Difference between revisions of "Lecture - Limb Development"

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<div style="background:#F5FFFA; border: 1px solid #CEF2E0; padding: 1em; margin: auto; width: 95%; float:left;"><div style="margin:0;background-color:#cef2e0;font-family:sans-serif;font-size:120%;font-weight:bold;border:1px solid #a3bfb1;text-align:left;color:#000;padding-left:0.4em;padding-top:0.2em;padding-bottom:0.2em;"> Lecturer : Dr Steve Palmer</div></div>
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{{Header}}
 +
==Introduction==
 +
[[File:Appendicular_skeleton_developmental_regions.jpg|thumb|right|Appendicular skeleton]]
 +
This lecture is an introduction to the events in limb development.  
  
  
==Introduction==
+
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.  
[[File:Appendicular skeleton.jpg|thumb|400px]]
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This lecture is an introduction to the events in limb development. Initially somites develop and then begin to differentiate forming sclerotome, dermomyotome and then dermatome and myotome. The lateral portion of the hypaxial 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.
+
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).
 
The appendicular skeleton consists of: Shoulder girdle, Upper limb (arm, hand), Pelvic girdle, Lower limb (leg, foot).
  
  
[[:File:Human_Carnegie_stage_1-23.jpg|Carnegie stage 1-23]]
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[[Media:ANAT2341_Lecture_21_-_2018_-_Beverdam_-_Limb_development.pdf|'''2018 Lecture PDF''']]
 +
 
  
==Lecture Objectives==
 
[[File:Stage14 sem2b-limb.jpg|thumb|Human Embryo stage 14 SEM]]
 
  
* Understanding of limb positioning
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{| class="wikitable mw-collapsible mw-collapsed"
* Understanding of differences in developmental timing of upper and lower limbs
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! Recent Reviews &nbsp;
* Understanding of regions and factors determining limb axes
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|-
* Understanding of limb rotation
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| 2017
* Understanding of limb muscle, blood vessel, bone and nerve formation
+
<pubmed>28283405</pubmed>
* Brief understanding of limb molecular factors and cell death
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<pubmed>28131856</pubmed>
* Brief understanding of limb abnormalities
 
  
 +
2015
  
==Textbooks==
+
<pubmed>28283405</pubmed>
  
{|
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2011 '''Developmental Dynamics''' - Special Issue: [http://onlinelibrary.wiley.com/doi/10.1002/dvdy.v240.5/issuetoc Special Issue on Limb Development] May 2011 Volume 240, Issue 5
| [[File:The Developing Human, 8th edn.jpg|80px]]
+
|}
| Moore, K.L. &amp; Persuad, T.V.N. (2008). <i>The Developing Human: clinically oriented embryology</i> (8<sup>th</sup> ed.). Philadelphia: Saunders.
 
* [http://www.mdconsult.com/books/linkTo?type=bookPage&amp;isbn=978-1-4160-3706-4&amp;eid=4-u1.0-B978-1-4160-3706-4..50019-0 Chapter 16 - The Limbs]
 
  
::(chapter links only work with a UNSW connection).
+
{| class="wikitable mw-collapsible mw-collapsed"
 +
! 2016 Lecture Video Recording &nbsp;
 
|-
 
|-
| [[File:Larsen's human embryology 4th edn.jpg|80px]]
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| This 2016 lecture video recording is similar in content to the current 2017 online lecture.
| Schoenwolf, G.C., Bleyl, S.B., Brauer, P.R. and Francis-West, P.H. (2009). <i>Larsen’s Human Embryology</i>  (4<sup>th</sup> ed.). New York; Edinburgh: Churchill Livingstone.
+
 
 +
<html5media height="600" width="800">File:2016Lecture-Limb.mp4</html5media>
 +
 
 +
Click to play new window - [[Media:2016Lecture-Limb.mp4|'''2016 Lecture Video''']] (50 MB)
 +
|}
 +
 
 +
[[:File:Human_Carnegie_stage_1-23.jpg|Carnegie stage 1-23]]
  
