MCB3023S
Limb development
Module 1. Introduction to development
Introduction to developmental biology
Development
- difficult to work on the brain or heart as they are essential whereas a limb is something you can manipulate without killing
the embryo
- inheritance from mother cell can influence the fate of the cell (maternal inheritance model)
- or can adopt a fate as a result of signaling molecules from neighboring cells (American model and signaling)
- how cells known when to stop diving: growth factor signals are important in controlling the rate of cell division and
morphogenic process
- a key question in biology is how to you build an embryo from a single fertilized cell
- multicellular organisms do not arrive fully formed in the world, a multicellular organism starts off as a single fertilized egg
(zygote) which divides mitotically to produce all the cells in the body
What is required
- cells need to multiply and differentiate into different types such as muesli, neurons and blood cells
- cells in an embryo contain the same set of genes
- a key question is how can the same set of genes produce so many different cell typexs in an embryo?
- the answer lies in understanding how different genes are activated in different cell types, much of research into cell
differentiation comes down to understanding how genes are turned on and off
- cells need to organize themselves, reproducibly into different tissues and structures (or organs), a process known as
morphogenesis
- communication between cells is critical for the formation of tissues and organs
- patterning of fields of cells by gradients of diffusing morphogens is a critical concept in development
Cell differentiation
Approaches to studying development
- there are a number of different ways to study developmental biology
- anatomical called descriptive embryology: describes the different stages of development in a species being studied
- comparative embryology compares development between different species
Approaches to studying development
1) Anatomical: describe the different stages of development, descriptive embryology
2) experimental: manipulation of embryos
3) genetic: alter gene function and see effects on development
1. Anatomical approaches
- describes the different stages of development and to align the same stages between different species
- stages of development have been clearly defined in vertebrate embryology for example the Carnegie stages of
development shows the first 60 days of the human embryo
- this is a standardized system of 23 stages based on the appearance of morphological features
- it provides a unified developmental chronology of vertebrate embryos, independent of the number of days of development
or the size of the embryo
- the Hamburger Hamilton stages are specific to chick development, and describes what developmental features appear after
a certain number of days after fertilization
- each stage will be accompanied by a complete anatomical description
- this allows us to compare stages of development between different species
,Hamburger Hamilton (HH) stages of chick development
- chick development is amenable to study development as they have very quick development and they embryo is very
accessible allowing for manipulation
- can use days after fertilization as an approximation of a developmental stage
- can study embryos and we stage the different stages of development
Comparative embryology
- reasoned that different vertebrate embryos all started from a similar basic
form and subsequently diverged into different looking organisms
- comparative compares the different embryos across different species,
looking for similarities and differences
- shows in early stages of development, the stages of development look
very similar (argument that all vertebrates go through a very similar process
of development)
- later found that the initial drawings showing the similarity was very exaggerated
- ray and fish, see same pharyngeal pouches as in the cat and possum
- even the less complex vertebrates still go through cell differentiation
- same processes but get variation because variation comes from different genetic mechanisms and new genes or gene
functions that gave new functions and the levels at which the master regulators are expressed are changes which influences
the morphological gradients
- for comparative: must compare different species but that are at the same stage
- the similarity between embryos at the tailbud stage is exaggerated to prove the point that human embryos showed
similarities to other vertebrate embryos, evidence for evolution and common descent
2. Experimental approaches
- includes doing microsurgery
- for example, ablating different regions of embryos and watching the effect on subsequent t development and transplanting
different tissues to different regions of the embryo
- most work done on amphibian and chick embryos which were easily accessible to manipulation
- another important technique is cell lineage tracing where founder cells were labelled with dyes and biologists observed in
which tissues the descendants of these cells ended up
- these cell lineages studies showed that some cells travel large distances during embryonic development
- for example, cells of the neural crest migrate from the dorsal part of the neural tube to different regions of the embryo
giving rise to diverse lineage’s including smooth muscle cells, melanocytes, cartilage in our faces, glia and neurons
- the vertebrate limb was one of the first organs to be studied, as it readily accessible to experimental manipulation
- importantly since successful limb development is not critical to survival of an embryo at early stages, the effects of these
manipulations can be followed at subsequent stages of development (unlike the heart which is essential so cannot be
manipulated)
3. Genetic approaches
- modern developmental biology has benefitted greatly from the development of techniques to identify genes and
manipulate their function
- these techniques include using reporter genes to trace in which cells a gene is expressed, to knocking a gene out and
observing the effect this has on development
- the phenotypes of mouse mutants where a specific gene has been knocked out has led to important advancements in our
understanding of the roles that the genes play during cell differentiation and the development of organs
Gene manipulation in mouse embryos
- can overexpress a particular gene of interest and see
how that affects development
- to do that, have a transgene contrast with a strong
promoter, take a fertilized cell and directly inject the
DNA into the fertilized cell and the contrast will
insert itself into a chromosome, the cell will davidite
and daughter cells will inherit the genes and can see
