin of embryonic disc.

Mesoderm cartoon 04.jpg Somites continue to form. The neural groove fuses dorsally to form a tube at the level of the 4th somite and "zips up cranially and caudally and the neural crest migrates into the mesoderm.

Somite Development

Mesoderm cartoon 05.jpg Mesoderm beside the notochord (axial mesoderm, blue) thickens, forming the paraxial mesoderm as a pair of strips along the rostro-caudal axis.
Mesoderm cartoon 06.jpg Paraxial mesoderm towards the rostral end, begins to segment forming the first somite. Somites are then sequentially added caudally. The somitocoel, is a cavity forming in early somites, which is lost as the somite matures.
Mesoderm cartoon 07.jpg Cells in the somite differentiate medially to form the sclerotome (forms vertebral column) and dorsolaterally to form the dermomyotome.
Mesoderm cartoon 08.jpg The dermomyotome then forms the dermotome (forms dermis) and myotome (forms muscle).

Neural crest cells migrate beside and through somite.

Mesoderm cartoon 09.jpg The myotome differentiates to form 2 components dorsally the epimere and ventrally the hypomere, which in turn form epaxial and hypaxial muscles respectively. The bulk of the trunk and limb muscle coming from the Hypaxial mesoderm. Different structures will be contributed depending upon the somite level.

Limb skeletal muscle arises from the hypomere region of the myotomes adjacent to the developing upper (C5-C8) and lower (L3-L5) limb buds.

Limb Axis Formation

Limb bud geometry and patterning[1]

Four Concepts - much of the work has been carried out using the chicken and more recently the mouse model of development.

  1. Limb Initiation
  2. Proximodistal Axis
  3. Dorsoventral Axis
  4. Anteroposterior Axis

Mouse limb Patterning Images

Mouse Limb Images: Tbx3 and Tbx2 forelimb E10 | Alx3 and Gli3 forelimb E10 | Fgf and Hox forelimb E10.5 | Bmp4 forelimb E11.5 | Bmp4 hindlimb E11.5 | Shh forelimb E11.5 | Fgf8 hindlimb E11.5 | Sox9 forelimb E12.5 | Msx2 forelimb E12.5 | Shh hindlimb E12.5
Links: Fgf | Hox | Shh | Sox | Limb Development | Mouse Development

Limb Initiation

  • Fibroblast growth factor (FGF) coated beads can induce additional limb
  • FGF10 , FGF8 (lateral plate intermediate mesoderm) prior to bud formation
  • FGF8 (limb ectoderm) FGFR2
  • FGF can respecify Hox gene expression (Hox9- limb position)
  • Hox could then activate FGF expression

Note that during the embryonic period there is a rostrocaudal (anterior posterior) timing difference between the upper and lower limb development

  • this means that developmental changes in the upper limb can precede similar changes in the lower limb (2-5 day difference in timing)

Limb Identity

Forelimb and hindlimb (mouse) identity appears to be regulated by T-box (Tbx) genes, which are a family of transcription factors.

  • hindlimb Tbx4 is expressed.
  • forelimb Tbx5 is expressed.
  • Tbx2 and Tbx3 are expressed in both limbs.

Related Research - PMID: 12490567 | Development 2003 Figures | Scanning electron micrographs of E9 Limb bud wild-type and Tbx5del/del A model for early stages of limb bud growth | PMID: 12736217 | Development 2003 Figures

Limb patterning factors 09.jpg

Tbx3 and Tbx2 expression in E9.75 to 10.5 wild-type mouse embryonic forelimb.[7]

Body Axes

  • Anteroposterior - (Rostrocaudal, Craniocaudal, Cephalocaudal) from the head end to opposite end of body or tail.
  • Dorsoventral - from the spinal column (back) to belly (front).
  • Proximodistal - from the tip of an appendage (distal) to where it joins the body (proximal).