* [http://www.mdconsult.com/books/linkTo?type=bookPage&isbn=978-0-443-06811-9&eid=4-u1.0-B978-0-443-06811-9..10018-1 Chapter 18 - Development of the Limbs]
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==Lecture Objectives==
 +
[[File:Stage14-sem2_limb.jpg|thumb|200px|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
  
::(chapter links only work with a UNSW connection).
+
==Lecture Resources==
 +
{| class="wikitable mw-collapsible mw-collapsed"
 +
! Movies &nbsp;
 
|-
 
|-
| [[File:Logo.png|80px]]
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| valign="bottom"|{{Embryo 10mm movie 1‎}}
| Hill, M.A. (2011) <i>UNSW Embryology</i> (11<sup>th</sup> ed.). Sydney:UNSW.
+
|}
 
+
{| class="wikitable mw-collapsible mw-collapsed"
*  [[Musculoskeletal System - Limb Development|Limb Development]]
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! References &nbsp;
*  [[Musculoskeletal System - Limb Abnormalities|Limb Abnormalities]]
 
 
|-
 
|-
| '''Developmental Dynamics'''
+
| {{Embryo logocitation}}
 
|  
 
|  
* Special Issue: [http://onlinelibrary.wiley.com/doi/10.1002/dvdy.v240.5/issuetoc Special Issue on Limb Development] May 2011 Volume 240, Issue 5
+
* [[Musculoskeletal System - Limb Development|Limb Development]]
 
+
* [[Musculoskeletal System - Limb Abnormalities|Limb Abnormalities]]
 +
* Archive - [[Media:Lecture 2016 - Limb Development.pdf|2016 PDF]] | [https://embryology.med.unsw.edu.au/embryology/index.php?title=Lecture_-_Limb_Development&oldid=203567 2015] | [[Media:2015ANAT2341_Lecture_18_-_Limb Development.pdf|2015 PDF]][https://embryology.med.unsw.edu.au/embryology/index.php?title=Lecture_-_Limb_Development&oldid=146702 2014] | [[Media:ANAT2341_Lecture_18_-_2014_Limb_Development.pdf|2014 PDF]]
 +
* [[ANAT2341 Lab 9]]
 +
{{Factor Links}}
 +
* '''Developmental Dynamics''' - Special Issue: [http://onlinelibrary.wiley.com/doi/10.1002/dvdy.v240.5/issuetoc Special Issue on Limb Development] May 2011 Volume 240, Issue 5
 +
|-
 +
| [[File:The Developing Human, 10th edn.jpg|90px]]
 +
| {{MPT2015APAcitation}}
 +
* [http://ebookcentral.proquest.com.wwwproxy1.library.unsw.edu.au/lib/unsw/reader.action?docID=2074364&ppg=480 Development of Limbs]
 +
|-
 +
| [[File:Larsen's human embryology 5th ed.jpg|90px]]
 +
| {{SBBF2015APAcitation}}
 +
* [http://ebookcentral.proquest.com.wwwproxy1.library.unsw.edu.au/lib/unsw/reader.action?docID=2074524&ppg=519 Development of the Limbs]
 
|}
 
|}
  
:'''Links:''' [[Embryology Textbooks]]
 
 
==Limb Buds==
 
==Limb Buds==
* Limbs are initially undifferentiated mesenchyme (mesoderm) with an epithelial (ectoderm) covering.
+
{|
* Blood vessels then begin forming, the largest (marginal vein) is adjacent to tip of the limbbud.
+
|
 +
* 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
 
* Positioning of the limbs is distant from final location
 +
[[File:Stage 13 image 077.jpg|400px]]
  
 +
Stage 13
 +
| valign=bottom|{{SlideStage14bf18}}
 +
|}
 
==Upper and Lower Limb==
 
==Upper and Lower Limb==
[[File:Stage20-23 limbs a.jpg]]
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'''Human Limb Development during week 6'''
 +
 
 +
[[File:Stage16-17-limbs01.jpg|600px]]
 +
 
 +
'''Human Limb Development during week 8'''
 +
 
 +
[[File:Stage20-23_limbs_a.jpg]]
 
[[File:Stage14_somites_limbbuds.png|thumb]]
 
[[File:Stage14_somites_limbbuds.png|thumb]]
 
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 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).
  
