Download Genetic mechanisms

DEVELOPMENT
AND
DISORDERS
It is amazing that embryonic development proceeds normally in most cases
 20-50% of human cleavage-stage embryos successfully implant in the
uterus
 Of the embryos that do implant only 40% survive to term.
 Among the babies that come to full term 2.5% have a recognizable
birth defect.
With so many genes, cells and tissues becoming organized
simultaneously during development it is not surprizing that some
events do not happen properly.
Three major pathways to abnormal development:
 Genetic mechanisms: Mutations in genes or changes in the
number of chromosomes can alter development.
Environmental mechanisms: Agents (usually chemicals) from
outside the body cause deletetrious changes by inhibiting or
enhancing developmental siganls.
Stochastic (random events): Chance plays a role in determining
the phenotype and some developmental anomalies are just bad
luck.
Developmental anomalies can be
the result of stochastic events
An actual case history of genetically identical female
foetuses (monozygotic twins):
Their phenotypically normal mother was heteroxygote for a lethal
mutant allele on the X chromosome.
Both twins inherited this mutant X chromosome from their mother
and a normal X chromosome from their father.
The phenotypically normal twin had a typical pattern of random Xinactivation, with the paternal and maternal X inactivated in about
50% of the cells.
The other twin had extensive developmental anomalies; by chance
the paternally derived X chromosome was in activated in almost all
of her cells.
Cell specification, developmental signaling and cell migration are
thought to be influenced by chance fluctuations in amounts of
transcription factors, paracrine factors and receptors produced at a
particular moment.
Thus genetically identical animals raised in the same environment
can have vastly different phenotypes.
Mathematical modelling has permitted scientists to study these
stochastic events enabling researchers to demonstrate that
development is a combination of stochastic and deterministic
events. Thus chance influences normal development.
Genetic Errors of Human Development
Congenital (“present at birth”) abnormalities and losses of the fetus prior to birth
have intrinsic and extrinsic causes.
Those abnormalities that are caused by genetic events may occur due to
mutations, aneuploidies and translocation.
Human birth defects which range from life-threatening to mild are often
linked to syndromes, where several abnormalities occur together.
Genetically based syndromes are caused either by :
1) A chromosomal event (such as aneuploidy) where several genes are
deleted or added
OR
2) Pleitropy –the production of several effects by a single gene or a pair of
genes.
Pleitropy: the production of several effects by a single gene or a pair of genes
In mosaic pleiotropy a gene is independently expressed
in multiple tissues. Each tissue independently needs
the gene product and develops abnormally in its
absence. The KIT gene is need for proliferation of blood
stem cells, pigment stem cells and germ stem cells.
Thus in mutants of this gene there is anemia, sterility
and albinism.
In relational pleiotropy, a gene is needed by only one particular
tissue. However a second tissue needs a signal from the first tissue
in order to develop properly. For example the failure of MITF in
the pigmented epithelium of the eye prevents this structure from
fully differentiating, further leading to malformation of the
choroid fissure and drainage of vitreous humor. Without this fluid
the eye fails to enlarge and micropthalmia or small eye is seen
where the lens and cornea are smaller even though they do not
express MITF themselves.
Mosaic syndromes can be the result of aneuploidies
(errors in the number of chromosomes)
 Downs syndrome caused by trisomy of chromosome
21.
 Even an extra copy of the tiny chromosome 21 disrupts
numerous developmental functions.
 Downs syndrome is characterized by facial pattern,
cognitive deficiencies, heart and gastrointestinal
defects etc.
 Certain genes on chromosome 21 are thought to
encode transcription factors and regulatory microRNAs.
 Extra copies of chromosome 21 probably lead to
Fluorescent in-situ
overproduction of these regulatory proteins and RNA.
hybridization with probes
 Such overproduction would cause the misregulation of
for chromosome 21 (pink)
genes necessary for heart, muscle and nerve formation.
and chromosome 13 (blue)  One such miR155, is encoded on chromosome 21 and
show that this person has
found throughout the human fetus. This miRNA
three copis of
downregulates the translation of messages encoding
chromosome 21 but two
certain transcription factors required for normal heart
copies of chromosome 13.
and neural development.
 miR155 is highly elevated in brains and hearts of
people with Downs syndrome.
