UNSW Embryology

Musculoskeletal - Limb Development

© Dr Mark Hill (2008)

Acknowledgements

Introduction

Somites form and then begin to differentiate forming sclerotome, dermomyotome and then dermatome and myotome. The lateral portion of the myotome edge migrates at level of limbs (upper limb first then lower) and mixes with somatic mesoderm. Meanwhile the dermotome continues to contribute cells to myotome.

Limb development occurs at different times for forelimbs and hindlimbs. In the mid-4th week, human upper limb buds first form and lower limbs about 2 days later. The limbs form at vertebra segmental levels C5-C8 (upper limbs) L3-L5 (lower limbs).

Upper Limb (Stage 23)	Changes in External Appearance of Upper Limb (embryonic stages, not to scale. More? Upper Limb Movie)

Page Links: Introduction | Some Recent Findings | Appendicular Skeleton | Limb Axes | Hands and Feet | Limb Ossification | Molecular Development | Carnegie Stage - Upper Limb | Limb Movies | Somite Development | Limb Development Abnormalities | References | Glossary

Other Pages: Stage 13/14 Limbbuds | Stage 22 | Stage 22 Upper Limb | Stage 22 Lower Limb

Some Recent Findings

Sun M, Ma F, Zeng X, Liu Q, Zhao XL, Wu FX, Wu GP, Zhang ZF, Gu B, Zhao YF, Tian SH, Lin B, Kong XY, Zhang XL, Yang W, Lo W, Zhang X. Triphalangeal thumb-polysyndactyly syndrome and syndactyly type IV are caused by genomic duplications involving the long-range, limb-specific SHH enhancer. J Med Genet. 2008 Apr 16;

Nissim S, Allard P, Bandyopadhyay A, Harfe BD, Tabin CJ. Characterization of a novel ectodermal signaling center regulating Tbx2 and Shh in the vertebrate limb. Dev Biol. 2007 Apr 1;304(1):9-21

"The data presented here identify the non-AER border of dorsal-ventral ectoderm as a new signaling center in limb development that localizes the ZPA to the limb margin. This finding explains the tight restriction of Shh expression to the posterior margin throughout limb outgrowth as well as the tight restriction of Shh expression to the anterior margin in many mutants exhibiting preaxial polydactyly."

Nissim S, Hasso SM, Fallon JF, Tabin CJ. Regulation of Gremlin expression in the posterior limb bud. Dev Biol. 2006 Nov 1;299(1):12-21. (More? OMIM - Gremlin)

"Proper outgrowth of the limb bud requires a positive feedback loop between Sonic hedgehog (Shh) in the zone of polarizing activity (ZPA) and Fgfs in the overlying apical ectodermal ridge. The Bmp antagonist Gremlin is expressed in a domain anterior to the ZPA and is thought to act as a signaling intermediate between Shh and Fgf."

Appendicular Skeleton

The appendicular skeleton consists of: Shoulder girdle, Upper limb (arm, hand), Pelvic girdle, Lower limb (leg, foot).

Limb Axes

The limb is initially mesenchyme with an ectodermal covering. Within this mesenchyme blood vessels begin forming, the largest (marginal vein) is adjacent to the tip of the limbbud.

At the tip of the limbbud lies a thickened ridge of ectoderm, the apical ectodermal ridge (AER), which has a role in patterning the structures that form within the limb. The majority of cell division (mitosis) occurs just deep to AER and this region is known as the progress zone. Another 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. (More? Molecular Development).

Hands and Feet

In the 5th week hand and footplates appear at the ends of limb buds and ridges form digital rays. Cells between the digital rays are removed by programmed cell death (apoptosis).

Late in Carnegie stage embryogenesis (Stage 20-23, 8th week) limb rotation occurs. Forelimbs and hindlimbs rotate in different directions, upper limb rotates dorsally, lower limb rotates ventrally, thumb and toe rostral, knee and elbow face outward.

Limb Ossification

Bones within the limb form by endochondrial ossification (begins Carnegie stage 18) throughout embryo. This process is the replacement of cartilage with bone (week 5-12).

For more information see Bone Development

Molecular Development

There is another page with an overview of Molecular Development of Musculoskeletal structures and for more specific information about factors involved in limb patterning look at the Molecular Development Page or use the links below.

Limb Initiation

• FGF beads can induce additional limb formation (More? see factors- FGF)
• 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 activate FGF expression

Limb Specification (Fore- Hind-)

• regulated by T-box genes (transcription factor) (More? see factors- TBX)
  • Tbx5- forelimb
  • Tbx4 - leg

Limb Patterning- Axes

• Wing has been used as Model of limb development as chick wing easy to manipulate: removal, grafting, additional ARER, ZPA etc

Limb Patterning- Axes

• Proximodistal Axis
  • AER formed by Wnt7a (More? see factors- Wnt7a)
  • then AER secretes FGF2, 4, 8
  • stimulates proliferation and outgrowth
• Dorsoventral Axis
  • somite provides dorsal signal to mesenchyme
  • which dorsalizes ectoderm
  • ectoderm then signals back (Wnt7a) to mesenchyme to pattern limb
• Anteroposterior Axis
  • ZPA zone of polarizing activity
  • mesenchymal posterior region of limb
  • addition of extra ZPA duplicated digits
  • signal is Shh (More? see factors- SHH)
• Limb Axes
• Limb Patterning- Axes (More? see Limb Axes)
  • Signals give positional information which is interpreted by Hox gene expression establishing programs of differentiation. (More? see factors- Hox)
    • Proximodistal Axis
    • Dorsoventral Axis
    • Anteroposterior Axis

Carnegie Stage

Carnegie stage 12 to 23 Human Forelimb Development

Limb Movies

Below are links to movies generated from the above Carnegie images. Note that the series of movie images are not to scale as the early images would be far too small to see any detail.

