Download 18. GENETIC REGULATION OF DEVELOPMENT.

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18. GENETIC REGULATION OF
DEVELOPMENT.
Chromosome imprinting. Maternal effects. Genetic
dissection of maternal effects. Homeosis and atavism.
The knockout mice. Cell-to-cell communication and
the embryonic induction. Limb development.
INTRODUCTION
As discussed in chapter 14, cells of the multicellular
organisms live their life in rather constant environment.
The most important task of their genes is to bring into
reality the developmental program encoded in the
genes: govern development of a single cell zygote to an
adult composed from billions of cells in harmony and
ensure its life in time and space. We shall first discuss
the maternal and the paternal contributions that
establish favorable preconditions for the formation of
the zygotes. Special attention will be devoted to
homeosis, the homeotic genes that determine segment
identity, and to atavism. The last paragraph will deal
with the significance of cell-to-cell communication,
embryonic induction and limb development.
Chromosome imprinting
It is the paternally derived X chromosome that becomes
inactivated in e.g. duckbill platypus and kangaroos. In
embryos of - among others - cats and humans either the
maternally or paternally-derived X chromosomes are
inactivated. The inactivated X chromosome forms the
Barr body, naturally in different cells. (See chapter 12;
Fig. 12.9.) Obviously, the mammalian embryonic cells
can somehow distinguish between the maternally and
the paternally derived X chromosomes. It appears that
the X chromosomes are somehow labeled, imprinted
prior to fertilization. Imprinting may involve entire
chromosomes, segments of chromosomes or single
genes, the sex as well as the autosomes. Chromosome
(gene) imprinting takes place both in males and in
females during germ cell formation. Imprinting often
means the inactivation of genes up to well defined
stages of embryogenesis. The basis of chromosome
(gene) imprinting is (i) methylation of DNA and/or (ii)
association of the DNA with specific protein
molecules. Gene imprinting plays a key role in e.g.
Wilms tumor formation.
The role of maternal effects in the regulation of
development
Maternal effect implies the presence of components in
the egg cytoplasm (mRNA and protein molecules
mostly) that are deposited into the egg cytoplasm
during oogenesis. (See chapter 8.) Synthesis of the
maternally-derived components is controlled by genes
of the mothers. The maternally provided substances
regulate early embryogenesis and play essential roles in
determining fates of the embryonic cells. (The so-called
mosaic development is a typical example of maternal
regulation of embryogenesis: in e.g. Cynthia partita, a
species of worms, differently colored compartments of
the egg cell cytoplasm lead to differentiation of
different cell types (see Fig. 8.6.). In several species the
zygotic genes are not expressed during early
embryogenesis and the embryos rely exclusively on the
maternally provided substances. For example, the
enucleated frog zygote, of which the nucleus and thus
the nuclear genes were removed, develops to a several
hundred "cell" stage and appears like a tadpole. (A few
genes of the human zygotes are expressed already at the
two cell stage. Of course, maternal effects last well
beyond the two cell stage in humans.)
Genes of the Drosophila embryos are first expressed
shortly before reaching the blastoderm stage when the
embryo is composed already from as many as six
thousand cells. Interestingly developmental program of
the blastoderm cells are specified by the time of
reaching the blastoderm stage. Apparently fate of the
Drosophila blastoderm cells is determined by
maternally provided factors. What are those factors?
When and how are they deposited into the egg
cytoplasm? How do they determine developmental
program of the cells?
When a few blastoderm cells of a Drosophila embryo
are destroyed by UV laser beam, the corresponding
cells will be missing form the developing larva. By
destroying systematically groups of blastoderm cells
and evaluating the resulting defects, a correlation can
be established between cell position and cell fate, and a
so-called blastoderm fate map can be constructed. The
blastoderm fate map is the topographic representation
of the blastoderm cells and their developmental fates
(Fig. 18.1).
Fig. 18.1. The Drosophila blastoderm fate map. Please
notice that the head and the thorax segments derive
from almost the middle regions of the blastoderm.