Genetic heterogeneity:
Mutations in different genes can produce the same phenotype. If several genes are part
of the same signal transduction pathway a mutation in any one of them will often
produce the same phenotype.
For example:
1) The syndrome of albinism, anaemia and sterility caused by absence of the Kit protein
can also be caused by the absence of its paracrine ligand the stem cell factor (SCF).
2) Cyclopia, which is produced by mutations in the sonic hedgehog gene, can also
results from mutations in genes activated by Hedgehog or the genes controlling
cholesterol synthesis.
Phenotypic heterogeneity:
The same mutation can produce different phenotypes in different individuals. This is
because genes are not autonomous agents they interact with other genes and gene
products. For example the same mutation FGFR3 in 10 different unrelated families can
lead to phenotypes ranging from phocomelia (absence on limbs) to a mild abnormality
of the thumb. The severity of a phenotype thus depends on other genes, environmental
and stochastic factors.
Teratogenesis: Environmental Assaults On Human Development
In 1962 there were two key discoveries to show that the embryo was vulnerable to environmental agents:
1) The pesticide DDT was destroying birds eggs and preventing reproduction in several species.
2) Thalidomide, a sedative given to manage pregnancies could cause limb and ear abnormalities in foetuses.
http://www.bbc.co.uk/schools/gcseb
itesize/science/aqa/drugs_use/drugs
rev2.shtml
http://pixgood.com/ddt-eggs.html
http://radiopaedia.org/images/4669603
In 1964 an epidemic of rubella (German measles) spread
across the US. More than 20,000 foetuses of mothers infected
with rubella born blind, deaf or both. Many also had heart
defects and/or mental retardation.
Weeks of gestation and sensitivity of embryonic organs to environmental agents
 The period of maximum
susceptibility is between
3-8 weeks when most
organs are forming.
 The nervous system
remains vulnerable
throughout
development.
 Prior to week 3 there is
not much of an effect
because either there is
effect on too many cells
which kills the embryo
or it affects only a few
cells which die and the
rest of the embryo
compensates and
develops normally.
 Although there are
variations of effects of
chemicals on different
species, animal models
have been used to screen
for teratogens.
 Xenopus and zebrafish
use the same basic
molecular mechanisms as
humans and thus have
often been used for such
screens.
Water-soluble crude oil components from the Deepwater Horizon oil spill
were teratogenic in zebrafish. There were reduction in size of the head, gill
and thorasic cartilages associated with cranial neural crest migration.
 The largest class of teratogens includes
drugs and chemicals, but viruses,
radiation, high body temperature and
metabolic conditions in the mother can
also act as teratogens.
 Some chemicals found naturally in the
environment can cause birth defects
such as jervine and cyclopamine (plant
products) cause cyclopia.
 Nicotine, a natural product
concentrated in cigarette smoke
impairs lung and brain development.
Alcohol (ethanol) as a teratogen
 The most devastating teratogen is alcohol.
 Babies born with fetal alcohol syndrome
(FAS) have small head size, indistinct
philtrum, low nose bridge etc. Occuring in 1
out of every 650 children born in the US.
 The brain of such a child maybe dramatically
smaller and shows poor development due to
deficiencies in neural and glial migration.
 The term fetal alcohol spectrum disorder
(FASD) has been coined to encompass all of
the alcohol induced malformation and
functional deficiencies that occur.
 In many FASD children behavioural
abnormalities exist without any changes in
head size or deficits in IQ. However, subtle
abnormlities that correlate with altered
mental processing speed and executive
functioning such as planning, memorizing
have been identified using recent techniques.
Alcohol induced craniofacial and brain
abnormalities in mice.
B) Anterior neural tube
failed to close, exposing
the brain tissue.
C) Small nose abnormal
upper lip.