Proximodistal Axis

Limb proximodistal developmental regions
Mouse limb (E14.5)
  • Apical Ectodermal Ridge (AER) formed by Wnt7a
  • then AER secretes FGF2, 4, 8
  • stimulates proliferation and outgrowth

The developing limb can be described along the proximodistal axis as having three main regions:

  1. Stylopod - the proximal region the limb, the skeletal component of the upper limb (forelimb) is the humerus, and for the lower limb (hindlimb) is the femur.
  2. Zeugopod - the mid-section of the limb , the skeletal components of the upper limb (forelimb) are the radius and ulna, and for the lower limb (hindlimb) are the tibia and fibula.
  3. Autopod - the distal region the limb, the musculoskeletal component of the upper limb (forelimb) is the hands, and for the lower limb (hindlimb) is the foot.

Dorsoventral Axis

  • Somites - provides dorsal signal to mesenchyme which dorsalizes ectoderm
  • Ectoderm - then in turn signals back (Wnt7a) to mesenchyme to pattern limb

Wnt7a

  • name was derived from 'wingless' and 'int’
  • Wnt gene first defined as a protooncogene, int1
  • Humans have at least 4 Wnt genes
  • Wnt7a gene is at 3p25 encoding a 349aa secreted glycoprotein
  • patterning switch with different roles in different tissues
  • mechanism of Wnt and receptor distribution still being determined (free diffusion, restricted diffusion and active transport)

One WNT receptor is Frizzled (FZD)

  • Frizzled gene family encodes a 7 transmembrane receptor

Fibroblast growth factors (FGF)

  • Family of at least 17 secreted proteins
  • bind membrane tyrosine kinase receptors
  • Patterning switch with many different roles in different tissues
  • FGF8 = androgen-induced growth factor, AIGF

FGF receptors

  • comprise a family of at least 4 related but individually distinct tyrosine kinase receptors (FGFR1- 4) similar protein structure
    • 3 immunoglobulin-like domains in extracellular region
    • single membrane spanning segment
    • cytoplasmic tyrosine kinase domain

Anteroposterior Axis

Shh expression in ZPA mouse forelimb (E11.5)[10]
  • Zone of polarizing activity (ZPA)
  • a mesenchymal posterior region of limb
  • secretes sonic hedgehog (SHH)
    • note digit 1 (thumb/big toe) is the only digit that forms independent of SHH activity.
  • apical ectodermal ridge (AER), which has a role in patterning the structures that form within the limb
  • majority of cell division (mitosis) occurs just deep to AER in a region known as the progress zone
  • A second region at the base of the limbbud beside the body, the zone of polarizing activity (ZPA) has a similar patterning role to the AER, but in determining another axis of the limb

Week 5

Stage13 bf2.jpg Stage14 somites limbbuds.png Stage15 bf1.jpg
Carnegie stage 13 Carnegie stage 14 Carnegie stage 15
Stage14 sem2l.jpg


Links: Week 5 | Carnegie stage 13 | Carnegie stage 14 | Carnegie stage 15

Week 6

Stage16-17-limbs01.jpg

Digital rays become visible on the upper limb.

Links: Week 6 | Carnegie stage 16 | Carnegie stage 17

Week 7

Stage18 bf1.jpg Stage18 bf1.jpg
Carnegie stage 18 Carnegie stage 19

Digital rays become visible on the lower limb.

Links: Week 7 | Carnegie stage 18 | Carnegie stage 19

Week 8

Stage20-23 limbs.jpg


Links: Week 8 | Carnegie stage 20 | Carnegie stage 21 | Carnegie stage 22 | Carnegie stage 23

Limb Rotation

Stage19- limb rotation.jpg

Human Embryo (stage 19) showing direction of limb rotation.

Interdigital Apoptosis

Interdigital apoptosis in the mous hindlimb.[11]

Early development of both the hand and foot appear initially as "paddles" at the end of the upper and lower limb respectively. As they continue to grow the digits (fingers and toes) are initially "webbed" together and the cells in the webbing die by programmed cell death to form the separate digits, this process is described as interdigital apoptosis.

Interdigital apoptosis, like general limb growth, occurs first in the upper limb and then later in the lower limb.

Links: Apoptosis

Fetal Growth

Fetal growth icon.jpg
 ‎‎Fetal Development
Page | Play
Embryonic period - the external appearance of both the upper and lower limb has been formed.


Fetal period - the limbs continue to grow significantly in length (elongate).


Play the associated animation to observe the relative change in limb dimensions.