Arm Movie Page or Movie on new page (179Kb)

Somite Development

		Mesoderm beside the notochord (axial mesoderm) thickens, forming the paraxial mesoderm as a pair of strips along the rostro-caudal axis. 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.
		Cells in the somite differentiate medially to form the sclerotome (forms vertebral column) and laterally to form the dermomyotome. The dermomyotome then forms the dermotome (forms dermis) and myotome (forms muscle).
		Pig Embryo Serial Sections (st13/14) Serial section D3 (rotated 90 degrees) Mouseover to identify structures see also links below
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. Note these are very simplified figures.

Limb Abnormalities

Limb Abnormalities both genetic and environmentally derived and now covered in more detail on a page Limb Abnormalities).

References

Nissim S, Allard P, Bandyopadhyay A, Harfe BD, Tabin CJ. Characterization of a novel ectodermal signaling center regulating Tbx2 and Shh in the vertebrate limb. Dev Biol. 2006 Dec 9;

McKee PR, et al.   [See Related Articles] Skeletal relationships in the human embryonic foot based on three-dimensional reconstructions. Acta Anat (Basel). 1987;129(1):34-42.

Search PubMed: Feb 2007 "limb bud development" 1,264 reference articles of which 132 were reviews.

Search PubMed Now: limb bud development | limb development | musculoskeletal development

(More? PubMed- Medline)

Selected Lists of References from PubMed March 1999 search results are available for Department of Anatomy computers without internet access: Somite Reviews | Somitogenesis Abstracts | Mesoderm Review List

Computers with internet access can search from either Below or directly from PubMed Internet Access

