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The Problem Of development: embryogenesis

1.1 The problem of development
All sexual organisms begin as a single cell-the feltilized egg or
zygote-which, by growth and cell division, ultimately gives rise to the
whole mature organism. Because these cell division are all mitotic, every
cell in the mature organism should have the same genetic constitution as
the zygote. Despite this, cells differentiate into different type of cells,
tissues, and organs ( see the general reading section at the end of the
chapter). Not only do cells develop along different pathways, they do so
only in particular places in the organism. The different cell types,
tissues, and organs bear characteristic spatial relationships to each other.
This there-dimensional form is specified in some way by the genes,
since there are genes for leaf shape, for example, and the form of the
flower is used by taxonomists as the most reliable index of the genetic
differences between species.
The problem of development and differentiation is, threrefore, how
is gene expression modified or changed to produce different cell, tissue,
and organ types? How are genes expressed selectively, not only at
different times during development (i.e. sequentially activated or
repressed) but also in different places within the organism? And how do
genes which ultimately regulatethe synthesis of proteins and which as
enzymes determine only the rates of process translated into changes in
shape? Or, to put this another way : how is the scalar information of the
DNA transformed into the vectorial processes involved in the
construction of a three- dimensional multicellular organism? The
implication is that we need to be able to explain directions and planes of
growth in terms of the rates of the underlying metabolic processes.
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1.2 Changes in gene expression
1.2.1 Genes are potentially active
Selective gene activity could be because genes are selectively
deactivated or lost during development so that the developmental
potential of different cells becomes progressively less in different ways.
Nuclear transfer experiments in animals (Amphibia) suggest that nuclei,
even from quite highly differentiated cells sech as those of the gut, can
sometimes produce a whole new organism if they are transplanted into
an egg whose original nucleus has been removed, showing that genes are
inactivated rather than lost or irrevocably damaged. In plants, this is seen
much more easily because cells from differentiated tissues, such as leaf
mesophyll, epidermis, secondary phloem (admittedly rather
undifferentiated pholoem), pith, cambium, petals, ovules, and nucleus,
have all been made to regenerate whole plants in culture. The plant cell,
at least while it still has a nucleus (which is lost in differentiation of
some xylem and phloem cells), is therefore totipent, i.e. capable of
producing all other cell types and so re-creating the whole organism
under the right codition. During development, the genes must therefore
be activated or repressed selectively (but not lost), so that as the
organism develops only some genes are active at any particular time and
in any particular time and in any particular part of the organism.
1.2.2 Genes are switched on at specific times and places and show
scalar and vectorial expression
Molecular techniques have shown that genes for processes
occurring during fruit ripening are switched on only during ripening.
The transfer of genes from one species of plant to another by modern
techniques has shown that genes coding for seed proteins are still
expressed in the genetically engineered transgenic plants, so there must
be controls of gene activity during development that are common to
different species (see ch.9). But the control of development is
presumably more complex than the switching on or off single genes
from time to time. Genes controlling the process of cell division are
inactive in won-dividing cells. Here, there seems to be a whole group of
genes whose activity is coordinately switched on and off together. Even
more complex examples are where genes controlling shape are switched
on or off as organs are initiated and then begin to mature. Another
problem in the scalar control of genes expression: genes are not
necessarily just switched on or off, but may be switched on to a very
small extent and then up-regulated, or if they are being actively
expressed they may become down-regulated. Evidence is now becoming
available (Ch.9) of the sort controls at the

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Molecular level that may achieve this. But this pushes the problem one
stage further back- how are the regulator genes themselves regulated and
in a coordinated manner? A major problem of development is to explain
how genes are expressed vectorially to specify the shape of an organ or
organism. At the molecular level, we can begin to see how activities of
individual genes or groups ogf genes may be regulated. At the other
extreme, we can see what happens to the whole organism as it develops.
In between, we need to see what is happening at the cellular level, which
can explain the organization of the tissues, the organs, and the whole
palnt, and which in turn has to be explained by the changes ingene
expression. It is this in-between area that this book is about.