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Following pricking of the anterior tip of a newly
deposited Drosophila egg, a small drop of cytoplasm
will leak out. The head and the thorax segments are
missing from the embryo that develops inside the egg
(Fig. 18.2). Results of the pricking experiments show
(i) the presence of a maternally derived factor in the
anterior tip of the eggs. (ii) The factor must have a long
range effect since it organizes formation of the distantly
located head segments. Obviously, synthesis and
deposition of the head factor into the egg cytoplasm
is under control of the maternal genes. Function of the
genes may be eliminated by mutations. Females
homozygous for a head factor eliminating mutation are
expected to deposit eggs. However, embryos without
head should develop in every of the eggs. The headless
embryos will die and consequently the females
homozygous for the mutation will be sterile. That is to
say, the genes that code for the head-determining factor
may be identified by female sterile (fs) mutations.
Females homozygous for some of the fs mutations
deposit normal looking eggs in which the developing
embryos are headless. There are three fs mutations in
Drosophila that fulfil the above expectations: bicoid
(bcd), exuperantia and swallow (Fig. 18.2). Of the three
mutations bicoid is the key gene. Note that the bcd
mutations identify the normal bicoid gene.
before the blastoderm stage, at the time when fate of
the blastoderm cells is determined. Concentration of the
Bicoid protein is high in the anterior tip of the eggs and
decreases, along a concentration gradient, towards the
middle of the egg. (See fig. 8.10.) The Bicoid protein is
located inside the nuclei, suggesting nuclear function.
Indeed, the Bicoid protein is a transcription factor that
regulates expression of a few genes of the zygotes. The
Bicoidc protein is an example of the morphogens,
molecules that directly determine cell fate.
The zygotic genes
If the Bicoid morphogen does indeed control
expression of the zygotic genes, function of the
regulated zygotic genes can be eliminated by
mutations. It may be expected that headless embryos
develop in absence of the Bicoid-regulated zygotic
gene function. However, the headless phenotype
should depend on genotype of the zygote. Among all
the zygotic lethal mutations of Drosophila, only the
hunchback (hb) mutations lead to zygotic headless
phenotype (Fig. 18.2). The hb mutations identify the
normal hunchback gene. (The hb mutant alleles are
typical examples of the zygotic lethal mutations that
bring about death during embryogenesis.)
It is thus to be expected that the Bicoid protein
regulates expression of the hunchback gene. The
hunchback gene is expressed in an anterior band of the
blastoderm stage embryos, in those cells that will give
rise to the head and the thorax (Fig. 18.3). The
hunchback gene is not expressed in embryos that derive
from the bcd/bcd mutant females that lack Bicoid
protein) indicating that the Bicoid protein turns the
zygotic hunchback gene on, i.e. the Bicoid protein is a
positive regulator of the hunchback gene. In wild type
embryos the zygotic Krüppel gene is expressed just
posterior to the head segments, showing that function
of the Krüppel gene is required for the formation of
those segments (Fig. 18.3). However, in eggs of the
bcd/bcd mutant females the Krüppel gene is expressed
over the entire anterior region showing that the Bicoid
protein is negative regulator of the Krüppel gene over
the anterior embryonic territories (Fig. 18.3).
Fig. 18.2. Headless embryos - shown on (b) - develop
(i) in the anteriorly pricked wild type Drosophila eggs,
(ii) in eggs of the bcd/bcd mutant females. (iii)
Embryos homozygous for the hunchback (hb) mutation
are also headless. A wild type embryo is shown on the
left side (a).
The bicoid gene has been cloned and is known on the
level of nucleotide sequence. It has turned out that the
bicoid mRNA is localized in the anterior cytoplasm of
the wild type eggs. (The bicoid mRNA-containing part
of the egg cytoplasm leaks out following pricking.)
Translation of the bicoid mRNA takes place shortly
Fig. 18.3. Expression of the hunchback and the
Krüppel genes in embryos of wild type (+/+) and
bcd/bcd mutant females.
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Since large sections (gaps) of the embryo body are
missing in both the hunchback and in the Krüppel
homozygous mutant embryos, the hunchback and the
Krüppel genes (and a few others) are called gap
genes (Fig. 18.4). Products of the gap genes are
transcription factors and regulate expression of the socalled pair-rule genes. The pair-rule genes regulate
establishment of segment pairs. Products of the pairrule genes are also transcription factors and regulate
expression of the so-called segment polarity genes.