E) Absence of olfactory
bulbs and the cerebral
hemispheres are
abnormally united in the
midline.
 Mice exposed to alcohol at the time of
gastrulation, defects in the face and brain
comparable to those in humans with FAS can
be observed.
 As in humans in these pups the nose and
upper lips fail to develop properly, and
nervous system defects include failure of
neural tube closure and incomplete
development of the forebrain.
 This mouse model has been use to study the
mechanism by which alcohol causes these
defects.
 Alcohol affects several processes including cell
migration, proliferation, adhesion and survival.
 Neural crest cells on alcohol exposure
prematurely differentiate into cartilage
instead of migrating and dividing.
 Several genes are misregulated which are in
volved in cytoskeleton reorganization and cell
movement.
 Expression of Shh is downregulated in
embryos exposed to alcohol. The placement
of Shh secreting cells in the head mesenchyme
rescues the death of neural crest cells.
Cell death caused by exposure to alcohol
A) Head region of day 9 control mouse embryo.
B) Head region of day 9 mouse embryo exposed to alcohol with nile blue staining
showing cell death.
C) The alcohol induced cell death is rescued by superoxide dismutase.
 In later stage mouse embryos
exposure to alcohol induces
cell death in neural crest
derived structures as early as
12 hours following exposure.
 When alcohol exposure is at a
stage that corresponds to 3-4
week of humans, the cells
that should form the median
forebrain, upper mid-face and
cranial nerves are killed.
 In early chick embryos,
transient exposure to ethanol
causes cell death throughout
the head region and
decimates migrating neural
crest cells.
 One reason for this cell death
is the alcohol induced
production of superoxide
radicals that can damage the
cell membrane.
Inhibition of L1 mediated
cell adhesion by alcohol
 As little as 7mM alcohol (blood
levels) produced by a single
drink can block the adhesive
function of L1 protein in vitro.
 Moreover mutations in the
human L1 gene cause a
syndrome of mental retardation
and malformation similar to
that seen in severe FAS cases.
Retinoic acid (RA) as a teratogen
Retinoic acid is a derivative of Vitamin A that is very important for A-P axis specification as well as the development
of many organs and structures in the embryo.
 The exposure to excess RA can happen through the use of
medication used to treat acne (Accutane) that contains
high levels of Isoretinoins.
 Anomalies are largely caused due to the failure of cranial
neural crest cell migration.
 One mechanism that has been proposed to explain the
teratogenic effects of RA states that exposure to excess RA
activates the negative feedback pathway activating RA
catabolic enzymes which lead to a long lasting decease in
RA levels. It is this deficiency in RA that results in the
malformations
A paradoxical teratogenic mechanism for retinoic acid
Lee et al , 13668–13673|PNAS|August 21, 2012|vol. 109|no. 3
Other teratogenic agents:
In addition of natural chemicals hundreds of new artificial compounds come into use each year in our society and all are
not tested for their potential as teratogens. This is because standard screening tests are expensive, long and subject to
interspecies differences. Thus there is no consensus on how best to test a substance’s teratogenicity for human embryos.
Heavy metals
Conjoined trout hatchlings from the Great lakes
 Heavy metals such as Zinc, Mercury and lead are powerful teratogens
 In the former Soviet union unregulated industrialization lead to a lot
of birth defects.
 In Khazakstan heavy metals are found in high concentrations in air,
vegetables and water.
 In the US lax antipollution laws has led to contamination of the lake
water by the discharge of heavy metal containing slag into streams
and lakes.
 Pregnant women are warned not eat fish caught in the great lakes in
the US and Canada.
 Mercury and lead can damage the developing nervous system.
 Mercury causes damage to the cerebral cortex, exposure in mice led
to small brains and eyes
 Lead damages the brain in fetal and childhood stages and contributes
to developmental delays and mental retardation. Lead-based paints
are banned.
Other teratogenic agents:
Pathogens
 In 1941 it was first documented that rubella the virus causing german
measles is a teratogen. This virus enters cells and produces a protein that
blocks mitosis causing cell death.