Links: Fetal Development

Limb Bone

Table Of Ossification Of The Bones Of The Superior Extremity
(Days and weeks refer to the prenatal, years to the postnatal period.)
Bone Centres Time of appearance of centre Union of primary and secondary centres; remarks.
Clavicle Diaphysis 6th week There are two centres in the shaft, a medial and a lateral. These blend on the 45th day (Mall). Shaft and epiphysis unite between the 20th and 25th years.
Sternal epiphysis 18th to 20th year
Scapula Primary centres: The chief centre appears near the lateral angle. The subcoracoid centre appears at the base of the coracoid process and also gives rise to a part of the superior margin of the glenoid fossa. The coracoid process joins the body about the age of puberty. The acromial epiphysis centres (two or three in number) fuse with one another soon after their appearance and with the spine between the 22nd and 25th years (Quain); 20th year (Wilms). The subcoracoid and the epiphysis of the coracoid process, the glenoid fossa, the inferior angle, and the vertebral margin join between the 18th and 24th years in the order mentioned (Sappey).
1. That of the body, the spine, and the base of the glenoid cavity. 8th week (Mall)2
2. Goraooid process 1st year
3. Subcoracoid 10th to 12th year
Epiphyses:
Acromial epiphyses 15th to 18th year
Epiphysis of the inferior angle. 16 to 18th year
Epiphyses of the vertebral border. 18th to 20th year
Epiphyses of upper surface of coracoid. 16th to 18th year.
Epiphysis of surface of glenoid fossa. 16th to 18th year.
Humerus Diaphysis 6th to 7th week (Mall) The epiphyses of the head, the tuberculum majus and the tuberculum minus (the last is inconstant) unite with one another in 4th-6th year and with the shaft in 20th-25th year. The epiphyses of the capitulum, lateral epicondyle, and trochlea unite with one another and then in the 16th-17th year join the shaft. The epiphysis of the medial epicondyle joins the shaft in the 18th year.
Epiphyses:
Head 1st to 2d year
Tuberculum majus 2d to 3d year
Tuberculum minus 3d to 5th year
Capitulum 2d to 3d year
Epioondylus med 5th to 8th year
Lateral margin of trochlea 11th to 12th year
Epicondylus lat 12th to 14th year
Radius Diaphysis 7th week (Mall) The superior epiphysis and shaft unite between the 17th and 20th years. The inferior epiphysis and shaft about the 21st year (Pryor); M 21st year, F 21st-25th year (Sappey). Sometimes an epiphysis is found m the tuberosity (R. and K.) and in the styloid process (Sappey).
Epiphyses:
Carpal end F 8th month - M 15th month (Pryor)
Humeral end 6th-7th year
Ulna Diaphysis 7th week The centre for the shaft of the ulna arises a few days later than that for the radius. The proximal epiphysis is united to the shaft about the 17th year; the inferior epiphysis between the 18th and 20th years; F 20th - 21st years, M 21st - 24th years (Sappey). There is sometimes an epiphysis in the styloid process (Sohwegel) and in the tip of the olecranon process (Sappey).
Epiphyses:
Carpal end F 6th-7th year - M 7th-8th year (Pryor)
Humeral end 10th year
Carpus Os capitatum F 3d-6th month M 4th-10th month The navicular sometimes has two centres of ossification (Serres. Rambaud and Renault). Serres and Pryor have described two centres of ossification in the lunatum. Debierre has described two centres in the pisiform, one in a girl of eleven, the other in a boy of twelve. The OS hamatum may have a special centre for the hamular process. Pryor has found two centres in the triquetrum. Pryor (1908), describes the centres of ossification of the carpal bones as assuming shapes characteristic of each bone at an early period.
Os hamatum F 5th-10th month M 6th-12th month
Os triquetrum F 2d-3d year M about 3 years
Os lunatum F 3rd-4th year M about 4 years
Os naviculare F at 4 years, or early in 5th year M about 5 years
Os mult. maj. F 4th-5th year M 5th-6th year
Osmult. min. F 4th-5th year M 6th-6th year
Os pisiforme F 9th-10th year M 12th-3th year
Metacarpals Diaphyses 9th week (Mall) The centres for the shafts of the second and third metacarpals are the first to appear. There may be a distal epiphysis for the first metacarpal and a proximal epiphysis for the second. Pryor (1906). found the distal epiphysis of the first metacarpal in about 6 per cent, of cases. It is a family characteristic. It arises before the 4th year and unites later. Pryor found the proximal epiphysis of the second metacarpal in six out of two hundred families. It unites with the shaft between the 4th and 6th-7th year; sometimes, however, not until the 14th year. In the seal and some other animals all the metacarpals have proximal and distal epiphyses (Quain). The epiphyses join the shafts between the 15th and 20th years. There may bean independent epiphysis for the styloid process of the 5th metacarpal. The epiphysis of the metacarpal of the index finger appears first. This is followed by those of the 3d, 4th, 5th, and 1st digits.
Proximal epiphysis of the first metacarpal 3d year
Distal epiphyses of the metacarpals 2d year
Phalanges Diaphyses 9th week (Mall)
First row Proximal epiphyses 1st-3rd year (Pryor) The shafts of the phalanges of the second and third fingers are the first to show centres of ossification. The phalanges of the little finger are the last, the epiphysis in the middle finger is the first to appear. This is followed by those of the 4th, 2d, 5th, and 1st digits.
Middle row Diaphyses 11th-12th week (Mall) The centres in the shafts of this row are the last to appear. The epiphysis of the phalanx of the middle finger is the first to appear. This is followed by those of the ring, index, and little finger (Pryor).
Proximal epiphyses 2nd-3rd year
Terminal row Diaphyses 7th-8th week The terminal phalanx of the thumb is the first to show a centre of ossification in the shaft. This is the first centre of ossification in the hand. It is developed in connective tissue while the centres of the other phalanges are developed in cartilage (Mall). The epiphysis of the ungual phalanx of the thumb is followed by those of the middle, ring, index, and little fingers. The fusion of the epiphyses of the phalanges with the diaphyses takes place in the 18th-20th year.
Proximal epiphyses 2nd-3rd year
Sesamoid bones Ossification begins generally in the 13th - 14th years, and may not take place until after middle life (Thilenius). For table of relative frequency in the embryo and adult see p. 385.
  1. According to Poirier, Traite d'Anatomie, p. 138, two centres appear in the eighth week, and unite in the third month to form a centre of ossification for the body of the scapula.
  2. F. Keibel and F.P. Mall Manual of Human Embryology (1910) Philadelphia & London: J. B. Lippincott Company.