1.3 Embrogenesis the problem exemplified

Many of the problem of development are highlighted by the processes
occurring during embryogenesis. The development of the embryo of
capsella (Shepherds purse) is often taken as typical (fig, 1.1)
1.3.1 polarity of zygote
The entire embryo arises by growth and cell divisionfrm the
fertilized egg, which is called the zygote. In seed plants, the egg, and
consequently the zygote, is in a polar environment, i.e. the environment
of the two ends of the egg differ, one end being against the embryo sac
wall next to the micropyle, where the pollen tube enters, and the other
end projecting into the fluid-filled embryo sac, containing high
concentration of growth substances and other metabolites.
The zygote itself displays polarity, i.e. it has an axis with the ends
being different. It has a large vacoele at the micropylar end and dense
cytoplasm, with the nucleus, at the embryo sac end. This is an
aaccentuation of the polarity that already existed in the unfertilized egg,
so it is not the result of the position at which the feltilizition nucleus
enters-form the pollen tube which is what would be expected by
anology with animal egg. Whether the polarity of the egg results from
the polarity of its environment in the embryo sac is not known. The
polarity of the egg is translated into the structure of thw embryo because
the first division of the zygote is normally transverse, cutting off a
densely cytoplasmic terminal cell, that gives rise to the embryo proper,
and a highly vacuolated basal cell that gives rise to the suspensor. In the
embryos of some species, the first division is longitudinal; whether this
correspond to a lateral polarization of the zygote is not known, but it
seems doubtful.
Evidence that the polarityof the zygote and the subsequent embryo
is the result of the physical contraints of its immediate environment
come

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 Polarity can arise within the embryo itself in free-living embryos,
as in the seaweed focus, as a result of many different external stimuli
(see Ch. 6, table 6.1) Embryos can, however, from and develop in an
apparently apolar enpironment, as shown by the development of
embryos from zygote of the alga homosira in shake culture and from
continually agitated angiosperm cell suspension cultures. In angiosperm
cell suspensions, where the cells are presumably in an apolar
environment, the cells nevertheless usually, or always, become polarized
by the development of along axis. Wheather this depens on being
triggered by some external stimulus (however fleeting) or wheather it
develops spontaneously within the cells, and whether te development of
polarity is esentitial for embryo development or can arise as a
consequence of it, is not known. Morphological and physiological
polarity is characteristic of all plants and can be studied at the cellular
level. Only when cells form unorganized callus is polarity absent
1.3.2 formation of embryo and suspensor- a division of labour
The first cell division in the embryo gives the terminal cell, which
develops into the proembro, or embryo proper, and the basal cell, which
gives rise to the suspensor. (the division of labour is not always as clear
cut as this, e.g. in capsella some derivatives of the basal cell give rise to
part of the embryo; see fig. 1.1). The elongating suspenshor pushes the
developing embryo into the the embryo sac and the endosperm, which is
a metabolite source for the embryo. The polarity of the embryos
environment is therefore maintained because one end of the embryo is
free in the embryo sac or endosperm and the other is attached to the
susupensor. Is the role of the suspensor to maintain the embryo in a
polarized environment, or does it have other functions as well?
It seems an attractive idea that the polarity of the embryo might be
partly the result of the opposing sources of nutrients that presumably
result from a supply from the embryo sac at one end and from the young
embryo are covered by a cuticle, suggesting that the surface in contact
with the embryo sac and endosperm may not (in the early stages,
anyway) be an absorptive surface, but that the preferred transport
channels to the embryo are via the suspensor. Also, the basal cell of the
suspensor soon develops into a transfer cell (see Ch. 8, section 8.3.11),
suggesting that there is an important flux of material into and through it.
In phaseolus, the suspensor is relatively large and can be dissected out,
with its young embryo attached, in an apparently undamaged state. Up to
the heart stage of development the suspensor acts as a supplier of
substances to the embryo, but once the cotyledons have formed they take
over as the major absorptive organs for the embryo (table 1,1). Before
the.

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Major axis and main of the embryo have been mapped out, the embryo
probably depend mostly on the suspensor for its supply of nutrients
The Suspensor may not be esessential, for suspensor-less adventive
embryos in culture grow successfully as they also do in citrus, which
shows polyembryony and in which only the zygotic embryo has a
suspensor and the accessory asexually derived embryo development,
exceptperhapsto supply the young embryo with growth substances and
to push it into the nutrient-rich medium in the embryo sac. It may
contribute gibberellin to the developing embryo up to the heart stage,
since lack of a suspensor in young cultured embryos can be compensated
for by a supply of gibberellin in the culture medium (table 1.2). Transfer
of materials from the suspensor to the embryo does not only occour
when theyare in organic contact-placing them next to each other in
culture is also effective. Continuity of the symplast of the suspensor and
embryo via the plasmodesmata linking them may normally facilitate
development but there is no symplasmic contact of the basal cell with
the embryo sac. The base of the basal cell may have no plasmodesmata
but develops instead as a transfer cell. The lack of plasmodesmata here
also implies that the embryo and its suspensor are an isolated symplast
entity within

