These pages have been left in this location as a service to the numerous websites around the world which link to this content. The original authors are no longer at the University of Leeds, and the former Centre for Human Biology became the School of Biomedical Sciences which is now part of the Faculty of Biological Sciences.
Lecture 15 How many toes, and would sir
like them webbed?(OVERHEAD)
If the limb bud is too small from the start, as opposed to
growing less than it should, digits simply do not appear in the
foot. When digits are simply removed like this it is commoner
to lose postaxial digits. This is unexpected, because if the
ZPA organises from the region of digit V and we might guess that
anterior toes might be more vulnerable. However, there are plenty
of examples: (OVERHEAD) postaxial hemimelia, described
by Searle, has a postaxial mesodermal deficiency and loses posterior
Another set of mutations seem to have the opposite effect:
they increase the size of the limb bud and the number of digits.
Once again polydactyly may be pre or postaxial, but more commonly
preaxial. Preaxial polydactyly is often associated with an apparently
contradictory loss of more proximal preaxial elements. As an
example lets look at one of the best described mutants Carter's
luxate (lx, OVERHEAD). This is a semidominant gene i.e.
in the heterozygotes the hind feet only are mildly affected,
the homozygotes more so. In heterozygotes the limb may be normal,
or the hallux may have three phalanges (a common indication of
low grade polydactyly) or be joined by a pre-hallux. In the homozygote
there may be up to seven toes. The polydactyly is accompanied
in homozygotes by a progressive reduction of the preaxial side
of the limb skeleton: the tibia is first reduced then replaced
by a ligament, the fibula is thickened: in more extreme cases
the femur and even the pelvis are affected. (OVERHEAD).
In the most extreme individuals this is accompanied not by polydactyly
but by loss of preaxial digits down to four or three.
The limb buds here are reduced in size at 11 days, with a
small AER. At 12.5 days the AER is larger than normal preaxially
(OVERHEAD). A little later the tibial blastema is seen
to be reduced, with concomitant extra digital rays, or absent,
in which case preaxial digits are reduced.
Several similar mutations are known, all showing the same
features, an initially narrow bud with later preaxial excess.
Tibial reduction followed by digital excess can be explained
like this. The initial limb is small, probably due to delay or
absence of the anterior part of the AER. If the AER does not
recover from this initial setback then the tibia is reduced and
there are no extra preaxial digits - in fact there may be losses.
Frequently in these mutants, however, we see a recovery of the
preaxial part of the AER, which is present for the normal length
of time: but because it starts late it also finishes late. Mesoderm
perhaps normally destined for the tibia is generated at a time
when digits are being formed, and preaxial polydactyly ensues.
If extra digits are added to the row what will they be? How
does the limb bud control system cater for this sort of event?
In polydactylous limbs (OVERHEAD) it is quite clear that
we often have examples of mirror image duplications, just like
those produced by graftiing an additional anterior ZPA (OVERHEAD).
If we allow that simple preaxial polydactyly is a low grade of
mirror image duplication (as it certainly is in luxate and many
other cases) then we know of a couple of dozen separate genes
which insert an anterior ZPA. This seems rather a lot. Why should
all these genes, on different chromosomes and with different
effects all produce mirror image duplication?
Interestingly early limbed animals had more than five toes,
six, seven or eight (OVERHEAD). The tetrapod limb is often
referred to as the pentadactyl limb because it has five digits,
and we assumed that five was the primitive number. But is it?
We know that many species (horses, sheep, birds) have less, some
(whales, dolphins) have more. Are we biased because we have five,
or do most species have five? Certainly the retention of the
'primitive' five fingers is regarded as a prerequisite for tool
Now the five fingered limb doesn't exist in fish, because
fins are made rather differently, broad based and symmetrical:
it is first seen in early land dwelling tetrapods. The earliest
of these were found in Greenland inn 1929 by a Danish expedition.
They are Devonian, 390-340mya. On the death of their discoverer
Jarvik took over the study of the fossils in the 50s. Although
no specimens of the earliest, Ichthyostega and Actanthostega
had a full hand or foot Jarvik, of course, reconstructed them
with a 'primitive' pentadactyl limb.
All was well until 1984 when another early fossil Tulerpeton
was shown to have six digits. Then in 1990 Coates and Clack showed
that Acanthostega had seven digits and Ichtyosega
seven although the upper parts of the limbs coincided closely
with Jarvik's material. So the primitive condition was not five:
three intact feet are known, non with five toes, one six, one
seven, one eight.