M = male F = female.


Links: Historic - Upper limb ossification table | Historic - Chapter 11 Development of the Skeleton | Upper limb table | Upper limb collapsible table | Lower limb table | Bone Development Timeline | Limb Development
Table Of Ossification Of The Bones Of The Inferior Extremity
(Days and weeks refer to the prenatal, years to the postnatal period.)
Bone Centres Time of appearance of centre Time of fusion: general remarks
Os coxae Os ilium 56th day (Mall) The rami of the ischium and the pubis are united by bone in the 7th or 8th year (Quain) ( 12-14 year Sappey). In the acetabulum the three hip bones are separated by a Y-shaped cartilage until after puberty. In this cartilage between the ilium and pubis the "os acetabuli" appears between the ninth and twelfth years. This bone, variable in size, forms a greater or less part of the pubic portion of the articular cavity. Leche (1884). Krause (1885), and many others consider it primarily an independent bone. About puberty between the ilium and ischium and over the acetabular surfaces of these bones small irregular epiphyseal centres appear. The os acetabuli becomes imited to the pubic bone about puberty and soon afterwards the acetabular portions of the ilium and ischium and the ischium and pubis begin to become united by bone. The acetabular portions of the pubis and ilium are unite a little later. Osseous union takes place earlier on the pelvic than on the articular surface of the acetabulum. The union of the several primary centres and the epiphyses is usually completed about the twentieth year.
Os ischii 105th day (Mall)
Os pubis 4th to 5th fetal month
Os acetabuli. 9th to 12th year
Epiphyses:

Those of the acetabulum

Soon after puberty
Crest of ilium Soon after puberty Fuses with main bone 20th to 25th year
Tuberosity of ischium Soon after puberty Fusion begins in the 17th year and is completed between the 20th and 24th years (Sappey)
Ischial spine Soon after puberty 18th to 20th year (Poirier).
Ant. inf. spine of ilium Soon after puberty 18th to 20th year (Poirier)
Symphysis end of os pubis (1 or 2 centres) 18th to 20th year (Sappey) After the 20th year
Femur Diaphysis 43d day (Mall)
Epiphyses:

Distal end

Shortly before birth1 20th to 24th year
Head 1st year 18th to 19th year
Great trochanter 3d to 4th year (Osseous granules soon after birth, (Poirier) 18th year
Small trochanter 13th to 14th year

8th year (Sappey)

17th year (Quain)

Proximal epiphysis 18th to 22d year (Poirier)

Patella 3d to 5th year The osseous patella reaches its definitive form soon before puberty
Tibia Diaphysis 44th day (Mall)
Epiphyses:

Proximal end

About birth 19th to 24th year (Sappey)
Distal end 2d year 16th to 19th year
Tubercle (occas.) 13th year Fuses with epiphysis of the proximal end and then with this to the diaphysis
Fibula Diaphysis 55th day (Mall).
Epiphyses:

Distal end

2d year 20th to 22d year
Proximal end 3d to 5th year 22d to 24th year
Calcaneus Chief centre 6th fetal month The chief nucleus is endochondral. A periosteal nucleus appears frequently in the 4-5 fetal month (Hasselwander)
Epiphysis (distal end) 10th year (Quain)

7th-8th year ( Sappey)

15th-16th year (Quain)

16th-18th year (Poirier)

M 17-21, average 20 years

F 13-17, average 16 years (Hasselwander)

Talus 6th fetal month (Hasselwander) In the 7th-8th year the posterior part of the talus, the os trigonum, is frequently ossified from a special centre (v. Bardeleben). It fuses about the 18th year.
Cuboid About birth
Cuneiform III 1st year
Cuneiform I 2d-3d year
Cuneiform II 3d-4th year
Navicular 4th-5th year
Metatarsals Diaphyses 8th-10th week According to v. Bardeleben a second centre of ossincation appears much later than the primary in the navicular, and finally about the time of puberty a medial epiphyseal centre arises.
Epiphyses 3d-8th year The centre for the 2d metatarsal usually appears first, then come the 3rd, 4th, 1st and 5th. The epiphysis of the 1st metatarsal appears at the proximal end of the bone: the other epiphyses arise at the distal ends of the metatarsals. There may be a distal epiphysis in the first metatarsal also.2 In some instances a proximal epiphysis is formed on the tuberosity of the fifth metatarsal (Gruber). The epiphyses unite with the shafts in the 17-21 year in males and in the 14-19 year in females. (Hasselwander).
Phalanges:
Terminal row Diaphyses 58th day (Mall)
Epiphyses (distal) 4th year M 13-23, average 16-21 year.

F 13-17, average 14-17 year (Hasselwander).

Middle row Diaphyses 4th-10th fetal month
Epiphyses 3d year M 15-19 year

F 13-16 year (Hasselwander)

Proximal row Diaphyses 3d fetal month
Epiphyses 3d year M 15-17 year.

F 14-15 year (Hasselwander)

The centres for the shafts of the phalanges often appear double, one for the dorsal and one for the plantar surface. The centres for the medial phalanges in each row usually appear before the more laterally placed centres. The centre for the 5th terminal phalanx appears much later than the other centres in this row (Mall). According to Rambaud and Renault the epiphyses arise each from two centres which fuse together. In the terminal phalanx of the great toe the ossification centre of the epiphysis often appears as early as the second or even the first year. (Hasselwander)

Sesamoid bones of the great toe M 14th year

F 12th-13th year

Ossification may begin in the 8th year in females, in the 11th in males (Hasselwander).
  1. Poirier, Traite d'Anatomie, vol. 1. page 227, gives a summary of the literature on the time of the appearance of this epiphysis. The epiphysis has some medico-legal importance, since its presence or absence has been utilized to determine whether a child is born at term. Schwegel found it to appear between birth and the third year; Casper in the ninth fetal month. Hartmann found it lacking in 12 percent, of cases at birth and in 7 per cent, of cases present as early as the eighth fetal month.
  2. Mayet has described two centres of ossification for the proximal epiphysis of the first metatarsal, one of which represents the real metatarsal of the first digit.

M = male F = female.

