Limb Development and Evo-Devo
Lecture 1
Development: how does a fertilised cell give rise to an adult body
- How does a single cell give rise to hundreds of different types of cells – yet contain
the same genetic information
o Genes can be activated and inactivated at different times
§ Temporal and spatial expression can be different
- How do two daughter cells adopt different fates
o Unequal inheritance (European Model)
§ Inherit different transcription factors
o Chemical gradients
o Epigenetic expression: environmental influence on development and signals
received by cells (American Model)
§ Impacted by different growth factors present in the environment
Morphogenesis: how are organs formed; eg. How is the limb or brain formed
Cell Differentiation
- Same genes involved in patterning multiple organs
- Start life as a zygote à blastula (inner cell mass = embryonic stem cells) à inner
cell mass can adopt three fates in the gastrula (ectoderm = outer layer, mesoderm
= middle layer, endoderm = internal layer)
Approaches to studying development
- Anatomical: describes the different stages of development in species being studied
§ Allow comparison between different species at equivalent stages
o Descriptive embryology
§ Eg. Carnegie Stages of human development
• Stages are defined by key appearances and changes in
appearance or development
§ Eg. Hamburger Hamilton Stages of chick development
o Comparative embryology
§ Need to compare across the same stage of development
• Use landmark appearances (eg. Appearance of the eye)
§ Controversy: embryos of different vertebrates have a similar structure at
very early stages of development (after gastrulation) as per Karl von Baer
(1828) VS Richardson saw vast differences (1997)
- Experimental: manipulation of embryos
o Can involve microsurgery
§ Ablation of different embryonic regions and watching the effect on
development
§ Tissue transplant
o Need accessible embryos
§ Chick embryos
§ Amphibian embryos
o Cell lineage training
§ Founder cells are labelled with dyes
§ Follow where these cell lineages end up
§ Some cells can migrate large distances
,- Genetic: alter gene function and see effects on development
§ Use of reporter genes, gene knockout
o Gene addition (over-expression)
§ Introduce transgeneic constructs into fertilised egg
§ Construct will randomly integrate into nucleus and chromosomes
§ Can have transgenic and wild type mice
o Gene targeting (knockout)
§ Edit embryonic stem cells
§ Reintroduce into blastocyst
§ Introduce blastocyst into mouse
Introduction to Limb Development
- Classical model for studying organogenesis
- Humerus, radius, ulna, autopod (hand) à seen across vertebrates
- Same limb patterning but different appearances
o Result of same genes being activated
§ Number and coding sequences of genes is largely the same in terms of
limb patterning
o Variation in appearance is a result of timing of gene expression (different
enhancer regions, regulatory regions differ)
Limb Conservation and Diversity
- All tetrapod limbs contain:
o Stylopod (proximal element)
§ Humerus (forelimb)
§ Femur (hindlimb)
o Zeugopod (intermediate element)
§ Radius and ulna (forelimb)
§ Tibia and fibula (hindlimb)
o Autopod (distal element)
§ Wrist and fingers (forelimb)
§ Ankle and toes (hindlimb)
Three axes in limb development
- Proximal- distal (shoulder to fingers/digits)
- Posterior- anterior (pinky to thumb)
- Dorsal-ventral (back of hand vs palm)
Temporal stages of limb skeletal development
Limb bud formation à limb bud growth à cartilage condensations
o Limb formation is initiated when fore/hindlimb buds protrude from the sides of
the embryo
o Limb buds grow and are patterned along the three axes to lage establish
cartilage templates that prefigure bones
o Mesenchymal cells aggregate to form prechondrocyte condensations
§ Joint formation begins
o Chondrocyte cells stop dividing and differentiate into hypertrophic chondrocytes
(secrete extracellular matrix proteins to be detected by Alcian blue staining)
o Upon cartilage element formation, hypertrophic chondrocytes undergo apotosis
o Hypertrophic chondrocyte death allows for blood vessels to enter and bring in
osteoblasts
, o Osteoblasts invade mineralized matrix laid down by hyperchondrocytes and
deposit bone matrix
o Bone matrix can be visualised with Alizarin red
o Osteoblasts replace hypertrophic chondrocytes at the centre of the cartilage
template but chondrocytes at the end continue to proliferate and form growth
plates at the end of bones
* use alcian blue to stain cartilage
* use alizarin red to stain bone
Current models for limb development are experimentally based on:
- Classical embryological studies (transplant studies in embryos)
- Identification of patterning molecules and cloning of genes
- Expression patterns of genes
- Functional analysis (deletion/overexpression)
Lecture 1 Questions
1) How do you think cells in a uniform field (for eg, at the early stages of limb bud
outgrowth) can be instructed to adopt different fates?