 +
[[File:Mouse limb skeleton cartoon.jpg|600px]]
  
 
==Limb Axis Formation==
 
==Limb Axis Formation==
 
+
[[File:Keith1902 fig233.jpg|thumb|Limbs 28 days]]
 
Four Concepts - much of the work has been carried out using the chicken and more recently the mouse model of development.
 
Four Concepts - much of the work has been carried out using the chicken and more recently the mouse model of development.
  
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* Anterior boundary of Hoxc6 expression coincides with the position of forelimb development
 
* Anterior boundary of Hoxc6 expression coincides with the position of forelimb development
  
 +
[[File:Limb induction-initiation signal 01.jpg|400px|alt=Limb induction-initiation signaling]]
 +
 +
Limb induction-initiation signal<ref name="PMID26212321"><pubmed>26212321</pubmed></ref>
  
 
[http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=dbio&part=A3941&rendertype=figure&id=A3953 Autoregulatory loop of induction between FGF10 and FGF8]
 
[http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=dbio&part=A3941&rendertype=figure&id=A3953 Autoregulatory loop of induction between FGF10 and FGF8]
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===Limb Identity===
 
===Limb Identity===
 
 
Forelimb and hindlimb (mouse) identity appears to be regulated by T-box (Tbx) genes, which are a family of transcription factors.
 
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.
 
* hindlimb Tbx4 is expressed.
* forelimb Tbx5 is expressed.
 
 
* Tbx2 and Tbx3 are expressed in both limbs.
 
* Tbx2 and Tbx3 are expressed in both limbs.
 +
 +
[[File:Limb_patterning_factors_09.jpg|600px]]
 +
 +
Tbx3 and Tbx2 expression in E9.75 to 10.5 wild-type mouse embryonic forelimb.<ref name="PMID20386744"><pubmed>20386744</pubmed>| [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851570 PMC2851570] | [http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1000901 PLoS Genet.]</ref>
  
 
'''Related Research''' - [http://www.ncbi.nlm.nih.gov/pubmed/12490567?dopt=Abstract PMID: 12490567] | [http://dev.biologists.org/cgi/content/figsonly/130/3/623 Development 2003 Figures] | [http://dev.biologists.org/cgi/content/full/130/3/623/FIG1 Scanning electron micrographs of E9 Limb bud wild-type and Tbx5del/del] [http://dev.biologists.org/cgi/content/full/130/3/623/FIG7 A model for early stages of limb bud growth] | [http://www.ncbi.nlm.nih.gov/pubmed/12736217?dopt=Abstract PMID: 12736217] | [http://dev.biologists.org/cgi/content/figsonly/130/12/2741 Development 2003 Figures]
 
'''Related Research''' - [http://www.ncbi.nlm.nih.gov/pubmed/12490567?dopt=Abstract PMID: 12490567] | [http://dev.biologists.org/cgi/content/figsonly/130/3/623 Development 2003 Figures] | [http://dev.biologists.org/cgi/content/full/130/3/623/FIG1 Scanning electron micrographs of E9 Limb bud wild-type and Tbx5del/del] [http://dev.biologists.org/cgi/content/full/130/3/623/FIG7 A model for early stages of limb bud growth] | [http://www.ncbi.nlm.nih.gov/pubmed/12736217?dopt=Abstract PMID: 12736217] | [http://dev.biologists.org/cgi/content/figsonly/130/12/2741 Development 2003 Figures]
Line 118: Line 165:
 
* bind membrane tyrosine kinase receptors
 
* bind membrane tyrosine kinase receptors
 
* Patterning switch with many different roles in different tissues
 
* Patterning switch with many different roles in different tissues
 
+
{| class="wikitable mw-collapsible mw-collapsed"
FGF receptors
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! FGF receptors &nbsp;
 +
|-
 +
|
 
* comprise a family of at least 4 related but individually distinct tyrosine kinase receptors (FGFR1- 4) similar protein structure
 