Links: Historic - Lower limb ossification table | Historic - Chapter 11 Development of the Skeleton | Lower limb table | Lower limb collapsible table | Upper limb ossification table | Bone Development Timeline | Limb Development


Limb sox9 and Wnt6 expression[12]
Chicken- limb bud chondrogenesis

Bone formation within the limb occurs by endochondral ossification of a pre-existing cartilage template. Ossification then replaces the existing cartilage except in the regions of articulation, where cartilage remains on the surface of the bone within the joint. Therefore bone development in the limb is initially about cartilage development or chondrogenesis.

In addition, there are two quite separate aspects to this development.

  1. Pattern - where the specific regions will commence to form cartilage, which will be different for each cartilage element.
  2. Chondrogenesis - the differentiation of mesoderm to form cartilage, which will be essentially the same program for all cartilage templates.

A recent study has identified that the overlying limb surface ectoderm potentially inhibits limb early chondrogenesis through Wnt6 signaling.[12]


Links: Cartilage Development | Bone Development | Bone Development Timeline

Shoulder and Pelvis

Hip bone

The skeletal shoulder consists of: the clavicle (collarbone), the scapula (shoulder blade), and the humerus. Development of his region occurs through both forms of ossification processes.

The skeletal pelvis consists of: the sacrum and coccyx (axial skeleton), and pelvic girdle formed by a pair of hip bones (appendicular skeleton). Before puberty, he pelvic girdle also consists of three unfused bones: the ilium, ischium, and pubis. In chicken, the entire pelvic girdle originates from the somatopleure mesoderm (somite levels 26 to 35) and the ilium, but not of the pubis and ischium, depends on somitic and ectodermal signals.[13]


Links: Shoulder Development | Pelvis Development

Limb Skeletal Muscle

Embryonic skeletal muscles arise from the somite myotome region giving rise to myogenic progenitor cells.

In the chicken, myotome growth initially occurs by a contribution of myogenic progenitor cells from the medial border of the dermomyotome. Then later by progenitors from all four borders of the dermomyotome, with the medial and the lateral borders of the somite generate the epaxial and hypaxial muscles.[14] Depending on the somite level within the body (trunk), limb or between the limbs, myotome cells will then give rise to limb and trunk muscles respectively.[15]

  • limb level somites - cells from the hypaxial (ventrolateral) lips of the dermomyotome delaminate and migrate into the limb bud to form the musculature of the limbs.
  • between the limbs somites - cells delaminate from the edges or lips of the epithelial dermomyotome to form the subjacent postmitotic myotome, which gives rise to trunk muscles.

Myogenesis

  • Early myogenic progenitor cells in the dermomyotome can be initially identified by the transcription factor Pax3.
  • Subsequent myogenic program development then depends on the myogenic determination factors (Myf5, MyoD, and MRF4), both Myf5 and MyoD are expressed in the limbs.
  • Final differentiation of these cells into post-mitotic muscle fibers in the limb bud is regulated by another myogenic determination factor, Myogenin.

(Some of the above text modified from[15])


Links: Muscle Development | 1910 Limb Muscles

Wing as Limb Model

Chicken - wing cartilage

  • chicken wing easy to manipulate
    • removal, addition and rotation of limb regions
    • grafting additional AER, ZPA
    • implanting growth factor secreting structures

Mouse Limb Model

Mouse limb skeleton cartoon.jpg Mouse limb tissue development.jpg
Mouse limb skeleton cartoon[16]

Fore-limb and hind-limb buds for stages E9.5 to E13.5. Hindlimbs are morphologically delayed by about half a day.

  • Light blue - indicate mesenchymal condensations.
  • Thick black lines - indicate cartilage as determined by alcian blue staining.
Change in cell types and tissue formation as a function of mouse developmental stage.[16]

Forelimb

Mouse forelimb gene expression.jpg

Hindlimb

Mouse hindlimb gene expression.jpg


Links: Mouse Development

Bat Limb Model

Bat limb 02.jpg

Bat limb 01.jpg

Images of the bat embryo Miniopterus schreibersii fuliginosus at embryonic Stages 13-17.[17]

(aer - apical ectodermal ridge; chp - chiropatagium; eb - elbow; kn - knee)


Links: Bat Limb Development | Bat Development

Molecular

Fibroblast Growth Factors

  • Fgf8 - morphogen gradient forms by a source-sink mechanism with freely diffusing molecules.[18]

T-box Transcription Factors

Hand2

The HAND2 gene encodes a basic helix-loop-helix (bHLH).