Glossary

A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | W | X | Y | Z

Terms

• anlage- (Ger. ) primordium, structure or cells which will form a future structure.
• apical ectodermal ridge- (=AER) specialized region of ectoderm at the tip of the growing limbbuds that specifies proximo/distal axss of limb development
• aponeurosis- thin flat sheet tendon.
• apoptosis- the process of programmed cell death. In development of the limbs occurs in the "paddle" if both the hand and foot, generating the separated digits. Occurs in many tissues of the embryo and adult.
• axillary fossa- the future "armpit" region
• brachial plexus- mixed spinal nerves innervating the upper limb form a complex meshwork (crossing).
• cartilage- connective tissue from mesoderm in the embryo forms initial skeleton replaced by bone. In adult, found on surface of bone joints.
• dermatome-
• dermomyotome- dorsolateral half of each somite that forms the dermis and muscle.
• dorsal root ganglia- (=spinal ganglia) sensory ganglia derived from the neural crest lying laterally paired and dorsally to the spinal cord (in the embryo found ventral to the spinal cord). Connects centrally with the dorsal horn of the spinal cord.
• dura mater-
• dystrophy-
• ectoderm- the layer (of the 3 germ cell layers) which form the nervous system from the neural tube and neural crest and also generates the epithelia covering the embryo.
• ectodermal ring- the thickened ring of ectoderm seen dorsally in the early (stage13/14) embryo adjacent to the dermatome. Ectoderm ventrally is relatively thin, gaining its dermatome component at a later stage.
• endochondrial ossification- the process of replacement of the cartilagenous framework by osteoblasts with bone.
• epaxial myotome- the dorsal portion of the myotome that generates dorsal skeletal muscles (epaxial muscles).
• extracellular matrix- material secreted by and surrounding cells. Consists if fibers and ground substance.
• extensor-
• fascia-
• fascicle- (=bundle)
• femur-
• fibroblast growth factors- (FGF) a family of at least 10 secreted proteins that bind membrane tyrosine kinase receptors. A patterning switch with many different roles in different tissues. (FGF8 = androgen-induced growth factor (AIGF)
• fibroblast growth factor receptor- receptors comprise a family of at least 4 related but individually distinct tyrosine kinase receptors (FGFR1- 4). They have a similar protein structure, with 3 immunoglobulin-like domains in the extracellular region, a single membrane spanning segment, and a cytoplasmic tyrosine kinase domain.
• flexor-
• fossa-
• growth factor- usually a protein or peptide that will bind a cell membrane receptor and then activates an intracellular signaling pathway. The function of the pathway will be to alter the cell directly or indirectly by changing gene expression. (eg shh)
• hox- (=homeobox) family of transcription factors that bind DNA and activate gene expression. Expression of different Hox genes along neural tube defines rostral-caudal axis and segmental levels.
• humerus-
• hypaxial myotome- the ventral portion of the myotome that generates ventral skeletal muscles (hypaxial muscles).
• inguinal fossa- the region of the lower limb ajacent to flexor surface (exuivilant to the axillary fossa of the upper limb).
• lumbar plexus- mixed spinal nerves innervating the lower limb form a complex meshwork (crossing).
• mesenchyme-
• mesoderm- the middle layer of the 3 germ cell layers of the embryo. Mesoderm outside the embryo and covering the amnion, yolk and chorion sacs is extraembryonic mesoderm.
• metacarpal cartilage-
• muscle- 3 main types of muscle (smooth, cardiac and skeletal) all derived from mesoderm but different regions.
• myotome-
• myoblast- the undifferentiated mononucleated muscle cells that will fuse together to form a multinucleated myotube, then mature into a muscle fibre.
• MyoD- transcription factor involved in the determination of muscle cells in the somite. A basic helix-loop-helix factor which binds DNA.
• myotome- the portion of the dermamyotome that generates skeletal muscle. Has 2 components epaxial (dorsal muscles ) hypaxial (ventral muscles).
• neural crest- cell region at edge of neural plate, then atop the neural folds, that remains outside and initially dorsal to the neural tube when it forms. These paired dorsal lateral streaks of cells migrate throughout the embryo and can differentiate into many different cell types(=pluripotential). Those that remain on the dorsal neural tube form the sensory spinal ganglia (DRG). Neural crest cells migrate into the somites.
• patella-
• Pax- name derived from Drosophila gene 'paired' (prd) the 'paired box' is a amino end 124 amino-acid conserved domain (signature aa 35-51: P-C-x(11)-C-V-S). Transcription factor of the helix-turn-helix structural family, DNA binding, and activating gene expression. In human, nine member proteins from Pax-1 to Pax-9. Regulate differentiation of many different tissues. Some members of the family (PAX3, PAX4, PAX6, PAX7) also contain a functional homeobox domain.
• sclerotome- ventromedial half of each somite that forms the vertebral body and intervertebral disc.
• segmentation- to break a solid structure into a number of usually equal size pieces.
• spinal nerve- mixed nerve (motor and sensory) arising as lateral pairs at each vertebral segmental level.
• somatic mesoderm- derived from lateral mesoderm closest to the ectoderm and separated from other component of lateral mesoderm (splanchnic, near endoderm) by the intraembryonic coelom.
• somite- segmental block (ball) of mesoderm formed from paraxial mesoderm adjacent to notochord (axial mesoderm). Differentiates to form initially sclerotome and dermamyotome (then dermotome and myotome).
• somitic mesoderm-
• somitocoel- a transient cavity that appears within each of the the early forming somites then is lost.
• somitogenesis- the process of segmentation of the paraxial mesoderm to form "mesoderm balls" beginning cranially (humans day20) and extending caudally at 1 somite/90 minutes until approx. 44 pairs have been formed.
• sonic hedgehog- (=shh) secreted growth factor that binds patched (ptc) receptor on cell membrane. SHH function is different for different tissues in the embryo. In the nervous system, it is secreted by the notochord, ventralizes the neural tube, inducing the floor plate and motor neurons. In the Limb it is secreted by the zone of polarizing activity (ZPA) organizing limb axis formation.
• syndactyly- fusion of digits.
• tarsal-
• Tbx- T-box genes (transcription factor) involved in mouse forelimb (Tbx4) and hindlimb (Tbx5) specification.
• tibia-
• transcription factor- a factor (protein or protein with steroid) that binds to DNA to alter gene expression, usually to activate. (eg steroid hormone+receptor, Retinoic acid+Receptor, Hox, Pax, Lim, Nkx-2.2).
• Wnt7a- The designation 'Wnt' was derived from 'wingless' and 'int'. The Wnt gene was first defined as a protooncogene, int1. Humans have at least 4 Wnt genes: Wnt7a gene is at 3p25 encoding a 349aa secreted glycoprotein. A patterning switch with different roles in different tissues. The mechanism of Wnt distribution (free diffusion, restricted diffusion and active transport) and all its possible cell receptors are still being determined. At least one WNT receptor is Frizzled (FZD). The Frizzled gene family encodes a seven-transmembrane receptor.
• zone of polarizing activity- (zpa) dorsal region with forming limbbud mesenchyme that secretes shh and regulates limb axis formation.

Quick Links

Development Notes ----------------- Menstrual Cycle Week 1 Week 2 Week 3 Week 4 Placenta Serial Images Genes/DNA Molecular Dev Abnormal Dev Postnatal Dev Notes Index

System Notes ----------------- Heart/Cardiovascular Respiratory Coelomic cavity Gastrointestinal Tract Head and Neck Neural Neural Crest Eye Ear Nose Endocrine Urogenital Musculoskeletal Integumentary

Movies ----------------- Embryo Growth Ultrasound Week 1 Week 2 Week 3 Week 4 Stage 13/14 Stage 22 Neural Head and Neck Heart Vasculature Gastrointestinal Urogenital Other Embryos Larsen Internet

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