* 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
 
* 3 immunoglobulin-like domains in extracellular region
Line 126: Line 175:
  
 
[http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=dbio&part=A1038&rendertype=figure&id=A1041 FGF receptors are paired proteins on the cell surface with an internal tyrosine kinase domain]
 
[http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=dbio&part=A1038&rendertype=figure&id=A1041 FGF receptors are paired proteins on the cell surface with an internal tyrosine kinase domain]
 
+
|}
 
===Dorsoventral Axis===
 
===Dorsoventral Axis===
 
* Important for patterning muscles - ventral muscles - flexors;  Dorsal muscles - extensors
 
* Important for patterning muscles - ventral muscles - flexors;  Dorsal muscles - extensors
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* name was derived from 'wingless' and 'int’
 
* name was derived from 'wingless' and 'int’
 
* Wnt gene first defined as a protooncogene, int1
 
* Wnt gene first defined as a protooncogene, int1
* Humans have at least 4 Wnt genes
+
* Humans have 19 Wnt genes
 
* Wnt7a gene is at 3p25 encoding a 349aa secreted glycoprotein
 
* Wnt7a gene is at 3p25 encoding a 349aa secreted glycoprotein
 
* patterning switch with different roles in different tissues
 
* patterning switch with different roles in different tissues
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=====Morphogen production from the ZPA - Sonic Hedgehog (SHH)=====
 
=====Morphogen production from the ZPA - Sonic Hedgehog (SHH)=====
 
+
[[File:Limb patterning factors 03.jpg|thumb|Shh expression in ZPA mouse forelimb (E11.5)<ref name="PMID17194222"><pubmed>17194222</pubmed>| [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1713256 PMC1713256] | [http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.0020216 PLoS Genet.]</ref>]]
 
* Sonic hedgehog (SHH) is a diffusible morphogen secreted from cells, the protein product of the SHH gene
 
* 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 protein is processed by cleavage of the preprotein and addition of a palmitate molecule to the amino terminus and cholesterol to the carboxy terminus
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===Limb cartilage and bone===
 
===Limb cartilage and bone===
 
* Derived from local proliferating mesenchyme derived from the somatic lateral plate mesoderm (somatopleure)
 
* 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
 +
 +
[[File:Mesoderm-cartoon4.jpg]]
 +
 +
[http://www.ncbi.nlm.nih.gov/books/NBK10085/figure/A3468/?report=objectonly - Differentiation of somitic mesoderm in the chick embryo]
  
 
===Limb muscle and dermis===
 
===Limb muscle and dermis===
* Skeletal muscle derived from somites, the hypaxial part of the myotome
+
{|
 +
|
 +
* 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  
 
* Pax3 positive migratory myoblasts invade the limb bud  
 
* Similarly, dermal cells also invade derived from the dermomyotome
 
* Similarly, dermal cells also invade derived from the dermomyotome
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* wing of chick sectioned a week later
 
* wing of chick sectioned a week later
 
* found muscle cells in chick wing derive from transplanted quail somites
 
* found muscle cells in chick wing derive from transplanted quail somites
 +
| [[File:Somite cartoon5.png]]
 +
|}
  
Dorsal/Ventral Muscle Mass
+
Dorsal/Ventral Muscle Mass - sometimes referred to as the anterior and posterior muscle compartments.
 
 
Forelimb Muscles
 
 
 
Limb Muscle - Differentiation, Skeletal muscle differentiates the same
 
  
 +
Limb Muscle - Differentiation of Skeletal muscle is the same as in the myotome blocks but involves an extra migratory step
 +
===Muscle Development===
 
# Muscle precursor cells migrate to the muscle location
 
# Muscle precursor cells migrate to the muscle location
 
# Form beds of proliferating myoblasts
 
# Form beds of proliferating myoblasts
# Myoblasts fuse together to form myotubes
+
# Myoblasts fuse together to form a syncitial structure called a myotube
 
# Myotubes begin to express contractile proteins, form sarcomeres
 
# Myotubes begin to express contractile proteins, form sarcomeres
# mature into myofibers, Innervation determines final muscle maturation
+
# mature into myofibers with tendon connections at each end, motor and sensory innervation.
 