Links: Fibroblast Growth Factor | Sonic hedgehog | Wnt | Hand2 | OMIM

References

  1. 1.0 1.1 Anna Kicheva, James Briscoe Limbs made to measure. PLoS Biol.: 2010, 8(7);e1000421 PMID: 20644713 | PMC2903596 | PLoS
  2. Alicia Mayeuf-Louchart, Mounia Lagha, Anne Danckaert, Didier Rocancourt, Frederic Relaix, Stéphane D Vincent, Margaret Buckingham Notch regulation of myogenic versus endothelial fates of cells that migrate from the somite to the limb. Proc. Natl. Acad. Sci. U.S.A.: 2014, 111(24);8844-9 PMID: 24927569
  3. Elena Kozhemyakina, Andreia Ionescu, Andrew B Lassar GATA6 is a crucial regulator of Shh in the limb bud. PLoS Genet.: 2014, 10(1);e1004072 PMID: 24415953
  4. Bea Christen, Alexandre Miguel Cavaco Rodrigues, Monserrat Barragán Monasterio, Carme Fabregat Roig, Juan Carlos Izpisua Belmonte Transient downregulation of Bmp signalling induces extra limbs in vertebrates. Development: 2012, 139(14);2557-65 PMID: 22675213
  5. Leila Taher, Nicole M Collette, Deepa Murugesh, Evan Maxwell, Ivan Ovcharenko, Gabriela G Loots Global gene expression analysis of murine limb development. PLoS ONE: 2011, 6(12);e28358 PMID: 22174793
  6. Bernd Boehm, Henrik Westerberg, Gaja Lesnicar-Pucko, Sahdia Raja, Michael Rautschka, James Cotterell, Jim Swoger, James Sharpe The role of spatially controlled cell proliferation in limb bud morphogenesis. PLoS Biol.: 2010, 8(7);e1000420 PMID: 20644711 | PMC2903592 | PLoS
  7. 7.0 7.1 Antonella Galli, Dimitri Robay, Marco Osterwalder, Xiaozhong Bao, Jean-Denis Bénazet, Muhammad Tariq, Renato Paro, Susan Mackem, Rolf Zeller Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development. PLoS Genet.: 2010, 6(4);e1000901 PMID: 20386744
  8. Akira Satoh, Aki Makanae, Naoyuki Wada The apical ectodermal ridge (AER) can be re-induced by wounding, wnt-2b, and fgf-10 in the chicken limb bud. Dev. Biol.: 2010, 342(2);157-68 PMID: 20347761
  9. Mengyao Dai, Yao Wang, Lu Fang, David M Irwin, Tengteng Zhu, Junpeng Zhang, Shuyi Zhang, Zhe Wang Differential expression of Meis2, Mab21l2 and Tbx3 during limb development associated with diversification of limb morphology in mammals. PLoS ONE: 2014, 9(8);e106100 PMID: 25166052 | PLoS One.
  10. Amitabha Bandyopadhyay, Kunikazu Tsuji, Karen Cox, Brian D Harfe, Vicki Rosen, Clifford J Tabin Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoS Genet.: 2006, 2(12);e216 PMID: 17194222 | PMC1713256 | PLoS Genet.
  11. Amitabha Bandyopadhyay, Kunikazu Tsuji, Karen Cox, Brian D Harfe, Vicki Rosen, Clifford J Tabin Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoS Genet.: 2006, 2(12);e216 PMID: 17194222 | PMC1713256
  12. 12.0 12.1 Poongodi Geetha-Loganathan, Suresh Nimmagadda, Bodo Christ, Ruijin Huang, Martin Scaal Ectodermal Wnt6 is an early negative regulator of limb chondrogenesis in the chicken embryo. BMC Dev. Biol.: 2010, 10;32 PMID: 20334703
  13. Yegor Malashichev, Bodo Christ, Felicitas Pröls Avian pelvis originates from lateral plate mesoderm and its development requires signals from both ectoderm and paraxial mesoderm. Cell Tissue Res.: 2008, 331(3);595-604 PMID: 18087724
  14. Jérôme Gros, Martin Scaal, Christophe Marcelle A two-step mechanism for myotome formation in chick. Dev. Cell: 2004, 6(6);875-82 PMID: 15177035
  15. 15.0 15.1 Julien Giordani, Lola Bajard, Josiane Demignon, Philippe Daubas, Margaret Buckingham, Pascal Maire Six proteins regulate the activation of Myf5 expression in embryonic mouse limbs. Proc. Natl. Acad. Sci. U.S.A.: 2007, 104(27);11310-5 PMID: 17592144
  16. 16.0 16.1 Leila Taher, Nicole M Collette, Deepa Murugesh, Evan Maxwell, Ivan Ovcharenko, Gabriela G Loots Global gene expression analysis of murine limb development. PLoS ONE: 2011, 6(12);e28358 PMID: 22174793
  17. Zhe Wang, Naijian Han, Paul A Racey, Binghua Ru, Guimei He A comparative study of prenatal development in Miniopterus schreibersii fuliginosus, Hipposideros armiger and H. pratti. BMC Dev. Biol.: 2010, 10;10 PMID: 20092640 | PMC: 2824742 | BMC Dev Biol.
  18. Shuizi Rachel Yu, Markus Burkhardt, Matthias Nowak, Jonas Ries, Zdenek Petrásek, Steffen Scholpp, Petra Schwille, Michael Brand Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules. Nature: 2009, 461(7263);533-6 PMID: 19741606