Both Acanthostrega and Ichthyostega have two
groups of digits, a main hand and a small group of digits in
the thumb region. i.e. they have mirror image polydactyly. Could
it be that early tetrapods had another ZPA anteriorly as well
acting variably to produce some more digits? And could it be
that a single developmental step that cuts out this second ZPA
stabilises the pattern at the five digits that develop from it,
instead of the 6,7, or 8 that develop from the pair? This scheme
is attractive because the change from 5 to 6 or 6+ is not just
quantitative, it is qualitative as well. Five digits may have
chosen itself as a maximum - perhaps because a signal from the
ZPA would only reach that far: and perhaps the next ZPA along
was for another of those seven pairs of limbs/fins we mentioned.
Yet some contrary animals do normally have six fingers: but
if so the sixth is always developed as a different entity from
the bunch of five, not as a continuation. In six toed frogs the
extra digit arises as an extension of the normally non-branching
radius or tibia. The panda's thumb is really an extension of
a wrist bone: so is that of the mole.
A sense of proportion
Most of the evolutionary change that we see works on the five
toed limb by changing its pattern in various ways. The most obvious
of these is by obliterating or adding digits by various means,
but we have seen that proportions and growth can also change
for power or speed. Lets look at mutations covering these as
The mouse mutant brachypod has a normal axial skeleton and
short legs. The interest in this particular mutation is that
the internal proportions of the legs are changed (OVERHEAD).
The ulna and tibia are mildly shortened, the femur and humerus
rather more and the manus and pes more still. Obviously this
is not a simple proximo-distal gradient.
The brachypod limb bud is of normal size until the digital
contours and blastemal condensations arise at 13 days. At this
point the digits are thin, and the M/P joint too proximal.
This misallocation of material may give us a clue as to how
non-proportional changes in limbs might be brought about. What
is brachypod doing? We are not sure, but interestingly one of
the earliest effects of the gene is known to be on cell adhesion
. If this is the cause, presumably by an effect on blastemal
shape and size, this might be the factor which determines the
relative length of the various segments of our limbs - an important
difference between, say, man and gorilla or dog and badger.
This is more common: many mutants are known which affect all
parts of the limb and result in short limbs attached to a relatively
normal body. We have already talked about achodroplasia, as in
the circus dwarf, which actually affects all cartilage replacement
In nature we might expect this not to be very useful: but
we should remember that evolutionary potential is exploited by
man. In the eighteenth century the Ancon sheep (OVERHEAD)
was developed. These have short legs and are less able to jump
over fences, so became very popular. Dogs, such as basset hounds
and dachshunds are selected for short legs, known to be due to
a single gene. The rest of the body is unaffected in these animals.
Changes in proportion between different species was one of
the problems tackled by D'Arcy Thompson (OVERHEAD) in
his classic On Growth and Form. He saw that it
was possible to apply a Cartesian transformation to bone outlines
Cartesian transformation means basically that you draw a grid
over a bone outline then deform it in a particular way to produce
another bone outline. The cannon bones of giraffe, sheep and
ox are thus related to each other by simple proportion: width
changes but length doesn't.
The same technique will not work on the whole limb: we need to apply a different degree of distortion or stretch to each segment, proportionally more distally than proximally to mimic the change. This could, of course be achieved by varying
the conditions for outgrowth in a non linear way: if this
happens we are looking at a more sophisticated change than that
seen in Ancorn sheep or beagles.
We have discussed many of the processes which shape the limb
bud: there is however one more, not yet mentioned, which is important
not only in the limbs but elsewhere in the body, both as a morphological
shape determinant and in a wider context.
Almost as important as growth in the developing embryo is
cell death or apoptosis (OVERHEAD). All cells, of course,
die sooner or later. But which is it to be? Once germ cells have
been separated from somatic cells the latter rapidly split into
one or other of two groups. Some cells, nerve cells, muscle fibres,
liver parenchyma, endothelium, fibrocytes slow down the process
of division almost to the point of stopping. In most cases, but
not all, these cells can be reactivated if the population is
depleted by accident or surgery. Others, like epithelial cells
in the gut, or blood forming cells have a short life in fully
differentiated form, and so are continually replaced from a population
of stem cells. Stem cells are potentially immortal: those from
the haemopoitic system of mice can be labelled and grafted into
other mice: this experiment was discontinued, with the cells
still healthy after five times the normal mouse lifespan. Stem
cells, and incidentally cancer cells show no sign of ageing or
The cell deaths which interest us happen during embryological
and fetal development. The idea that cell death might be planned
arose in the first half of the present century, because groups
of dying cells or areas of debris were observed at specific sites
within the embryo. In the last thirty years cell death has been
identified in the implanting mouse embryo, the limb, the palate,
the nervous system, the heart and the eye.
Cell death certainly varies between classes of vertebrates,
but also between species: the control of programmed cell death
may therefore be used as a developmental strategy, to change
the shape or extent of a morphological feature.
