Lecture - Limb Development

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Introduction

Appendicular skeleton

This lecture is an introduction to the events in limb development.


The limb has long been used as a model of how developmental patterning occurs by manipulation of the limb in animal models. This lecture will therefore also introduce some concepts and experiments that have identified patterning mechanisms within the limb.

The previous lecture covered the basics of bone, muscle and cartilage development, that can be applied to the same elements within the limb.

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).


2017 Lecture PDF


Recent Reviews  
2017

Irene Delgado, Miguel Torres Coordination of limb development by crosstalk among axial patterning pathways. Dev. Biol.: 2017; PubMed 28283405

Jamie M Verheyden, Xin Sun Embryology Meets Molecular Biology: Deciphering the Apical Ectodermal Ridge. Dev. Biol.: 2017; PubMed 28131856


2015

Irene Delgado, Miguel Torres Coordination of limb development by crosstalk among axial patterning pathways. Dev. Biol.: 2017; PubMed 28283405


2011 Developmental Dynamics - Special Issue: Special Issue on Limb Development May 2011 Volume 240, Issue 5

2016 Lecture Video Recording  
This 2016 lecture video recording is similar in content to the current 2017 online lecture.

Click to play new window - 2016 Lecture Video (50 MB)

Carnegie stage 1-23

Lecture Objectives

Human Embryo stage 14 SEM
  • Review of the subdivisions of mesoderm development.
  • Differentiation of somites
  • Limb patterning (axes)
  • Cartilage formation
  • Bone formation
  • Development of skeletal muscle

Lecture Resources

Movies  
Embryo 10mm surface icon.jpg
 ‎‎Embryo 10mm
Page | Play
References  
UNSW Embryology logo
Hill, M.A. (2017). UNSW Embryology (17th ed.) Retrieved November 23, 2017, from https://embryology.med.unsw.edu.au
Factor Links: hCG | BMP | Sonic hedgehog | HOX | FGF | Nanog | Nodal | Notch | FOX | PAX | Retinoic acid | SIX | Slit2/Robo1 | Sox | TBX | TGF-beta | VEGF | WNT | Hippo | NGF | Category:Molecular
The Developing Human, 10th edn.jpg Moore, K.L., Persaud, T.V.N. & Torchia, M.G. (2015). The developing human: clinically oriented embryology (10th ed.). Philadelphia: Saunders.
Larsen's human embryology 5th ed.jpg Schoenwolf, G.C., Bleyl, S.B., Brauer, P.R., Francis-West, P.H. & Philippa H. (2015). Larsen's human embryology (5th ed.). New York; Edinburgh: Churchill Livingstone.

Limb Buds

  • 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

Stage 13 image 077.jpg

Stage 13

Stage 14 - Lateral View

Stage14 bf18.jpg

 ‎‎Mobile | Desktop | Original

Stage 14 | Embryo Slides

Upper and Lower Limb

Human Limb Development during week 6

Stage16-17-limbs01.jpg

Human Limb Development during week 8

Stage20-23 limbs a.jpg

Stage14 somites limbbuds.png

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).

Mouse limb skeleton cartoon.jpg

Limb Axis Formation

Limbs 28 days

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

Limb Initiation

  • 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

Limb induction-initiation signaling

Limb induction-initiation signal[1]

Autoregulatory loop of induction between FGF10 and FGF8

Site of FGF10 expression in the chick embryo

Positioning of the limb on the rostrocaudal (anteroposterior) axis is determined by the expression of Hox genes

Examples of Hox gene expression boundaries in the mouse Hoxb2 and Hoxb4

Limb Identity

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

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

Limb patterning factors 09.jpg

Tbx3 and Tbx2 expression in E9.75 to 10.5 wild-type mouse embryonic forelimb.[2]

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

Tbx4 expression can turn an experimentally induced forelimb into a hindlimb

Axes and Morphogens

Limb bud geometry and patterning
  • 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).

Model of patterning signals in the vertebrate limb

Diffusible morphogens create a concentration gradient accross an embryonic field

Proximodistal Axis

  • 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

limb development at embryo images online

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
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

FGF receptors are paired proteins on the cell surface with an internal tyrosine kinase domain

Dorsoventral Axis

  • 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

Consequence of Wnt7a deficiency in the mouse forelimb

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

Anteroposterior Axis

  • Zone of polarizing activity (ZPA)
  • a mesenchymal posterior region of limb
  • secretes sonic hedgehog (SHH)

ZPA secretes SHH and determines the anteroposterior axis of the limb bud

Morphogen production from the ZPA - Sonic Hedgehog (SHH)
Shh expression in ZPA mouse forelimb (E11.5)[3]
  • 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.

