Greig cephalopolysyndactyly

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Genes in Development - November 5, 2000 Karen
B. Avraham, Instructor
Developmental malformation syndrome
Greig cephalopolysyndactyly Polydactyly - extra
digits Syndactyly - webbed digits
Mutation in GLI3 gene on chromosome 7 Zinc finger
gene Cranial, hand abnormalities
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Waardenburgs syndrome
Mutation in PAX3 gene on chromosome
2q35 Paired-box transcription factor
gene Deafness, white forelock, iris heterochromia
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Brief outline of human development
Fertilization Pre-embryonic stage first cell
division zygote reaches uterine
cavity formation of bilaminar disc formation of
trialaminar disc Embryonic stage cranio-caudal
and dorso-vental axes established cellular
aggregation and differentiation - tissue and
organ formation Fetal stage rapid growth and
development
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Developmental genes discovered through mutations
Frog
Fruitfly (Drosophila)
Zebrafish
Worm (C. elegans)
Mouse spontaneous ENU-induced transgenics knoc
k-outs
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Ways to study genes in development
In situ hybridization
Whole mount
Sections
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Life begins with a single cell Reaches maturity
with trillions of cells combined into complex
organism with many organ systems General body
plan Insect 6 legs mammals 4 legs All must
differentiate the anterior from the posterior end
and the dorsal from the ventral side
dorsal
late embryo adult
posterior
anterior
ventral
early embryo
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During establishment of body plan, cells adopt
specific cell fates Cell fates the capacity to
differentiate into particular kinds of
cells Determination process of commitment to a
particular fate As cells proliferate, decisions
are made to specify fate of cells Cells make
developmental decisions in context of decisions
made by their neighbors
Inner ear
Single fate
Totipotent uncommitted
Eye
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• Genetic dissection of cell fates
• 15 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

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Every stage of human (and other) development is
controlled by genes
The cell cycle Interphase G1, G2, S Cell
division (mitosis) prophase, metaphase,
anaphase, telophase Apoptosis (cell
death) Sperm development Ovum development Germ
cell formation (male and female) Fertilization Cle
avage and implantation Etc..
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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

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• 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

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Homeobox gene clusters in humans
cluster Hox 1 Hox 2 Hox 3 Hox 4
Chromosome 7p 17q 12q 2q
number of genes 11 9 9 9

• 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)

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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 Waardenburgs
  syndrome
• (rearrangements cause rare childhood
  tumor,alveolar rhabdomyosarcoma)

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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 (Wilms tumor gene)
• Increased risk of renal malignancy/
  Denyss- Drash syndrome (abnormal sexual
  differentiation and disordered renal
  development)

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Apoptosis
C. elegans
Drosophila
mammals
Suicide of supernumary, misplaced or damaged
cells Activation of evolutionarily conserved
molecular program Dysfunctions implicated in
developmental abnormalities and disease
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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
or
ON
OFF
Downstream regulatory factors induced
Default regulatory factors operate
New development pathway induced
Default developmental pathway maintained
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Example Sex Determination
Early Drosophila embryo

• Relies on regulation of one transcription factor
  by another
• Ratio of X chromosome to sets of autosomes (XA
  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

XA 1 XA 0.5
Sx/ON Sx/OFF
maintenance
tra/ON tra/OFF
dsx RNA dsx RNA splice
splice
dsx-F dsx-M protein protein
Repression of -specific structural genes
Repression of -specific structural genes
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Example Development of male germ cells
Germ cells highly specialized cells for
transmitting genetic information to the next
generation Separated from somatic lineages at
early stage of embryogenesis Germ cell
specification takes place during early
gastrulation Germ-line precursors give rise to
primordial germ cells (PGC) Germ-line precursors
located in rim of epiblast adjacent to
extra-embryonic ectoderm before
gastrulation PGC identified in the gastrulating
mouse embryo at 7.25 days postcoitum
(dpc) Proliferating PGCs migrate into genital
ridges around 10.5-11.5 dpc PGCs colonizing
genital ridge differentiate into precursor cells
of either male or female gametes under control
of cell interactions in developing gonad
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Genes involved in formation of germ cell
precursors

• Germ cell precursors - pole cells
• Genetic studies in Drosophila has led to
  discovery of genes involved
• Oskar, 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

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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 hybridization Pax6 expression
in developing mouse eye
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• Ectodermally derived eye imaginal disc
• Morphogenetic furrow moves from posterior to
  anterior
• Progress of furrow driven by wave of ommatidial
  differentiation

Drosophila
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Genetic pathway controlling eye development
BMP4/BMP7
Pax6 lens placode Dach
toy ey dac
dpp
Eya Six3/Optx2
so eya
Drosophila Mouse/Human
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Vertebrate genes Drosophila homolog
loss of function
Aniridia, small eye no lens placode no lens
placode no eye phenotype in BOR Eya1-/- Holopres
encephaly microphthalmia Anophthalmia
Pax6 Bmp4 Bmp7 Eya1 Six3 Optx2 Dach1
eyeless, twin of eyeless Dpp 60A eyes
absent sine oculis Optix dachshund
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The Human Genome Project

• As of June 26, 2000
• Finished sequence
• 24 of genome
• Draft sequence
• 85 of genome
• 38,000 predicted genes

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Comparative Mapping and Sequencing

• Saccharomyces cerevisiae (Bakers 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 Mb
• 5.8 of genome

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Genes in Disease and Development

• Cystic fibrosis (CFTR)
• Huntingtons disease (Huntingtin)
• Ataxia talengiesta (ATM)
• Retinoblastoma (RB1)
• Wilsons disease (ATP7B)
• Gauchers disease (2 genes)
• Deafness ( 100 genes)