The segment polarity genes divide the segments into
anterior and posterior parts. Products of the segment
polarity genes are also transcription factors. They
regulate expression of the so-called homeotic genes.
The homeotic genes determine identity of the segments
and will be discussed in some detail below. In
summary, the process of segment cell fate
determination originates step by step, throughout
coordinated expression of different classes of genes.
Using as probes of the above genes, mouse (and also
human) homologues of the above types of Drosophila
genes were isolated and their roles elaborated in
vertebrate development. (See below for details.)
The mutations identify a Drosophila homeotic gene that
determines the T3 thoracic segment identity.
Fig. 18.5. Halters develop from the 3rd thoracic
segment of the wild type Drosophila. The bithorax
mutations eliminate function the 3rd segment identity
homeotic gene, and consequently development follows
the pathway characteristic for the 2nd thoracic segment
Flies with two 2nd thoracic segments develop four
wings.
Fig. 18.4. The mechanism of stepwise determination of
segment identity through the action of gap, pair-rule,
segment polarity and homeotic genes.
Homeosis and atavism
Existence of homeotic genes was revealed, among
others, by the four-winged mutant fruit flies (Fig. 18.5).
In wild type flies a pair of halters develops in the third
thoracic (T3) segment. Due to mutations in the so-called
Bithorax gene complex (BX-C) characteristic T2
T3
transformation takes place and flies with two T2
segments - i.e. with four wings develop (Fig. 18.5).
Replacement of a structure by another structure that is
characteristic for a different body region is called
homeosis. (Homeo is a Greek word and means similar.)
Genes identified by homeotic transformation-causing
mutations are called homeotic genes. (It should be
mentioned in brackets that there are environmental
effects that result in the expression of a mutant
phenotype characteristic for known mutations and
induce phenocopy. Naturally, the phenocopy-related
mutant phenotype is not transmitted to the subsequent
generations. Phenocopy is one type of teratogenesis.
Teratogenesis is the science of abnormal development.)
The four-winged Drosophila flies are reminiscent of
evolutionary ancestor species from which the Dipterans
(including Drosophila) developed. Mutations which
result in four winged flies are classical examples of
atavism the recapitulation - through mutations - of
evolutionary ancient characters. Atavism is a rather
common phenomenon. Polythely (multiple nipples) and
uterus bicornis (uterus with two chambers) are typical
examples of atavism in humans (Fig. 18.6).
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Fig. 18.6. Polythely is an example of atavism in
humans.
Homeobox and homeodomain
Every homeotic gene of Drosophila has been
identified by homeotic mutations. The eight homeotic
genes are organized into two large gene complexes: the
Antannapedia (ANT-C) and the Bithorax (BX-C)
complexes. The ANT-C and the BX-C complexes
include five and three genes, respectively (Fig. 18.7).
That segment of the chromosome which includes the
ANT-C and the BX-C complexes is referred to as HomC, the homeotic gene complex. It is rather peculiar that
homeotic genes are organized in the same successive
order as the segments that the homeotic genes identify
(Fig. 18.8).
Fig. 18.8. Expression pattern of the homeotic genes in
Drosophila and in mice embryos.
Every of the Drosophila homeotic genes have been
cloned and sequence of the composing nucleotides
were determined. Comparison of the nucleotide
sequences of the Drosophila homeotic genes revealed
that every homeotic gene contains an evolutionary
highly conserved 180 base pair long section, the socalled homeobox. (Box = evolutionary conserved
stretch of DNA. Homeo- because it is part of the
homeotic genes.) The homeobox encodes the
homeodomain, a 60 amino acid long functional motif in
the so-called homeo proteins (Fig. 18.9). The
homeodomain is that section of the homeo proteins
with which they bind to DNA. (Naturally, the homeo
proteins are also transcription factors.) One part of the
homeodomain forms a helix-turn-helix motif. (See
chapter 14.) The variable parts of the homeo proteins
ensure homeotic gene-specific activities. The homeotic
proteins regulate expression of those genes of the
zygotes that are required for attainment and
maintenance of segment identity.
