Greig cephalopolysyndactylyPolydactyly - extra digitsSyndactyly - webbed digits

Developmental malformation syndrome

Mutation in GLI3 gene on chromosome 7Zinc finger geneCranial, hand abnormalities

Genes in Development - November 5, 2000Karen B. Avraham, Instructor

Waardenburg’s syndrome

Mutation in PAX3 gene on chromosome 2q35Paired-box transcription factor geneDeafness, white forelock, iris heterochromia

Brief outline of human development

FertilizationPre-embryonic stagefirst cell divisionzygote reaches uterine cavityformation of bilaminar discformation of trialaminar discEmbryonic stagecranio-caudal and dorso-vental axes established

cellular aggregation and differentiation -> tissue and organ formationFetal stagerapid growth and development

Developmental genes discovered through mutations

Fruitfly (Drosophila)

Zebrafish

Worm (C. elegans)MousespontaneousENU-inducedtransgenicsknock-outs

Frog

Ways to study genes in development

In situ hybridization

Sections

Whole mount

Life begins with a single cellReaches maturity with trillions of cells combined into

complex organism with many organ systemsGeneral body planInsect 6 legsmammals 4 legsAll must differentiate the anterior from the posterior end and the dorsal from the ventral side

ventral

posterioranterior

dorsal

earlyembryo

late embryo

adult

During establishment of body plan, cells adopt specific cell fates

Cell fates: the capacity to differentiate into particular kinds of cellsDetermination: process of commitment to a particular fateAs cells proliferate, decisions are made to specify fate of cellsCells make developmental decisions in context of decisions made by their “neighbors”

Totipotentuncommitted

Single fate

Inner ear

Eye

Genetic dissection of cell fates15 years ago

• Description of mutant phenotypes• Microsurgical manipulations of embryos

Today• Combination of genetics and recombinant DNA

techniques• Can now identify protein products contributing to these

developmental events• Can fish out related genes from different organisms

• Same basic set of regulatory proteins govern major developmental events in all higher animals

Every stage of human (and other) development is controlled by genes

The cell cycleInterphaseG1, G2, SCell division (mitosis)prophase, metaphase ,

anaphase, telophaseApoptosis (cell death)Sperm developmentOvum developmentGerm cell formation (male and female)FertilizationCleavage and implantation

Etc..…

Genes involved in early development: Transcription factors

• Control RNA transcription from DNA template by binding to specific regulatory DNA sequences

• Switch genes on and off by activating or repressing gene expression

• Control many genes involved in segmentation, induction, migration, differentiation, and apoptosis (programmed cell death)

• Three gene families in vertebrates• homeotic genes• paired box genes• zinc finger genes

Homeotic mutation• Homeosis - replacement of one body part by another

• In place of normal antennae, an Antennapedia mutation causes antennal precursor cells to develop into a leg

Homeobox gene clusters in humans

cluster

Hox 1 Hox 2 Hox 3

Hox 4

number of genes

11999

Chromosome

7p17q12q2q

• Conserved 180 bp sequence - homeobox• In each Hox cluster, there is direct linear correlation between

position of gene and its temporal and spatial expression• CHX10 (14q) micropthalmia (congenital blindness) in humans• Hand-foot-genital syndrome (HFGS), 7p, HOXA13 in humans• Transgenic mice have multiple severe abnormalities (face &

skull)

Paired-box (PAX) genes

• Highly conserved DNA sequence that encodes ~130 aa• First identified in Drosophila• Encode DNA binding proteins

• 8 Pax genes identified in mice and humans

• Mutations in Pax1 cause vertebral malformations in mice• Mutations in Pax3 cause pigmentary abnormalities in mice

• Mutations in Pax6 cause small eyes in mice

• In humans, mutations in PAX6 cause aniridia (no iris)• In humans, mutations in PAX3 cause Waardenburg’s

syndrome(rearrangements cause rare childhood tumor,alveolar

rhabdomyosarcoma)

Zinc finger genes

• Finger-like projection formed by amino acids between 2 separated cysteine residues which form complex with zinc ion

• Many DNA binding proteins contain zinc fingers• GLI3 - Greig cephalopolysyndactyly

• WT1 (Wilm’s tumor gene) Increased risk of renal malignancy/ Denyss-

Drash syndrome (abnormal sexual differentiation and disordered renal

development)

C. elegans

Drosophila

mammals

Apoptosis

Suicide of supernumary, misplaced or damaged cellsActivation of evolutionarily conserved molecular programDysfunctions implicated in developmental abnormalities and disease