2) The photograph on the right, shows Alcian blue stained images of the developing
forelimb and hindlimb in different stages (CS14-CS16L) of bat embryo development.
i) What do alcian blue and alizarin red stain?
ii) Why is there is minimal Alcian blue staining in the CS14 embryo ?
iii) What can you say about the sequence of stylopod, zeugopod and autopod development from
this data?
iv) In the adult bat wing, digits are twice as long as the zeugopod elements. What can you
conclude about the development of digits that form the bat hand-wing from this data?
Lecture 2
Which genes pattern forelimbs vs hindlimbs
Genetics of limb development
- Largely based on chick and mouse embryos and experimental evidence
- Classical embryological studies (transplant studies)
- Identification of the patterning molecules (cloning of genes à advent of molecular
genetics)
- Expression patterns of genes (in situ hybridisation and ICC)
- Functional analysis (deletion/overexpression of genes in transgenic mice and in
chicken embryos)
o Overexpression via injection into fertilised mouse oocyte
o Knockout via embryonic stem (ES) cells
Manipulating gene function in a mouse
1. Gain of function; ectopic expression of genes
a. Pronuclear injection into male pronucleus of a fertilised mouse egg
b. Eg. Induce gene expression in cells /tissues it is not normally expressed in
i. Need limb specific enhancer/promotor and gene of interest to create
a transgenic construct
, 1. Does not matter where it integrates into the mouse genome
(occurs via random insertion event = high efficiency)
2. Inject construct into male pronucleus of fertilised mouse
oocyte
3. Transplant into pseudopregnant mouse
2. Loss of function; knock out mutations of genes in transgenic mice
a. Gene locus is interrupted in embryonic stem cells à stem cells then injected
into host blastocyst
b. Manipulation of embryonic stem (ES) cells
c. Requires homologous recombination at the site of the gene of interest (low
efficiency/rare event)
i. Select for successful integration with antibiotic neomycin and antiviral
ganciclovir
3. Conditional loss of function in transgenic mice
a. ES cells: Crelox system
4. Loss of function in transgenic mice via CRISPR in ES cells
1. Pronuclear Injection into a fertilized oocyte
- Require a specific promotor upstream of a transgene that can be:
o Strong viral or
§ Transgene will be induced in all cells
o Tissue specific or
§ Transgene will be induced only in specific tissue
o Inducible
§ Requires a system in which expression can be induced
§ Eg. Using nuclear receptors (eg. ER)
o Pronuclear injection of the transgene into the male pronucleus of a fertilised oocyte
(totipotent cell à all cells in a future mouse are derived from this oocyte)
o Transgene randomly integrates into the host genome at a single site
o Selected oocytes are then implanted into the oviducts of foster females
(pseudopregnant mice) à the injected oocyte will develop into a mouse pup
o Generate transgenic offspring
- Every cell (including germ cells) will have the integration in the same position if
successful: all cells are derived from the injected oocyte + will contain the foreign
DNA randomly inserted at the same position
- No selectable markers
- Highly efficient
*technique is useful for Cre-recombinase expression under a tissue specific promotor
*because the foreign DNA is randomly inserted in genome à need its own promotor to
induce overexpression of a particular protein or direct the expression to a particular tissue
*need to do lots of insertion experiments
- each oocyte will have the DNA integrated at a di;erent site by chance
Gene Targeting in Embryonic Stem Cells
- Embryonic stem cells = single cell that can
o Be maintained in culture
o Replicate itself or
o Di;erentiate into many cell types
Lecture 1
Development: how does a fertilised cell give rise to an adult body
- How does a single cell give rise to hundreds of different types of cells – yet contain
the same genetic information
o Genes can be activated and inactivated at different times
§ Temporal and spatial expression can be different
- How do two daughter cells adopt different fates
o Unequal inheritance (European Model)
§ Inherit different transcription factors
o Chemical gradients
o Epigenetic expression: environmental influence on development and signals
received by cells (American Model)