H2N
Fig. 18.7. Organization of the homeotic genes which
determine segment identity in Drosophila (top) and in
mouse (bottom). Homeotic genes in Drosophila are
clustered into the ANT-C and the BX-C gene
complexes. Homeotic genes in mice (also in humans)
are organized into four (A D) complexes. One Hox
complex is divided into 13 regions. (Contents of the
empty boxes are not known yet.)
Fig. 18.9. Organization of a homeo protein. The
homeobox encodes the evolutionary highly conserved
homeodomain. Part number 3 of the homeodomain
forms the helix-turn-helix DNA-binding motif.
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Homeotic genes of the vertebrates
When the homeobox was used as probe for the
screening of genomic libraries made from vertebrates
(among others Xenopus, chicken, mouse, and humans),
the vertebrate so-called Hox genes were fished out
right away. The vertebrate homeo gene complex is
called HOX-C. The HOX-C contains, more or less, four
copies of the complex described for Drosophila (Fig.
18.7). It is rather remarkable that the gene arrangement
in the HOX-C and the expression pattern of the Hox
genes are the same as in Drosophila (Fig. 18.8),
suggesting an evolutionary highly conserved
mechanism for segment formation control.
In vitro mutagenezis, knockout mice and
developmental abnormalities
Elaboration of the so-called knockout mice
technique greatly facilitated an understanding of
homeotic gene function in vertebrates. (See chapter
17.) Characteristic developmental abnormalities
appeared in mice mutant for homeotic mutations
(Fig. 18.10). Since position of the mutation is know
(it was made in a test tube at desired position during
in vitro mutagenesis) in the knockout mice,
correlation can be established between the site of
mutation and the type of defect. Experts have long
been familiar several types of the defects created in
the knock out mice, however molecular bases of the
mutations were unknown.
Fig. 18.11. The role of cell communication in
development of sea urchin embryos.
Fig. 18.10. In the mouse on the right side, promoter
of the Hox A1 gene regulates expression of the Hox
D4 gene. Expression of the Hox D4 gene at extopic
(unusual) regions leads to the formation of abnormal
skeleton. (A wild type mouse skeleton is shown on
the left side.)
Cell-to-cell communication and embryonic
induction
Cell-to-cell communication is an essential
component of development of the multicellular
organisms. Halved sea urchin embryos develop only
when their body contains cells form both the animal
and the vegetal poles. Development of the half
embryos with only animal or with only vegetal pole
cells is abnormal (Fig. 18.11). The animal pole is
determined by the polar body cells attached to the
egg cell and to the zygote.
Hans Spemann and Hilde Mangold (1924)
elegantly showed the importance of cell-to-cell
communication in transplantation experiments with
newt embryos. Spemann and Mangold used darkly
pigmented embryos of Triturus taeniatus and nonpigmented embryos of Triturus cristatus. When
transplanted into unusual (ectopic) positions, some
groups of newt embryo cells develop according to
their new position: they are told to respond to the
positional information in their new position (Fig.
18.12). Other groups of cells not only develop
autonomously at their new position but also instruct through the process called embryonic induction the neighboring cells to execute a developmental
program they would never do. One of the best known
examples is the dorsal lip cells. When transplanted
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into ectopic position the dorsal lip cells induce the
formation of an additional neural tube (Fig. 18.13).
Another well-know example of embryonic induction
is the formation of eye lens (Fig. 1814). The basis of
embryonic induction is the presentation on the cell
surface or the release of ligand molecules which will
instruct through signal transduction neighboring
cells to follow well defined pathway of development.
(See chapters 14 and 20.)
Formation of the limb bud
Limb formation is another classic example of cell
communication and cell differentiation. The decision
where along the embryonic anterior posterior axis
should limb buds form is governed by the Hox genes.
For example, wing buds in chickens form around the
region where expression of the Hox6 gene begins.