 Early infection with the Cytomegalo virus is lethal for the embryo and
late infection can lead to blindness, deafness, cerebral palsy and metal
retardation.
 Bacteria and protists are rarely teratogenic except Toxoplasma gondii a
protist present in cat feces can cause brain and eye defects. The
bacterium that causes syphilis Treponema pallidum can kill early
foetuses and produce congenital deafness and damage in older foetuses.
Endocrine disrupters: The embryonic origins of adult disease
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Endocrine disrupters are external agents that interfere with the function of hormones during development.
There are usually no obvious defects like the ones produced by classic teratogens.
The anatomical defects can only be detected microscopically and most of the effects are physiological.
The functional changes are subtle and often manifest later in adult life.
Sometimes the effects may persist for generations after the exposure to the disrupter.
How do these chemicals interfere with hormonal functions?
 They can mimic the effect of a natural hormone e.g. Diethylstilbesterol (DES) which mimics estradiol
by binding to the estrogen receptor.
 They can act as antagonists and inhibit the hormone from binding to its receptor or block the
sysnthesis of the hormone. DDE the metabolic product of DDT the insecticide can act as an antitestosterone.
 They can affect the synthesis, transportation or elimination of a hormone. The herbicide atrazine
elevates synthesis of estrogen and can convert testes into ovaries.
 Some endrocrine disrupters can prime the organism to be more sensitive to the hormone later in life.
Exposure to bisphenol A makes breast tissue more responsive to steroid hormones during puberty.
Endocrine disrupters differ from teratogens in several ways
1. Their pathological effects do not have to be congenital but may show
themselves in adulthood.
2. The effects are usually manifested as physiological problems rather than
anatomical ones.
3. Given the paradigm of teratogens it was thought that there are only a few
“bad” agents and the only people who receive these are pregnant women
who inadvertently expose themselves to these chemicals. We now know
that endocrine disrupters are everywhere in our technological society.
4. One is usually exposed to multiple endocrine disrupters and not just one.
5. More damage maybe done by a “moderate” dose than by a high dose as a
high dose may activate mechanisms that detoxify and eliminate the
harmful agent.
DES as an endocrine disrupter
Diethylstilbestrol or DES was prescribed to pregnant women as it was
though to ease pregnancy and prevent miscarriages. However, it had
not benficial effects on pregnancy, rather it lead to defects in the
reproductive tracts of female foetuses whose mothers took this drug.
 DES interferes with sexual and gonadal
development by causing cell type changes
in the female reproductive tracts.
 In many cases DES causes the junction
between the uterus and oviduct to be lost
resulting in infertility or subfertility.
 Distal mullerian ducts fail to come together
to form a single cervical canal.
 Symptoms similar to human DES occur in
the mice exposed to DES in utero allowing
the dissection of the mechanism by which
DES acts.
Effects of DES exposure on the female reproductive system
In situ hybridization of a Hoxa10 probe shows that DES
exposure represses Hoxa10.
Bisphenol A (BPA)
In the early days it was difficult to isolate the steroid hormones so chemists manufactured synthetic
analogues that would accomplish the same function. BPA was one of these analogues. Later polymer
chemists realized that BPA could be used in plastic production. It is widely used to line plastic bottles,
resin lining of most cans, polycarbonate plastic in baby bottles, children’s toys and as a dental sealant.
Human exposure comes mostly from BPA leached from food containers.
 BPA causes meiotic defects in
maturing mouse oocytes.
 BPA causes chromosomes to
randomly align on the spindle. This
causes different numbers of
chromosomes to enter the egg and
may result in aneuploidy and
infertility.
BPA and reproductive health
In model organisms BPA at environmentally
relevant concentrations can cause abnormalities
in fetal gonads, prostrate enlargement, low sperm
counts and behavioural changes when these
foetuses become adults.
Male mice exposed to BPA in utero had enlarged
prostate glands and female mice exposed to BPA
in utero had reduced fertility as adults. They also
had alterations in the organization of their uterus,
breast tissue, ovaries and altered estrous cycles.