§ Impacted by different growth factors present in the environment
Morphogenesis: how are organs formed; eg. How is the limb or brain formed
Cell Differentiation
- Same genes involved in patterning multiple organs
- Start life as a zygote à blastula (inner cell mass = embryonic stem cells) à inner
cell mass can adopt three fates in the gastrula (ectoderm = outer layer, mesoderm
= middle layer, endoderm = internal layer)
Approaches to studying development
- Anatomical: describes the different stages of development in species being studied
§ Allow comparison between different species at equivalent stages
o Descriptive embryology
§ Eg. Carnegie Stages of human development
• Stages are defined by key appearances and changes in
appearance or development
§ Eg. Hamburger Hamilton Stages of chick development
o Comparative embryology
§ Need to compare across the same stage of development
• Use landmark appearances (eg. Appearance of the eye)
§ Controversy: embryos of different vertebrates have a similar structure at
very early stages of development (after gastrulation) as per Karl von Baer
(1828) VS Richardson saw vast differences (1997)
- Experimental: manipulation of embryos
o Can involve microsurgery
§ Ablation of different embryonic regions and watching the effect on
development
§ Tissue transplant
o Need accessible embryos
§ Chick embryos
§ Amphibian embryos
o Cell lineage training
§ Founder cells are labelled with dyes
§ Follow where these cell lineages end up
§ Some cells can migrate large distances
,- Genetic: alter gene function and see effects on development
§ Use of reporter genes, gene knockout
o Gene addition (over-expression)
§ Introduce transgeneic constructs into fertilised egg
§ Construct will randomly integrate into nucleus and chromosomes
§ Can have transgenic and wild type mice
o Gene targeting (knockout)
§ Edit embryonic stem cells
§ Reintroduce into blastocyst
§ Introduce blastocyst into mouse
Introduction to Limb Development
- Classical model for studying organogenesis
- Humerus, radius, ulna, autopod (hand) à seen across vertebrates
- Same limb patterning but different appearances
o Result of same genes being activated
§ Number and coding sequences of genes is largely the same in terms of
limb patterning
o Variation in appearance is a result of timing of gene expression (different
enhancer regions, regulatory regions differ)
Limb Conservation and Diversity
- All tetrapod limbs contain:
o Stylopod (proximal element)
§ Humerus (forelimb)
§ Femur (hindlimb)
o Zeugopod (intermediate element)
§ Radius and ulna (forelimb)
§ Tibia and fibula (hindlimb)
o Autopod (distal element)
§ Wrist and fingers (forelimb)
§ Ankle and toes (hindlimb)
Three axes in limb development
- Proximal- distal (shoulder to fingers/digits)
- Posterior- anterior (pinky to thumb)
- Dorsal-ventral (back of hand vs palm)
Temporal stages of limb skeletal development
Limb bud formation à limb bud growth à cartilage condensations
o Limb formation is initiated when fore/hindlimb buds protrude from the sides of
the embryo
o Limb buds grow and are patterned along the three axes to lage establish
cartilage templates that prefigure bones
o Mesenchymal cells aggregate to form prechondrocyte condensations
§ Joint formation begins
o Chondrocyte cells stop dividing and differentiate into hypertrophic chondrocytes
(secrete extracellular matrix proteins to be detected by Alcian blue staining)
o Upon cartilage element formation, hypertrophic chondrocytes undergo apotosis
o Hypertrophic chondrocyte death allows for blood vessels to enter and bring in
osteoblasts
, o Osteoblasts invade mineralized matrix laid down by hyperchondrocytes and
deposit bone matrix
o Bone matrix can be visualised with Alizarin red
o Osteoblasts replace hypertrophic chondrocytes at the centre of the cartilage
template but chondrocytes at the end continue to proliferate and form growth
plates at the end of bones
* use alcian blue to stain cartilage
* use alizarin red to stain bone
Current models for limb development are experimentally based on:
- Classical embryological studies (transplant studies in embryos)
- Identification of patterning molecules and cloning of genes
- Expression patterns of genes
- Functional analysis (deletion/overexpression)
Lecture 1 Questions
1) How do you think cells in a uniform field (for eg, at the early stages of limb bud
outgrowth) can be instructed to adopt different fates?