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The parent plant. Whetherthis isolation is necessary for embryo
development is a point that is still debated. Nevertheles, the embryo and
suspensor in the normal plant are an example of differentiation, from
adjacent sister cells of the same parent cell, of two groups of cellsthat
complement each other in their growth and fuctions, even though it may
be mostly a one-side complementation with the embryo contributing
little r nothing to the suspensor.
1.3.3 rates and planes of cell division
The cell in the suspensor do not increase in number as in the
proembly and are limited in number, unlike the proembryo in which cell
number is potentially indefinite as it grows into the adult plant. The rate
and duration of cell division therefore differ in the proembryo and
suspensor. In a mutant of arabidopsisin which the young globular
embryo ceases growing and aborts, the growth potential of the suspensor
grows on to form a chain of two or more files of 15-150 cells. The
growth potential of the suspensor is therefore revealed only when the
inhibitory action of the proembryo is removed. Whether embros is
general have this inhibitory effect on the suspensor, and whether this is
the explanation for the different division rates in adjacent tissues, is not
known.
As the embryo develops it also changes in shape from globular to
heart shaped when the cotyledons are formed. Cell divisions are
apparently more frequent in the developing cotyledon than in the centre
of the embryo. After the first division of the zygote, usually transverse as
in capsella, the next division in the terminal cell is usually in a different
plane, normal to the first and therefore longitudinal (fig. 1.1). However.
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in the basal cell, which forms the suspensor, the second and subsequent
divisions are all in the same plane as the first and are therefore
transverse. The terminal group of cells goes on to divide in other planes
to form a mass of cell, called the globular embryo, which is anchored to
the embryo sac wall by the file of cells that is the suspensor (fig 1.1).
The various planes of division in the proembryo reflect its generally
isotropic (equal in all directions) growth, and the transverse divisions in
the suspensor reflect its linear growth. The problem is do the direction of
growth dictate the planes of division or do the planes of division dictate
the direction of growth? In some embryos the suspensor is elongated, as
usual, but is noncellular and multinucleate (e.g orobus or pisum, in
which there are several such cells). This suggests that the plane of cell
division is probably the result of the general direction of growth and not
its cause (see also Ch.5)
in the capsella proembryo the successive cell divisions are in
precise and predictable palnes, at least for the first live or so divisions,
after which divisions are harder to follow. What causes the divisions to
be orientated so precisely? If the overall shape of the globular embryo is
the result of controls on the embryo as a whole rather than on each
individual cell, then the positions of the cell walls might simply be those
that are the consequence of physical or structural restraints in the
embryo. The partitioning of a cell and its daughters according to simple
rules could conceivably produce the pattern seen in globular embryos.
The actual algorithm operated by the capsella embryo seems to specify
that each division is normal to the previous one until the formation of the
protoderm (the embryo epidermis), which is formed at the 16-cell stage
(fig 1.1) and which thereafter divides only anticlinally (I.e) divisions
normal to the surface) until the mature embryoor seedling is formed.
In the suspensor, however, divisions are all transverse, giving rise
to a string of cell one cell wide in casella. The plane of divisions
therefore alters in successive divisions in the proembryo but remains
constant in the suspensor. The control of the plane of division of
therefore intimately linked not only to the shape of the resulting tissues
(or organ) mass but also to its development fate. The changes in division
plane in the embryo pose the broader question: to what degree is the
shape of an organ caused by differential palnes of divisions rather than
by rates of growth? Is the plane of cell division a cause or a consequence
of organ shape? Is the growth rate in meristems controlled by cell
divisions cycle or is the cell cycle controlled by the general rate of
growth? These are questions that will be considered in later chapters (see
Chs 5 dan 2).
Since the rates and planes of cell division are obviously involved
in the generation of plant form, the cell wall is a commensurately
important organelle. In the plant, the walls not only encapsulate the
developmental history of the organism but also provide the permanent
structural.
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Framework that is the basis for all subsequent growth. Since plant form
is inherited and is therefore the result of gene action, gene activity is
expressed partly in the positions and planes of cell walls which depend
on the previous orientation of the mitotic spindle. Three-dimensional
cellular architecture, wall positioning, and the differential properties of
the cell wall within the same celi require explanations in terms of gene
actions. The plane of cell divisions is ultimately the result of gene
actions, but now?
1.3.4 Determination and differentiation
The cotyledons from from the upper part of the embryo but, more
than that, the lineage of the cell from which they are formed can be
traced back to the 8 celled stage of the proembryo,and their development
fate can be predicted (fig.1.1). This can be done in capsella because of
the regular pattern of cell division in the young embryo and the visible
persistence of the cell wall boundaries between the different parts of the
embryo as it grows. This is equivalent to the fate maps constructed for
animal embryos. The implication is that the fate of the cells is
predictable as the 8-celled stage according to their position in the
embryo, and so the embryos show mosaic development. But this does
not necessarilymean that the fate of the cell has become fixed as this
stage. Even if the cells were visibly different in the early embryo, this
would not necessarily imply that their development fates are fixed at this
point , any more than they are in animal embryos. Whether or not the
cells are determined, i.e. fixed in their fate, can only be measured by
experiments that can cause them to follow some different fate if they are
not yet determined.
1.3.4.1 When do cells become determined
Detemination is the process whereby the development fate of cells
become fixed. Before determination the cell, tissue, or organ can be
diverted into other development pathyways, but once determined its fate
is sealed and it becomes imposible to alter the developmental pathway
on which the cell have embarked. The only way to find out whether the
cells have become determined is to try to provoke the cells to develop in
a different manner by placing them under different experimental
conditions. If they cannot be made to develop in a different way, no
matter how they are treated, they are said to be determined. Test like this
have been made with animal embryos that are amenable to
experimentations because the eggs are laid external to the organism and
are fertilized outside the animal. Amphibian embryos have been
favoured because all the cells of the embryo contain yolk and therefore
are not dependent on other cells for their continued nourishment. Such

