DEVELOPMENTAND

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 X-inactivation, 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 DevelopmentCongenital (“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

OR2) 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 no 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, heat 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 overproduction of these regulatory proteins and RNA.

Such overproduction would cause the misregulation of genes necessary for heart, muscle and nerve formation.

One such miR155, is encoded on chromosome 21 and found throughout the human fetus. This miRNA downregulates the translation of messages encoding certain transcription factors required for normal heart and neural development.

miR155 is highly elevated in brains and hearts of people with Downs syndrome.

Fluorescent in-situ hybridization with probes for chromosome 21 (pink) and chromosome 13 (blue) show that this person has three copis of chromosome 21 but two copies of chromosome 13.

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 DevelopmentIn 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://radiopaedia.org/images/4669603http://www.bbc.co.uk/schools/gcsebitesize/science/aqa/drugs_use/drugsrev2.shtml

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.

http://pixgood.com/ddt-eggs.html

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.

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 (ethanol) as a teratogen

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.

Alcohol induced cranio-facial 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.

Cell death caused by exposure to alcohol 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 damsge the cell membrane.

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.

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 teratogenRetinoic 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.

A paradoxical teratogenic mechanism for retinoic acidLee et al , 13668–13673|PNAS|August 21, 2012|vol. 109|no. 3

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

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 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.

Conjoined trout hatchlings from the Great lakes

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 prouces 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• 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 anti-testosterone.

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 disrupterDiethylstilbestrol 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.

BPA induces altered mammary gland developmentMammary glands from newborn female rhesus

monkeys (A) control (B) exposed in utero to BPA

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 tertocarcinoma 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 pf 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.Stroma is not a major target in DMBA-mediated tumorigenesis of mouse mammary preneoplasia KittreDaniel Medina* and Frances ll, Journal of Cell Science 118, 123-127 (2004).

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) ppopulatopulation 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 preceeded 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 breat 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 regulationMethylation 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 my 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 expresse 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.