Hox genes and dynamic patterning of the limb

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

Mesoderm-cartoon4.jpg

- Differentiation of somitic mesoderm in the chick embryo

Limb muscle and dermis

  • Skeletal muscle derived from somites at the level of the limb buds (C3-C5; L3-L5),
    • 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
Somite cartoon5.png

Dorsal/Ventral Muscle Mass - sometimes referred to as the anterior and posterior muscle compartments.

Limb Muscle - Differentiation of Skeletal muscle is the same as in the myotome blocks but involves an extra migratory step

Muscle Development

  1. Muscle precursor cells migrate to the muscle location
  2. Form beds of proliferating myoblasts
  3. Myoblasts fuse together to form a syncitial structure called a myotube
  4. Myotubes begin to express contractile proteins, form sarcomeres
  5. mature into myofibers with tendon connections at each end, motor and sensory innervation.

Hand and Footplates

Depletion of BMP Signaling Causes Interdigital Syndactyly
  • 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

Mouse interdigit apoptosis 01.jpg

Interdigital apoptosis in the mous hindlimb.[4]

Links: hand growth

Apoptosis

Cell Biology - Apoptosis Lecture

Fluorescent staining of cells undergoing apoptosis in the limb

Limb Rotation

Stage19- limb rotation.jpg

Stage20-23 limbs a.jpg
  • 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

Limb Innervation

brachial plexus
Adult Dermatomes
  • 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

brachial plexus origin

Mouse limb tissue development.jpg

Mouse Limb Timeline 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 PubMed 22174793

| PMC3235105 | PLoS One.

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 Abnormalities

Limb Abnormalities

Congenital Hip Dislocation

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

Maternal

  • thalidomide Phocomelia
  • short ill-formed upper or lower limbs
  • hyperthermia
Links: Thalidomide

Genetic

  • Trisomy 21 - Downs syndrome
    Trisomy21 hand.jpg
  • Human Gene Mutations - mutation of any of the patterning genes will result in limb abnormalities

Type II syndactyly- HoxD13

Syndactyly

Syndactyly

Fusion of fingers or toes (Greek, syn = together, dactyly = digit) which may be single or multiple and may affect: skin only, skin and soft tissues or skin, soft tissues and bone. The condition is unimportant in toes but disabling in fingers and requires operative separation and is frequently inherited as an autosomal dominant. The presence of this additional "webbing" reflects preservation of the developmental tissues that in normal development are removed by programmed cell death (apotosis).

Talipes Equinovarus

Talipes equinovarus

(Latin, talipes = ankle bone, pes = foot, equinus = horse) Abnormality of the lower limb which begins in the embryonic period (first trimester of pregnancy) resulting in the foot is then turned inward and downward at birth, described as "club foot". Occurs in approximately 1 in 1,000 births, postnatally it affects how children walk on their toes with the foot pointed downward like a horse.

Muscle Development

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

References

  1. Satoko Nishimoto, Susan M Wilde, Sophie Wood, Malcolm P O Logan RA Acts in a Coherent Feed-Forward Mechanism with Tbx5 to Control Limb Bud Induction and Initiation. Cell Rep: 2015; PubMed 26212321
  2. 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 PubMed 20386744 | PMC2851570 | PLoS Genet.
  3. 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 PubMed 17194222 | PMC1713256 | PLoS Genet.
  4. 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 PubMed 17194222 | PMC1713256

Online Textbooks

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Images

Stage13

Stage13 bf1c.jpg Stage13 sem1c.jpg

Stage14

Stage14 bf2cl.jpg Stage14 sem1c.jpg

Historic Images

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


External Links

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 2017 ANAT2341 - Timetable | Course Outline | Group Projects | Moodle | Tutorial 1 | Tutorial 2 | Tutorial 3

Labs: 1 Fertility and IVF | 2 ES Cells to Genome Editing | 3 Preimplantation and Early Implantation | 4 Reproductive Technology Revolution | 5 Cardiac and Vascular Development | 6 CRISPR-Cas9 | 7 Somitogenesis and Vertebral Malformation | 8 Organogenesis | 9 Genetic Disorders | 10 Melanocytes | 11 Stem Cells | 12 Group

Lectures: 1 Introduction | 2 Fertilization | 3 Week 1/2 | 4 Week 3 | 5 Ectoderm | 6 Placenta | 7 Mesoderm | 8 Endoderm | 9 Research Technology | 10 Cardiovascular | 11 Respiratory | 12 Neural crest | 13 Head | 14 Musculoskeletal | 15 Limb | 16 Renal | 17 Genital | 18 Endocrine | 19 Sensory | 20 Fetal | 21 Integumentary | 22 Birth | 23 Stem cells | 24 Revision

 Student Projects: 1 Cortex | 2 Kidney | 3 Heart | 4 Eye | 5 Lung | 6 Cerebellum