 
Dermomyotome MyoD
 
  
 
==Hand and Footplates==
 
==Hand and Footplates==
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* 3-5 day difference between hand and foot development
 
* 3-5 day difference between hand and foot development
  
 +
[[File:Mouse interdigit apoptosis 01.jpg|400px]]
  
[http://www.med.unc.edu/embryo_images/unit-mslimb/mslimb_htms/mslimb024.htm hand growth]
+
Interdigital apoptosis in the mous hindlimb.<ref><pubmed>17194222</pubmed>| [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1713256 PMC1713256]</ref>
  
 +
:'''Links:''' [http://www.med.unc.edu/embryo_images/unit-mslimb/mslimb_htms/mslimb024.htm hand growth]
 
===Apoptosis===
 
===Apoptosis===
  
[http://php.med.unsw.edu.au/cellbiology/index.php?title=2009_Lecture_18 Cell Biology - Cell Death Lecture] |  [http://php.med.unsw.edu.au/cellbiology/index.php?title=2009_Lecture_18 Cell Biology - Apoptosis Lecture]
+
[http://php.med.unsw.edu.au/cellbiology/index.php?title=2009_Lecture_18 Cell Biology - Apoptosis Lecture]
  
 
[http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4&part=A3245&rendertype=figure&id=A3246 Fluorescent staining of cells undergoing apoptosis in the limb]
 
[http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4&part=A3245&rendertype=figure&id=A3246 Fluorescent staining of cells undergoing apoptosis in the limb]
  
 
==Limb Rotation==
 
==Limb Rotation==
 +
[[File:Stage19-_limb_rotation.jpg|400px]]
 
[[File:Stage20-23 limbs a.jpg|thumb]]
 
[[File:Stage20-23 limbs a.jpg|thumb]]
 
* 8th week limbs rotate in different directions (Humans Stage 20-23)
 
* 8th week limbs rotate in different directions (Humans Stage 20-23)
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[http://www.med.unc.edu/embryo_images/unit-mslimb/mslimb_htms/mslimb019.htm brachial plexus origin]
 
[http://www.med.unc.edu/embryo_images/unit-mslimb/mslimb_htms/mslimb019.htm brachial plexus origin]
 +
 +
[[File:Mouse_limb_tissue_development.jpg|600px]]
 +
 +
Mouse Limb Timeline<pubmed>22174793</pubmed>| [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3235105 PMC3235105] | [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0028358 PLoS One.]
 +
 +
{|
 +
|-
 +
| valign="bottom"|{{Human development movie 2}}
 +
| '''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==
 +
[[Musculoskeletal_System_-_Limb_Abnormalities|Limb Abnormalities]]
  
 
===Congenital Hip Dislocation===
 
===Congenital Hip Dislocation===
 
[[File:Congenital dislocation hip.jpg|thumb|Congenital Hip Dislocation]]
 
[[File:Congenital dislocation hip.jpg|thumb|Congenital Hip Dislocation]]
* Instability: 1:60 at birth;  1:240 at 1 wk: Dislocation untreated; 1:700
+
* 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
 
* congenital  instability of hip, later dislocates by muscle pulls or gravity
 
* familial predisposition female predominance
 
* familial predisposition female predominance
 
* Growth of femoral head, acetabulum and  innominate bone are delayed until the femoral head  fits firmly into the acetabulum
 
* Growth of femoral head, acetabulum and  innominate bone are delayed until the femoral head  fits firmly into the acetabulum
 
[http://embryology.med.unsw.edu.au/Notes/skmus72.htm limb abnormalities]
 
  
 
===Maternal===
 
===Maternal===
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* hyperthermia
 
* hyperthermia
  
 +
:'''Links:''' [[Abnormal_Development_-_Thalidomide|Thalidomide]]
 
===Genetic===
 
===Genetic===
 
* Trisomy 21 - Downs syndrome [[File:Trisomy21_hand.jpg|thumb]]
 