2) The photograph on the right, shows Alcian blue stained images of the developing
forelimb and hindlimb in different stages (CS14-CS16L) of bat embryo development.
i) What do alcian blue and alizarin red stain?
ii) Why is there is minimal Alcian blue staining in the CS14 embryo ?
iii) What can you say about the sequence of stylopod, zeugopod and autopod development from
this data?
iv) In the adult bat wing, digits are twice as long as the zeugopod elements. What can you
conclude about the development of digits that form the bat hand-wing from this data?
Lecture 2
Which genes pattern forelimbs vs hindlimbs
Genetics of limb development
- Largely based on chick and mouse embryos and experimental evidence
- Classical embryological studies (transplant studies)
- Identification of the patterning molecules (cloning of genes à advent of molecular
genetics)
- Expression patterns of genes (in situ hybridisation and ICC)
- Functional analysis (deletion/overexpression of genes in transgenic mice and in
chicken embryos)
o Overexpression via injection into fertilised mouse oocyte
o Knockout via embryonic stem (ES) cells
Manipulating gene function in a mouse
1. Gain of function; ectopic expression of genes
a. Pronuclear injection into male pronucleus of a fertilised mouse egg
b. Eg. Induce gene expression in cells /tissues it is not normally expressed in
i. Need limb specific enhancer/promotor and gene of interest to create
a transgenic construct
, 1. Does not matter where it integrates into the mouse genome
(occurs via random insertion event = high efficiency)
2. Inject construct into male pronucleus of fertilised mouse
oocyte
3. Transplant into pseudopregnant mouse
2. Loss of function; knock out mutations of genes in transgenic mice
a. Gene locus is interrupted in embryonic stem cells à stem cells then injected
into host blastocyst
b. Manipulation of embryonic stem (ES) cells
c. Requires homologous recombination at the site of the gene of interest (low
efficiency/rare event)
i. Select for successful integration with antibiotic neomycin and antiviral
ganciclovir
3. Conditional loss of function in transgenic mice
a. ES cells: Crelox system
4. Loss of function in transgenic mice via CRISPR in ES cells
1. Pronuclear Injection into a fertilized oocyte
- Require a specific promotor upstream of a transgene that can be:
o Strong viral or
§ Transgene will be induced in all cells
o Tissue specific or
§ Transgene will be induced only in specific tissue
o Inducible
§ Requires a system in which expression can be induced
§ Eg. Using nuclear receptors (eg. ER)
o Pronuclear injection of the transgene into the male pronucleus of a fertilised oocyte
(totipotent cell à all cells in a future mouse are derived from this oocyte)
o Transgene randomly integrates into the host genome at a single site
o Selected oocytes are then implanted into the oviducts of foster females
(pseudopregnant mice) à the injected oocyte will develop into a mouse pup
o Generate transgenic offspring
- Every cell (including germ cells) will have the integration in the same position if
successful: all cells are derived from the injected oocyte + will contain the foreign
DNA randomly inserted at the same position
- No selectable markers
- Highly efficient
*technique is useful for Cre-recombinase expression under a tissue specific promotor
*because the foreign DNA is randomly inserted in genome à need its own promotor to
induce overexpression of a particular protein or direct the expression to a particular tissue
*need to do lots of insertion experiments
- each oocyte will have the DNA integrated at a di;erent site by chance
Gene Targeting in Embryonic Stem Cells
- Embryonic stem cells = single cell that can
o Be maintained in culture
o Replicate itself or
o Di;erentiate into many cell types
