Lecture - Limb Development
This lecture is an introduction to the events in limb development. Cells of the ectoderm, cells derived from the dermatome and the hypaxial portion of the myotome mix with somatic component of the lateral plate mesoderm to give rise to the fore and hind limbs.
The appendicular skeleton consists of: Shoulder girdle, Upper limb (arm, hand), Pelvic girdle, Lower limb (leg, foot).
- Understanding of limb positioning
- Understanding of differences in developmental timing of upper and lower limbs
- Understanding of limb patterning - regions and factors determining the limb axes
- Understanding of limb rotation
- Understanding of limb muscle, blood vessel, bone and nerve formation
- Brief understanding of limb molecular factors and cell death
- Brief understanding of limb abnormalities
|Lectopia Lecture Audio|
The Developing Human: Clinically Oriented Embryology
|Citation: The Developing Human: clinically oriented embryology 9th ed. Keith L. Moore, T.V.N. Persaud, Mark G. Torchia. Philadelphia, PA: Saunders, 2011. (chapter links only work with a UNSW connection).|
Larsen's Human Embryology
- Limbs are initially undifferentiated mesenchyme (mesoderm) with an epithelial (ectoderm) covering. Uniform paddle shaped structures that grow outwards gradually.
- One the first noticeable changes is the development of a large blood vessel (marginal vein) which runs just underneath a thickening of the ectoderm at the tip of the limb bud called the Apical Ectodermal Ridge (AER).
- Positioning of the limbs is distant from final location
Upper and Lower Limb
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).
Limb Axis Formation
Four Concepts - much of the work has been carried out using the chicken and more recently the mouse model of development.
- Limb Initiation
- Proximodistal Axis
- Dorsoventral Axis
- Anteroposterior Axis
- Fibroblast growth factor (FGF) coated beads can induce additional limb
- FGF10 is expressed in lateral plate mesoderm prior to bud formation induces expression of FGF8 in the overlying ectoderm. FGF8 induces continued growth in the underlying mesoderm - thus a positive feedback loop
- Anterior boundary of Hoxc6 expression coincides with the position of forelimb development
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
Axes and Morphogens
- 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).
- Apical Ectodermal Ridge (AER) initially formed at the site of FGF10 induction
- then AER secretes FGF8 and FGF4 slightly later
- FGFs stimulate proliferation and outgrowth in the underlying mesenchyme
Morphogen production from the AER - The Fibroblast Growth Factors (FGFs)
- 22 FGF genes identified in humans
- bind membrane tyrosine kinase receptors
- Patterning switch with many different roles in different tissues
- 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
- Important for patterning muscles - ventral muscles - flexors; Dorsal muscles - extensors
- Early grafting experiments showed that the D/V signalling centre resided in the dorsal ectoderm
- Wnt7a is a diffusible morphogen that is secreted by dorsal ectoderm cells
- Wnt7a induces the expression of the homeobox gene Lmx1 in the underlying mesoderm adjacent to the dorsal surface
- The homeobox gene Engrailed (En1) is expressed in the opposite ventral ectoderm
Morphogen production from the dorsal ectoderm - Wnt7a
- name was derived from 'wingless' and 'int’
- Wnt gene first defined as a protooncogene, int1
- Humans have 19 Wnt genes
- Wnt7a gene is at 3p25 encoding a 349aa secreted glycoprotein
- patterning switch with different roles in different tissues
- One WNT receptor is called Frizzled (FZD) - named after a drosophila phenotype
- Frizzled gene family encodes a G protein-coupled receptor with 7 transmembrane domains
- Zone of polarizing activity (ZPA)
- a mesenchymal posterior region of limb
- secretes sonic hedgehog (SHH)
Morphogen production from the ZPA - Sonic Hedgehog (SHH)
- Sonic hedgehog (SHH) is a diffusible morphogen secreted from cells, the protein product of the SHH gene
- The protein is processed by cleavage of the preprotein and addition of a palmitate molecule to the amino terminus and cholesterol to the carboxy terminus
- The SHH receptor is a cell surface protein called Patched which interacts with another cell surface protein Smoothened.
- Binding of SHH to Patched blocks its inhibitory effect on Smoothened and allows it to initiate an intracellular signaling cascade
The Time Axis - Dynamic development and temporal gene expression
- Different Hox genes are expressed at different times in the developing limb bud and pattern the fine structure of the limb.