* Trisomy 21 - Downs syndrome [[File:Trisomy21_hand.jpg|thumb]]
 
* Human Gene Mutations - mutation of any of the patterning genes will result in limb abnormalities  
 
* Human Gene Mutations - mutation of any of the patterning genes will result in limb abnormalities  
 
Type II syndactyly- HoxD13
 
Type II syndactyly- HoxD13
 +
 +
=== Syndactyly ===
 +
[[File:Syndactyly.jpg|thumb|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 ===
 +
[[File:Talipes equinovarus 01.jpg|thumb|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===
 
===Muscle Development===
Line 261: Line 345:
 
Becker Muscular Dystrophy
 
Becker Muscular Dystrophy
 
* milder form, adult onset
 
* milder form, adult onset
 
 
 
==Online Links==
 
* UNSW Embryology [http://embryology.med.unsw.edu.au/Notes/skmus7.htm Limb Development]
 
* Embryo Images [http://www.med.unc.edu/embryo_images/unit-mslimb/mslimb_htms/mslimbtoc.htm Limb Unit]
 
* International J. Dev. Biology Vol 46 [http://www.ijdb.ehu.es/0207contents.htm Special Issue- Limb Development  2002]
 
* Research Labs - [http://pages.unibas.ch/anatomie/zeller/seiten/seite1.html Rolf Zeller University of Basel Medical School]
 
  
 
== References ==
 
== References ==
===Textbooks===
+
<references/>
* '''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 - 
 
 
 
 
===Online Textbooks===
 
===Online Textbooks===
 
* '''Developmental Biology''' by Gilbert, Scott F. Sunderland (MA): Sinauer Associates, Inc.; c2000 [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=dbio.section.3928 Formation of the Limb Bud] | [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=dbio.section.3941 Generating the Proximal-Distal Axis of the Limb]
 
* '''Developmental Biology''' by Gilbert, Scott F. Sunderland (MA): Sinauer Associates, Inc.; c2000 [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=dbio.section.3928 Formation of the Limb Bud] | [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=dbio.section.3941 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 [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=limb_development&rid=mboc4.figgrp.3815 Figure 21-13. Sonic hedgehog as a morphogen in chick limb development]
 
* '''Molecular Biology of the Cell''' Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002 [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=limb_development&rid=mboc4.figgrp.3815 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)
 
 
 
===Search ===
 
===Search ===
  
 
* '''Bookshelf'''  [http://www.ncbi.nlm.nih.gov/sites/entrez?db=Books&cmd=search&term=limb_development limb development]
 
* '''Bookshelf'''  [http://www.ncbi.nlm.nih.gov/sites/entrez?db=Books&cmd=search&term=limb_development limb development]
 
 
* '''Pubmed''' [http://www.ncbi.nlm.nih.gov/sites/gquery?itool=toolbar&cmd=search&term=limb_development limb development]
 
* '''Pubmed''' [http://www.ncbi.nlm.nih.gov/sites/gquery?itool=toolbar&cmd=search&term=limb_development limb development]
  
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===Stage14===
 
===Stage14===
 
[[File:Stage14_bf2cl.jpg]] [[File:Stage14_sem1c.jpg]]
 