Reviews

Justin Cotney, Jing Leng, Jun Yin, Steven K Reilly, Laura E DeMare, Deena Emera, Albert E Ayoub, Pasko Rakic, James P Noonan The evolution of lineage-specific regulatory activities in the human embryonic limb. Cell: 2013, 154(1);185-96 PMID: 23827682

Günter P Wagner, Alexander O Vargas On the nature of thumbs. Genome Biol.: 2008, 9(3);213 PMID: 18341703

Robert E Hill How to make a zone of polarizing activity: insights into limb development via the abnormality preaxial polydactyly. Dev. Growth Differ.: 2007, 49(6);439-48 PMID: 17661738


Articles

Akira Satoh, Aki Makanae, Naoyuki Wada The apical ectodermal ridge (AER) can be re-induced by wounding, wnt-2b, and fgf-10 in the chicken limb bud. Dev. Biol.: 2010, 342(2);157-68 PMID: 20347761

Antonella Galli, Dimitri Robay, Marco Osterwalder, Xiaozhong Bao, Jean-Denis Bénazet, Muhammad Tariq, Renato Paro, Susan Mackem, Rolf Zeller Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development. PLoS Genet.: 2010, 6(4);e1000901 PMID: 20386744

Emily K Stefanov, Jordan M Ferrage, Nicholas F Parchim, Christine E Lee, Angela D Reginelli, Mara Taché, Rosalie A Anderson Modification of the zone of polarizing activity signal by trypsin. Dev. Growth Differ.: 2009, 51(2);123-33 PMID: 19207183


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Search April 2010

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Search Pubmed: Limb Development | apical ectodermal ridge | zone polarizing activity | limb bud skeletal muscle

Additional Images

Historic Images

Limb Images: 274-278 Spinal Column and Lower Limb | 279-284 Lower Limb | 285-288 Knee | 289 Os Coxae | 290 Femur | 291 Tibia | 292 Fibula | 293 Foot | 294 | 295 | 296 | 297 | 298-299 | 300 Forearm and Hand | 301 Upper Limb Joints | 302 Clavicle | Upper Limb Ossification 1 | Upper Limb Ossification 2 | Bone Development Timeline


Skeleton and Connective Tissues: Connective Tissue Histogenesis | Skeletal Morphogenesis | Chorda Dorsalis | Vertebral Column and Thorax | Limb Skeleton | Skull Hyoid Bone Larynx

Keith, A. (1902) Human Embryology and Morphology. London: Edward Arnold. Chapter 20. The Limbs

External Links

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Cite this page: Hill, M.A. (2014) Embryology Musculoskeletal System - Limb Development. Retrieved November 28, 2014, from https://php.med.unsw.edu.au/embryology/index.php?title=Musculoskeletal_System_-_Limb_Development

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© Dr Mark Hill 2014, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G