Greig cephalopolysyndactyly Polydactyly - extra digits Syndactyly - webbed digits Developmental...
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Transcript of Greig cephalopolysyndactyly Polydactyly - extra digits Syndactyly - webbed digits Developmental...
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
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