Glucksmann (OVERHEAD) considered cell death to be of three kinds.
morphogenetic: involved in the shaping of organs e.g. neural tube closure, palatal fusion, limb shaping.
hisogenetic: associated with the differentiation of tissues e.g. the degeneration of Wolffian and Mullerian ducts in the development of the reproductive system
phylogenetic: regression of structures in higher vertebrates
which have a definite function in lower vertebrates, such as
the ductus arteriosus, pronephros and mesonephros.
These categories clearly overlap, and talk of higher is hardly
PC. Truman gave a rather more sensible assessment, saying that
cell death was necessary to match the size of a population of
cells (such as nerves) to the tissues with which they must interact,
that it may be necessary as a modifier of phenotype and that
it may act as a waste disposal system for excess cells.
Saunders introduced other questions. Is cell death assassination
or suicide? Since some areas of cell death are highly predictable
is it essential for those processes.
In any case we can distinguish this cell death from necrosis,
which happens when cells are exposed to non physiological conditions:
here nuclei swell and appear to burst. In apoptosis the cells
shrink or condense and may separate from their neighbours before
being phagocytosed or fragmenting. Also necrosis often leads
to an area of tissue damage: apoptosis never does.
The obvious place to study cell death is nematodes. Remember
that we know all about cell lineages in these animals and that
111 of the cells in the male die. In fact sexual dimorphism is
brought about by cell death. Nematodes are a bit kinky in that
there are two sexes male and hermaphrodite: at about 470 hours
of development in males hermaphrodite specific neurones die,
and in hermaphrodites male specific neurones do. Two mutations
are known which affect cell death: in one phagocytosis is blocked,
but cells die on time anyway. In the other cell death is inhibited
and the escaped cells go on to differentiate into recognisable
In leeches development includes a process of formation of
parallel columns of cells, 32 of which will make up a leech segment.
Cell death occurs in columns 33 and above, at the rear of the
embryo. If you swap the cells around, putting anterior ones to
the back columns 33 et seq still die - so its not mitotic order
or age which determines their fate.
The best examples of cell death in vertebrates come, perhaps
predictably, from our test system, the limb. Limb bud shaping
by differential cell death occurs in both ectoderm and mesoderm
of developing limb buds in most amniotes, although it seems to
be absent in amphibia. Mesodermal death is more extensive and
differs between fore and hindlimb, between species and between
normal and mutant individuals.
Four loci of cell death have been regularly seen:(OVERHEAD)
the anterior and posterior necrotic zones (ANZ,PNZ) which shape
the limb, the central opaque patch and later the interdigital
necrotic zones (INZ). all these are present in the chick and
are easily mapped by vital staining with Nile blue or neutral
red, which are picked up by macrophages which are inevitably
present in areas with significant cell death.
In the chick limb the ANZ is seen as a proximal wave of death
followed by a distal one, whilst the posterior necrotic zone
seems to be a single entity. The opaque patch perhaps has something
to do with separation of the radius and ulna.
The extent and presence of these zones of cell death varies
between species of birds and is characteristically absent in
rats and mice, which have more digits, and where there is only
a small necrotic area adjacent to digit 1.. The ANZ and PNZ may
therefore limit the amount of distal mesenchyme which is available
for digit formation in these species with a reduced set. However
Saunders found that if the ANZ is suppressed experimentally in
the chick no extra digits are formed. Against this the small
necrotic area found in mouse and rat is absent in mole, which
has a rather extraordinary prehallux or sixth digit anteriorly.
In mutants we find that talpid, which has up to eight digits
has no necrotic zones, whilst wingless has a very large ANZ.
The opaque patch is situated at a point which corresponds
to the proximal end of the radius and ulna: we already know that
chondrogenesis depends on increased cell adhesion: dead cells
don't stick together - they round up. Might this be something
to do with the switch from one skeletal element to two?
A little later in development further areas of cell death
(OVERHEAD) appear in the footplate in what will be the
interdigital areas. These follow a well defined timetable. They
have been found in all amniotes studied including man, rat, mouse,
birds turtles. The INZs are suppressed in web footed birds, and
in a number of mutants and teratological conditions where feet
The idea of cell death as a morphological mechanism is unusual:
we usually think of specific production of a protein or a peptide
as an indicator of differentiation, not death. So how does a
cell prepare for its own death? Scattered cells seem to be destined
for death, and become vacuolated, condense their chromatin and
round up. Adjacent cells phagocytose these victims and in fact
convert to macrophages, containing the remains of up to 10-12
of their neighbours.
The dying cells contain an active Golgi apparatus which produces additional lysosomal enzymes. So perhaps our ideas of differentiation being the result of novel or amplified gene products are not far off the mark. Since only some cells in a field die it seems likely that they are pre-programmed in some way. Do they form a pattern? Do they have positional information? Have they undergone a fixed number of mitotic cell divisions and then died as some cells do in culture?
This page is maintained by Steve Paxton