- Structures are determined in a proximal>distal direction with time, i.e. proximal structures such as the humerus bone are laid down first.
Cellular origins of the limb
Limb cartilage and bone
- Derived from local proliferating mesenchyme derived from the somatic lateral plate mesoderm (somatopleure)
- BMP2 and BMP4 play crucial roles in the development of cartilage - sufficient BMP must be present to achieve chondrogenesis. However, the main role is in later bone formation. Loss of BMP2 and 4 leads to a severe impairment of osteogenesis
Limb muscle and dermis
- Skeletal muscle derived from somites, the hypaxial part of the myotome
- Pax3 positive migratory myoblasts invade the limb bud
- Similarly, dermal cells also invade derived from the dermomyotome
- Both maintain the identity of the somite from which they were derived so that innervation corresponds to the same spinal nerve root.
- Note that dermatomes are rotated due to embryonic limb rotations
Origin of limb muscle cells - Migrations traced by grafting cells from a quail embryo into a chick embryo
- two species very similar in development
- quail cells recognizable by distinctive nucleoli
- Quail somite cells substituted for somite cells of 2 day chick embryo
- wing of chick sectioned a week later
- found muscle cells in chick wing derive from transplanted quail somites
Dorsal/Ventral Muscle Mass - sometimes referred to as the anterior and posterior muscle compartments. The posterior compartment of the lower hindlimb is mainly made up of the gastrocnemius muscles, the plantaris muscle and the soleus muscle.
Limb Muscle - Differentiation of Skeletal muscle is the same as in the myotome blocks but involves an extra migratory step
- Muscle precursor cells migrate to the muscle location
- Form beds of proliferating myoblasts
- Myoblasts fuse together to form a syncitial structure called a myotube
- Myotubes begin to express contractile proteins, form sarcomeres
- mature into myofibers with tendon connections at each end, motor and sensory innervation.
Hand and Footplates
- 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)
- 3-5 day difference between hand and foot development
- 8th week limbs rotate in different directions (Humans Stage 20-23)
- thumb and toe rostral
- knee and elbow face outward
- upper limb rotates dorsally
- lower limb rotates ventrally
- spinal cord segmental nerves form a plexus adjacent to each limb
- Brachial (upper) lumbar (lower)
- Plexus forms as nerves invade the limb bud mesechyme
- Fetal period - touch pads become visible on hands and feet
Congenital Hip Dislocation
- Instability of the femoral head in the acetabulum - ligaments may stretch: 1:60 at birth
- congenital instability of hip, later dislocates by muscle pulls or gravity
- familial predisposition female predominance
- Growth of femoral head, acetabulum and innominate bone are delayed until the femoral head fits firmly into the acetabulum
- thalidomide Phocomelia
- short ill-formed upper or lower limbs
- Trisomy 21 - Downs syndrome
- Human Gene Mutations - mutation of any of the patterning genes will result in limb abnormalities
Type II syndactyly- HoxD13
Duchenne Muscular Dystrophy
- X-linked dystrophy
- large gene encoding cytoskeletal protein- Dystrophin
- progressive wasting of muscle, die late teens
Becker Muscular Dystrophy
- milder form, adult onset
- UNSW Embryology Limb Development
- Embryo Images Limb Unit
- International J. Dev. Biology Vol 46 Special Issue- Limb Development 2002
- Research Labs - Rolf Zeller University of Basel Medical School
- The Developing Human: Clinically Oriented Embryology (8th Edition) by Keith L. Moore and T.V.N Persaud - Moore & Persaud
- Larsen’s Human Embryology by GC. Schoenwolf, SB. Bleyl, PR. Brauer and PH. Francis-West -
- Developmental Biology by Gilbert, Scott F. Sunderland (MA): Sinauer Associates, Inc.; c2000 Formation of the Limb Bud | Generating the Proximal-Distal Axis of the Limb
- Molecular Biology of the Cell Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002 Figure 21-13. Sonic hedgehog as a morphogen in chick limb development
- Madame Curie Bioscience Database Chapters taken from the Madame Curie Bioscience Database (formerly, Eurekah Bioscience Database)
- Bookshelf limb development
- Pubmed limb development
- 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 | Numbers | Symbols
- Dr Mark Hill 2013, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G