Regulatory cascades: complex network of genes coordinate developmental pathways

• Cells achieve different roles through series of “on-off” decisions

• Conditions within cell allow a master switch to be regulated

• Once master switch is activated, it sets in motion a cascade of “downstream” regulatory events

• In absence of activation of master switch, set of default signals remain in place

MASTER SWITCH

ON OFF

New development pathway induced

Default developmental pathway maintained

or

Downstream regulatory factors induced

Default regulatory factors operate

Example: Sex Determination

• Relies on regulation of one transcription factor by another

• Ratio of X chromosome to sets of autosomes (X:A ratio) in early embryo establishes whether fly becomes male or female

• Sexual differentiation carried out by master regulatory switch and several downstream sex-specific genes

Early Drosophila

embryo

Repression of -specific

structural genes

Repression of -specific

structural genes

X:A = 1 X:A = 0.5

Sx/ON Sx/OFF

tra/ON tra/OFF

dsx-F dsx-Mprotein protein

dsx RNA dsx RNA splice splice

maintenance

Example: Development of male germ cellsGerm cells

highly specialized cells for transmitting genetic information to the next generation

Separated from somatic lineages at early stage of embryogenesisGerm cell specification takes place during early gastrulationGerm-line precursors give rise to primordial germ cells (PGC)

Germ-line precursors located in rim of epiblast adjacent to extra-embryonic ectoderm before gastrulationPGC identified in the gastrulating mouse embryo at 7.25 days

postcoitum (dpc)Proliferating PGCs migrate into genital ridges around 10.5-11.5 dpcPGCs colonizing genital ridge differentiate into precursor cells of

either male or female gametes under control of cell interactions in developing gonad

Genes involved in formation of germ cell precursors

Germ cell precursors - pole cellsGenetic studies in Drosophila has led to discovery of genes

involvedOskar, Nanos, Tudor Vasa

• member of DEAD-box family of genes encoding ATP-dependent RNA helicase

• required for assembly and function of pole plasm• identified in many animal species, where it is expressed

specifically in germ-cell lineages• C. elegans - P-granules of eggs

• Xenopus - germinal granules of eggs• zebrafish • mouse - Mvh

Knock-out

Example: Vertebrate eye development• E8.5: the optic vesicle

forms as out-pouching of forebrain

• E9.0: optic vesicle contacts endoderm of head

• E9.5: signals from optic vesicle induce lens placode

• E10.0: lens placode invaginates to lens pit; optic vesible inaginates to create optic cup

• E10.5: invagination of lens pit to form lens vesicle complete. Lens vesicle detaches from overlying ectoderm

• E12.5: differentiation of optic cup into neuroretina and epithelium

The mouse

Whole mount in situ hybridizationPax6 expression in developing mouse eye

• Ectodermally derived eye imaginal disc

• Morphogenetic furrow moves from posterior to anterior

•Progress of furrow driven by wave of ommatidial differentiation

Drosophila

Genetic pathway controlling eye development

toy

ey

dac

dpp

so eya

Pax6

lens placode

Dach

BMP4/BMP7

Eya Six3/Optx2

Drosophila Mouse/Human

Vertebrate genes Drosophila homolog loss of function

Pax6

Bmp4

Bmp7

Eya1

Six3

Optx2

Dach1

eyeless, twin of eyeless

Dpp

60A

eyes absent

sine oculis

Optix

dachshund

Aniridia, small eye

no lens placode

no lens placode

no eye phenotype in BOREya1-/-

Holopresencephalymicrophthalmia

Anophthalmia

As of June 26, 2000

• Finished sequence24% of genome

• Draft sequence85% of genome

• 38,000 predicted genes

The Human Genome Project

Comparative Mapping and Sequencing

Saccharomyces cerevisiae (Baker’s yeast)• 1996• 15 Mb

• 6000 genes

Caenorhabditis elegans (nematode)• 1998• 99 Mb

• 19,000 genes

Drosophila melanogaster (fruitfly)• 1999

• 120 Mb euchromatic genome• 13,000 genes

Sequencing of the Mouse Genome• Finished sequence 20.3 Mb

0.65 % of genome• Draft sequence

180 Mb5.8 % of genome

Genes in Disease and Development

• Cystic fibrosis (CFTR)• Huntington’s disease (Huntingtin)

• Ataxia talengiesta (ATM)• Retinoblastoma (RB1)

• Wilson’s disease (ATP7B)• Gaucher’s disease (2 genes)

• Deafness (> 100 genes)