Mammary glands from newborn female rhesus
monkeys (A) control (B) exposed in utero to BPA
BPA induces altered mammary gland
development
Endocrine disrupters as “obesogens”
DES induced obesity in mice
The mother of the mouse on the left was injected with
carrier solution while the mother of the mouse on the
right was injected with DES. Weight gain is seen in 8 weeks.
The mice become sensitized by DES by this early exposure.
Later when large amounts of estrogen associated with
sexual maturity are secreted, the mice became obese.
Possible mechanism by which DES works:
Biases mesenchymal cells to become
adipocytes and activates fat-storing enzymes in
these cells.
Transgenerational inheritance of Developmental Disorders
Epigenetic transmission of testicular dysgenesis
syndrome:
If an endocrine disrupter, vinclozolin, is administered
to a pregnant female the F1 generation male is
exposed in utero to it and develops testicular digenesis
later in life.
However, the male offspring of the F1 males also
develop testicular disgenesis and so do the males in
the next two generations.
The mechanism for this appears to be DNA
methylation. The promoters of more than hundred
genes have their methylation pattern changed in the
sertoli cells of the F1 mice and altered methylation
patterns can be seen in the sperm of at least three
subsequent generations.
Cancer as a disease of development
Carcinogenesis can be viewed as an aberration of the very processes
that underlie differentiation and morphogenesis.
The following are four ways by which malignancy and metastasis can be
viewed in terms of development:
Context dependent tumor formation.
Deficient stem cell regulation in tumor formation.
Reactivation of embryonic migration pathways.
Epigenetic reprogramming of cancer cells.
Context dependent tumor formation
Many tumor cells have normal genomes and whether or not they will
become malignant depends on their environment. Example of this is a
teratocarcinoma or a tumor of germ cells or stem cells. These resemble the
inner cell mass of the mammalian blastocyst and they can kill the organism.
However if the teratocarcinoma cells are placed in inner cell mass of the
mammalian blastocyst it will integrate into the ICM and lose its malignancy.
Thus the environment determines whether the cell will become a tumor or
will be a part of the embryo.
 The mechanism by which a stem cell
environment supresses tumor
formation maybe due to its secretion
of inhibitors of the paracrine pathways.
 For example many melanomas secrete
Nodal which helps in their proliferation
and also helps supply them with blood
vessels.
 However when malignant melanoma
cells are transplanted in the early chick
embryo they down regulate their Nodal
expression and migrate along the
neural crest cell pathways and form
sympathetic ganglia, facial cartilage
and normal melanocytes.
Tumors can arise due to:
1. Defects in cell-cell communication:
Although 80% of human tumors are from epithelial cells, these cells are not always the site of the cancer
causing lesion. Maffini and colleagues (2004) recombined and carcinogen-treated epithelia and
mesenchyme in rat mammary glands, tumorous growth of mammary epithelium occurred not in carcinogen
treated epithelial cells but only in epithelia placed in combination with carcinogen treated mammary
mesenchyme.
Maffini, M. V., Soto, A. M., Calabro, J. M., Ucci, A. A. and Sannenschein, C. (2004). The stroma as a crucial
target in rat mammary gland carcinogenesis. J. Cell Sci. 117, 1495-1502
2. Defects in paracrine pathways:
Several key signaling pathways such as Hh, Notch, Wnt and BMP are involved in processes during
development but they have a critical role in tumorigenesis when reactivated in adult tissues through
sporadic mutations or other mechanisms.
Mechanisms by which the Hedgehog pathway can lead to cancer
(A) When Shh is a mitogen (for cerebellar granule neuron progenitor cells or hemtopoietic stem cells), loss of function of
Patched or gain in function of Smoothened activate the Hh pathway, even in absence of Shh. (B) in the autocrine mode
tumor cells both produce and respond to Shh. (C) in the papracrine model tumor cells produce and secrete the Hh ligand
and the surrounding stromal cells receive the signal and respond by producing growth factors such as VEGF or IGF.