(Note that Hox genes of the different vertebrate
species fully substitute each other.) Upon activation
of the Hox6 gene lateral plate mesoderm cells secrete
the fibroblast growth factor FGF10. Mioblast and
mesoderm cells congregate beneath the epidermis
and form a limb bud primordium (Fig. 18.15). The
key role of FGF10 in limb bud formation is elegantly
shown by the fact that extra limb forms from the site
where FGF10 coated beads are implanted under the
epidermis of young vertebrate embryos. For
outgrowth of the limb buds retinoic acid is also
required. (See also chapter 14 and 20.)
Fig. 18.12. Fate of some grafted frog embryonic
tissue follows the fate characteristic to the position
where the donor tissue was transplanted.
Fig. 18.15.
Scheme showing the molecular
mechanism of limb bud formation.
Fig. 18.13. The dorsal lip acts as an embryonic
organizer of the neural tube. When grafted into
ectopic position, the dorsal lip organizes an extra
neural tube.
Fig. 18.14. Embryonic induction during eye lens
formation.
The decision whether forelimbs or hindlimbs form
is controlled by expression of two Tbx genes:
expression of the Tbx5 and the Tbx4 genes determine
forelimb and hindlimb formation, respectively. (The
Tbx proteins are transcription factors with
characteristic DNA binding motifs.) People
homozygous for loss-of-function mutant Tbx5 alleles
possess Holt-Oram syndrome with partial
development of the arms and also abnormalities in
heart development.
During development of the limb buds and the
limbs, cells possess characteristic differentiation
pattern along three axes: (i) the proximal-distal, (ii)
the anterior-posterior and (iii) the dorsal-ventral axes.
The molecular mechanisms of limb bud cell
differentiation were elucidated during the past few
years. Bases of a number abnormal limb development
are also understood by now.
(1) Differentiation along the proximal-distal axis.
Differentiation along the proximal-distal axis is
governed by AER, the apical ectodermal ridge. The
key role of AER in proximal-distal differentiation is
elegantly shown by two findings. (1) Following
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removal of AER from a young forelimb bud only the
upper arm forms (Fig. 18.16). The older the limb bud
is at the time of AER removal, the more arm
structures develop. (ii) Grafting different age limb
bud segments leads to the formation of arms with
different arm components (Fig. 18.17). It appears that
for the longer time limb bud mesoderm cells spend
under AER exposure the more limb structures they
develop. What molecular mechanisms are associated
with AER activity?
Fig. 18.16. Differentiation of the arms and legs is
controlled by a morphogen released from the apical
ectodermal ridge.
Differentiation of AER is induced by FGF10 (Fig.
18.15). FGF10 also makes AER cells to secrete AER
cells FGF8 molecules. FGF8 stimulates mitoses and
prevents differentiation of the mesoderm-derived
limb bud cells to cartilage.
Hox genes determine once again differentiation of
cells along the proximal-distal limb axis (Fig. 18.18).
The role of Hox genes in arm and leg differentiation
was elucidated largely by two techniques. (i) In situ
hybridizations revealed that the different Hox genes
are expressed in different parts of the developing
limbs. (ii) Knock out mouse were produced next, in
which function of different Hox genes were knocked
out . Based on the expression pattern of the Hox
genes and the missing lib parts a correlation between
Hox gene function and arm structure formation was
elaborated (Fig. 18.18).
Fig. 18.18.
Relationship between Hox gene
expression and the formation of different arm
components. (The equivalent genes in each mouse
complexes such as Hox A1, Hox B1, Hox C1 and Hox
D1 comprise one paralogous group, see also Fig.
18.7.)
Several types abnormalities seen in knock out mice
are also known in humans. For example people
homozygous for loss of function HOX D13 mutant
alleles have abnormally developed digits as shown on
Fig., 18.19.
Fig. 18.19. The human synpolydactyly (many
fingers joined together) syndrome results from
homozygosity for mutant HOXD13 alleles.
Fig. 18.17. Grafting of limb buds of different ages
leads to the formation of abnormal appendages.
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(2) Differentiation along the anterior posterior axis.
Differentiation of cells in the developing limb
along the anterior posterior axis is regulated by ZPA,
the zone of polarizing activity, a battery of mesoderm
cells in the posterior corner of the limb bud (Fig.