[[File:Stage14_bf2cl.jpg]] [[File:Stage14_sem1c.jpg]]
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===Historic Images===
 +
Keith, A. (1902) [[Book - Human Embryology and Morphology|Human Embryology and Morphology]]. London: Edward Arnold. [[Book - Human Embryology and Morphology 20|Chapter 20. The Limbs]]
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<gallery>
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Keith1902 fig233.jpg|Fig. 233. Lateral view of a human embryo at the 28th day, showing the Limb Buds, Lateral Edges, and Primitive Segments.
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Keith1902 fig234.jpg|Fig. 234. Development of the Upper Limb.
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Keith1902 fig235.jpg|Fig. 235. Development of the Lower Limb.
 +
Keith1902 fig236.jpg|Fig. 236. The Corresponding Points {A, B, C, and D) in the Ilium and Scapula.
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Keith1902 fig237.jpg|Fig. 237. Section of the Arm Bud of a human embryo at the end of the 4th week.
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Keith1902 fig238.jpg|Fig. 238. Schematic Section showing the Origin and Arrangement of the Muscles and Nerves of the Limbs.
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Keith1902 fig239.jpg|Fig. 239. The Distribution of the Posterior Roots of the Spinal Nerves on the Flexor Aspect of the Arm.
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Keith1902 fig240.jpg|Fig. 240. Diagram to show the typical Manner in which the Posterior Nerve Roots are distributed in the Lower Limb.
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Keith1902 fig241.jpg|Fig. 241. Flexor Aspect of the Lower Limb showing the Sensory Distribution of the Segmental or Spinal nerves.
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Keith1902 fig242.jpg|Fig. 242. Diagram of the Pelvic Girdle of a Lizard.
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Keith1902 fig243.jpg|Fig. 243. The Pelvic Girdle of a Human Foetus at the 5th week.
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Keith1902 fig244.jpg|Fig. 244. The Shoulder Girdle of Ornithorynchus.
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Keith1902 fig245.jpg|Fig. 245. The Parts in the Shoulder Girdle of a human foetus which correspond with those of Ornithorynehus.
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Keith1902 fig246.jpg|Fig. 246. The Carpal Bones of a Tortoise.
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Keith1902 fig247.jpg|Fig. 247. The Os Trigenum and Bones of the Tarsus
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Keith1902 fig248.jpg|Fig. 248. The Foetal and Adult (in dotted outline) Forma of the Astralagus contrasted.
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Keith1902 fig249.jpg|Fig. 249. Latissimo-condyloideus Muscle.
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Keith1902 fig250.jpg|Fig. 250. The Morphology of the Short Muscles of the Digits.
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Keith1902 fig251.jpg|Fig. 251. Showing the Origin of the Ligamentum Teres and Reflected Bundle of the Capsular Ligament.
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Keith1902 fig252.jpg|Fig. 252. Showing the Origin of the Crucial Ligaments of the Knee.
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</gallery>
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==External Links==
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{{External Links}}
 +
* Embryo Images [https://syllabus.med.unc.edu/courseware/embryo_images/unit-mslimb/mslimb_htms/mslimb014.htm Limb Unit]
 +
* International J. Dev. Biology Vol 46 [http://www.ijdb.ehu.es/0207contents.htm Special Issue- Limb Development  2002]
 +
* Research Labs - [http://pages.unibas.ch/anatomie/zeller/seiten/seite1.html Rolf Zeller University of Basel Medical School]
  
  
{{Glossary}}
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{{2017ANAT2341 footer}}
{{Footer}}
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[[Category:Limb]]

Latest revision as of 09:20, 9 October 2018

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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).


2018 Lecture PDF


Recent Reviews  
2017

<pubmed>28283405</pubmed> <pubmed>28131856</pubmed>

2015

<pubmed>28283405</pubmed>

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. (2019). UNSW Embryology (19th ed.) Retrieved October 18, 2019, from https://embryology.med.unsw.edu.au
Factor Links: AMH | hCG | BMP | sonic hedgehog | bHLH | HOX | FGF | FOX | Hippo | LIM | Nanog | NGF | Nodal | Notch | PAX | retinoic acid | SIX | Slit2/Robo1 | SOX | TBX | TGF-beta | VEGF | WNT | 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<pubmed>22174793</pubmed>| 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. <pubmed>26212321</pubmed>
  2. <pubmed>20386744</pubmed>| PMC2851570 | PLoS Genet.
  3. <pubmed>17194222</pubmed>| PMC1713256 | PLoS Genet.
  4. <pubmed>17194222</pubmed>| PMC1713256

Online Textbooks

Search

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

External Links Notice - The dynamic nature of the internet may mean that some of these listed links may no longer function. If the link no longer works search the web with the link text or name. Links to any external commercial sites are provided for information purposes only and should never be considered an endorsement. UNSW Embryology is provided as an educational resource with no clinical information or commercial affiliation.


 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