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Ekperiments have shown that the first thing that becomes determined is
the regional pattern (i.e the differentiation into ectoderm, mesoderm, and
endoderm), and tissue transplantation has shown that determination
becomes progressive so thst the organ system and finally the cell types
become determined.
The development of plant embryos seem broadly similar, although
experiment comparable with those performed with amphibian embryos
have not been done because of the inaccessibility of the plant embryo,
enclosed as it is in an ovule (or, in the lower plants, an archegonium). It
has proved difficult to dissect out young globular embryos that will
recover and grow intact, and microsurgery does not seem to have been
attempted. However, older embryos have been cut up and the consistent
finding is that only the shoot portion will regenerate shoots, the
cotyledon portion produce only callus, and the root and hypocotyl
portions produce callus and roots. The cells are therefore determined as
root or shoot (and possibly cotyledon) and epidermis, cortex, or stele by
the time the seed has formed. Because plant cells show much greater
plasticity than animal cells, the whole embryo can be separated into its
component cells and each can apparently give rise to a whole new
embryo and ultimately to a whole plant. Because even mature plant cells
can often be made to dedifferentiateand grow into whole plant, plant
cells (except those that lose their nucleus in the course of
differentitation) are less determined until they are placed in conditions
that allow dedifferentiation and redevelopment into a whole plant,
although sometimes callus can then differentiate only into certain kind of
organs, e.g . roots or shoot (see Ch. 10).
We simply do not known whether the development of particular
cells of an 8-celled plant embryo into root or shoot (fig1.1) is because
they are already determined or, because of their position, they
necessarily end up in yhe root or the shoot and become determined later.
The same applies at the 16 cell stage when periclinal divisions (paralle)
to the surface) have formedthe protederm the embryonic epidermis.
Although the epidermis remains morphologically distinct form this
moment onwards, we do not known when he cells actually become
determined. The plant epidermis, in becoming distinct early in
development, is therefore comparable to an organ system in animal
embryos ratherthan to the animal epidermis, which can originate form
different organ system of the animal embryo
The early development of the plant embryo up to the establishment
of the root shoot axis and the cotyledon (s), and the formation of the
provascular tissue, seems to resemble the development of the animal
embryo in which cell fate epend on position in the embryo. In the plant
embryo, the factor that position the cell walls and dictate the planes and