18.20). Cell fate depends on their position in the limb
bud, on the positional information in the limb bud.
Some cells die through apoptosis, others develop e.g.
a finger. The type of developing finger is determined
by its distance from the ZPA. The role of ZPA is
elegantly shown by outcome of the experiment
illustrated on Fig. 18.20: when an additional ZPA is
implanted into the anterior corner of a limb bud,
digits arranged in mirror image symmetry develop.
Fig. 18.20. Digit differentiation along the anteriorposterior axis is controlled by ZPA. Digits arranged
in mirror image symmetry develop from limb buds
with two ZPAs, one in the posterior and one in the
anterior corner.
The Sonic hedgehog gene is expressed in the ZPA
cells. (Sonic hedgehog is the vertebrate homologue of
the Drosophila hedgehog gene, one of the segment
polarity genes.) Expression of the Sonic hedgehog
gene is induced by the FGF8 protein. FGF8 is
product of the AER (Fig. 18.15). Although every
AER cell releases FGF8, the Sonic hedgehog gene is
expressed in only those limb bud cells in which the
Hox8 gene is expressed, i.e. in only the ZPA cells
(Fig. 18.15). The shh protein make the ZPA cells
release BMP2 and BMP7 proteins. Diffusion of the
BMP2 and the BMP7 molecules determine positional
information and cell fates in the developing limb.
(3) Differentiation along the dorsal-ventral axis.
Differentiation along the dorsal-ventral axis, that is
the differentiation of knuckles-nails and pads-soles
are determined by the ectoderm encasing the limb
bud. If the ectoderm is rotated 180% with respect to
the limb bud mesenchyme, the dorsal-ventral axis is
reversed and the digits develop upside down .
Wnt7a is the key component of dorsal-ventral
differentiation. (Members of the Wnt family of
proteins are perhaps the most ancient types of
molecules engaged in cell-to-cell communication; see
also chapter 20. The Wnt genes received their name
from the wingless gene of Drosophila and its human
homologue called integrated.) The Wnta7 gene is
expressed in the dorsal but not in the ventral
ectoderm and knock out mice without functional
Wnta7 gene have sole pads on both surfaces of the
pawns.
The Wnt7a protein induces expression of the Lmx1
gene in the dorsal mesenchyme cells. The Lmx1
protein is a transcription factor that specifies dorsal
cell fate. Knock out mice without Lmx1 gene function
leads to the absence of dorsal limb cells. People
homozygous for Lmx1 mutant alleles possess the socalled nail-patella syndrome, a condition in which the
dorsal sides of the limbs have been ventralized (Fig.
18.21).
Fig. 18.21. Loss of Lmx1 gene function leads to
development of ventralized limb formation in
humans and the consequently the loss of nails and
patella.
Action of the above-described molecules leads to
limb cell fate specification along progression of
embryogenesis. The BMP proteins induce some cells
to die through apoptosis, others to form e.g. digits.
(The BMP bone morphogenetic protein molecules
received their name from their bone formation
stimulating abilities. They are known today to be
involved in cell cycle progression regulation,
apoptosis, cell migration and cell differentiation.)
Cells also die between the presumptive ulna and
radius establishing conditions for bone formation.
In summary, the genes and the encoded molecules
governing limb bud formation and differentiation
have already been identified. Of course, the above
short overview provides only a brief description of
the story.
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SUMMARY
Although life of the sexually reproducing
organisms begins with fertilization, the conditions for
normal embryogenesis are established already during
germ cell formation: chromosome imprinting
preprograms gene expression during embryogenesis
and maternal effects ensure developmental
programming of the embryonic cells. Determination
of cell features is achieved in a stepwise fashion
along cell differentiation. Cell-to-cell communication
is of basic importance in the regulation of
developmental programs. Mutations that disrupt body
pattern in Drosophila have opened the way to an
understanding of the molecular basis of cell fate
programming. Analysis of the homeotic mutations
revealed an evolutionary conserved mechanism for
the control of segmental organization and limb
formation. The knockout mice technique helped to
understand the molecular genetic basis of a number
of human developmental disorders.
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