the phenotype after
- reporter assay where you take a promoter and a
gene regulatory sequence you think is important, link
it to a reporter such a B-gal to see if the gene is
important in driving gene expression
,- can do a gene knockout experiment: take a gene, completely knock it out by homologous recombination (very targeted),
use embryonic stem cells so are pluripotent, cells placed into mouse blastocyst
- 2 different approaches, one uses pro-nuclear injection and the DNA randomly integrates and is used for over expression
studies; gene knockout happens at a particular gene locus using homologous recombination and embryonic stem cells
Introduction to limb development
- limb development is a classical model for studying organogenesis and has been studied for many years
- the diversity of vertebrate limbs is extraordinary between and within organisms; consider the bat wing and left, the seal
flipper, the chicken wing and the human arm
- within an organism, forelimbs and hindlimbs are substantially different
- despite these differences, the bones of the vertebrate limb are easily recognizable across species
- the stylopod (the humerus or femur) is found adjacent to the body wall, the zeugopod (radius/ulna or tibia-fibula) is found
in the middle region followed by the distal autopod (carpals-fingers/tarsals-toes)
- biologists have identifies a common set of genes that regulate development of vertebrate limbs
- a challenge is to understand how variation in the function of these genes can lead to the morphological diversity of
vertebrate limbs, for example, compare mouse forelimb to bat forelimb, the bat forelimb to the
bat hindlimb
Variation in limb morphology
- comparison of both forelimb and hind limb of the mouse and bat, see an extreme difference
- shows that there is a remarkable diversity seen in limbs but there is also commonality
between these limbs being that they all contain the same elements
- the common elements are all determined by the same genetic pathways
Limb conservation and diversity
- all tetrapod limbs contain:
a) a proximal element, the stylopod: humerus (forelimb), femur (hindlimb)
b) an intermediate element, the zeugopod radius and ulna (forelimb), tibia and
fibula (hindlimb)
c) a distal element, the autopod: wrist and fingers (forelimb) and ankle and
toes (hind limb)
Three axes in limb development
- the vertebrate limb is a complex organ which has 3 dimensions:
1. Proximal-distal axis (shoulder to digits)
2. Anterior-posterior axis (thumb [anterior] to pinkie [posterior])
3. Dorsal-ventral axis (palm to back of hand)
Temporal stages of limb skeletal development
- in addition to patterning along these 3 dimensions, there is a temporal
sequence of stages in the development of the limb skeleton
- limb bud formation is initiated when the forelimb and hindlimb buds protrude
from the sides of the vertebrate embryo
- in the second stage, the limb buds grow and are patterned along the 3 axes
culminating in the establishment of cartilage templates that prefigure the bones
- in stage 3, the mesenchymal cells aggregate to form pre-chondrocyte
condensations
Formation of cartilage templates that prefigure bone
- the mesenchymal cells receive a signal and differentiate and
aggregate to form pre-chondrocyte condensations
- the chondrocyte cells stop dividing and differentiate into
hypertrophic chondrocytes which begin to secrete extracellular
matrix proteins which can be detected by Alcian blue staining
- the hypertrophic chondrocytes are the first indication of where
the humerus is going to be as opposed to the radius and ulna and
the digits
, Endochondral bone formation
- once the cartilage elements have formed, the
hypertrophic chondrocytes undergo apoptosis
- their cell death allows blood vessels to enter,
bringing in osteoblasts
- the osteoblasts invade the mineralized matric laid
down by the hyperchondrocytes and deposit bone
matric
- this bone matrix can be visualized by staining
embryos with Alizarin red
- while the osteoblasts replace the hypertrophic
chondrocytes at the center of the cartilage template,
the chondrocytes at the end continue to proliferate
and form the growth plates at the end of the bones
Cartilage and bone formation in bat wing and leg
- limb development is one of the best understood examples of organogenesis, as it was possible to manipulate limb fields in
developing embryos without causing lethality
- in the developing chick embryo, developmental biologists found that is was easy to access developing limbs and surgically
manipulate them
- this rich embryology laid the foundation for the last decade in which genes involved in the development of limbs have
been identified
- many of the genetic interactions that pattern the limb are also important for the development of other organs
- thus, the development of the limb has become a model system for understanding the
development of other organs in vertebrates
Forelimb vs hindlimb
- compares the forelimb to the hindlimb because the hindlimb has very short digits so might
see a difference in patterning
- in stage 14, not much blue staining for both fore and hindlimb
- the sequence in the hindlimb is slightly delayed compared to the forelimb
Cartilage and bond formation in bat wing
- see the retained interdigital webbing and long fingers in the embryo
- experiment of Alizarin red staining and Alcian blue staining: can conclude
that the sequence of events of the condensation of the digits happens at a
typical timing of other vertebrate embryos but the difference is that once the
cartilaginous templates are formed and the osteoblasts enter, we form
growth plates and those carry on diving for a long time resulting in very
long digits
Genetics of limb development
- our understanding of limb development is largely based on experimental evidence from chick and mouse embryos
- many of current models for limb development are experimentally based on:
1. classical embryological studies (i.e. transplant studies in embryos)
2. identification of the patterning molecules (cloning of genes)
3. expression patterns of genes (in situ hybridization and ICC)
4. functional analysis (i.e. deletion/overexpression of genes in transgenic mice and in chicken embryos)
- we will look at several stages of limb development, and the genes that regulate them, namely:
1. the induction/formation of the limb bud: FGF10
2. specification of forelimb and hindlimb: Tbx5, Tbx4 and Pitx1
3. generation of proximal-distal axis (FGF8l, Hox genes) and formation of AER
4. specification of anterior-posterior axis (and formation of ZPA, RA and Shh signaling, Hox genes
5. Division of autopod into 5 digit-interdigit regions by a turning-like network of BMP-Sox9-Wnt interactions
6. generation of dorsal-ventral axis: Wnt7a
7. development of skeletal elements