The cancer stem cell hypothesis
 In 1971, Pierce and Johnson reported that “ malignant tissue, like normal tissue
maintains itself by proliferation and differentiation of its stem cells”.
 The existence of cancer stem cells was further confirmed By Pierce and Wallace (1971)
when they were found in rat carcinomas.
 In numerous cases such as glioblastomas, prostate cancer, melanomas and others there is
a rapidly dividing cancer stem cell (CSC) population which gives rise to more CSCs as well
as more slowly dividing differentiated cells. The CSCs thus can self-renew as well as
produce the more differentiated cell types of the tumor.
 When tumor cells are transplanted from one animal to another only CSCs give rise to new
heterogenous tumors.
 The origins of CSCs are not known it is speculated that they come from either normal
adult stem cells or progenitor cells.
Cancer stem cell and the epithelial-mesenchymal transition
 One of the most dangerous properties of CSC is their property to metastasize i.e. to migrate from the primary tumor
and form colonies in other tissues and organs.
 These migratory and colony-forming properties have been observed during development in neural crest cells as well
as when myotome cells from the somite migrate into the limb to form muscles.
 The beginning of such migration is epithelial-mesenchymal transition (EMT).
 EMT is caused by downregulation of cadherins on the surface of epithelial cells preceded by the upregulation of
expression of Slug, Snail and Twist transcription factors. Also there is reorganization of the cytoskeleton and
production of proteases.
 In the adult EMT may also lead to the production of CSCs. In vitro when breast cancer cells have undergone EMT they
express proteins characteristic of stem cells and also have the ability to seed new tumors.
 These cells also have another property similar to migrating embryonic cells i.e. their cell death pathways are blocked.
Normally epithelial cells on detachment undergo apoptosis but not these.
 Another phenomenon of metastasis involves the digestion of extracellular matrices my metalloproteinases. Migrating
embryonic cells use them to make clear path for migration. Metalloproteinases are reexpressed by cancer cells
allowing them to invade other tissues.
Cancer and epigenetic gene regulation
 Methylation patterns of mammalian genes change with age.
 What would happen if the random age-dependent patterns of gene methylation altered
the genes regulating cell division and cell signaling?
 An example is the estrogen receptor (ER). Estrogen stops the proliferation of cells in the
colon and hence ER acts as a tumor suppressor. Issa and colleagues (1994) showed that
in addition to the age related methylation of estrogen receptor loci there was much
higher level of DNA methylation of estrogen receptor genes in colon cancers.
 Jacinco and Esteller (2007) have provided evidence that the large number of mutations
that accumulate in cancer cells may have an epigenetic cause. In some cancer cells the
genes encoding DNA repair enzymes appear to be susceptible to inactivation by
methylation.
Interestingly, although environmental exposure to substances
such as cigarette smoke and endocrine disrupters can increase
DNA methylation, and certain mutations can predispose one
towards developing cancer, there is also a great deal of
random chance involved.
Rather than a dramatic reprogramming of cell fate
carcinogenesis could be the result of a slow accumulation of
hypermethylated promoter regions.
Development may thus link the genetic, environmental and
stochastic mechanisms of cancer.
Developmental therapies for cancer
 Cancers are often diseases of developmental
signaling.
 Several types of cancer cells can be normalized when
placed back into regions of embryos that express
some paracrine factors or their inhibitors.
 One new avenue for cancer treatment based on this
observation is differentiation therapy.
 In 1978 Pierce and colleagues hypothesized that
cancer cells should revert to normalacy if made to
differentiate.
 Treatment of acute promyelocytic leukemia patients
with all-trans RA results in remission in 90% of cases
because the additional RA is able to affect the
differentiation of the leukaemia cells into normal
neutrophils.
 In many tumors specific microRNAs are
downregulated which normally act as tumor
supressors by preventing changes in DNA
methylation. Adding these microRNAs to the tumor
cells may promote differentiation and hence block
cancer formation.