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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
Waardenburg’s syndrome
Mutation in PAX3 gene on chromosome 2q35
Paired-box transcription factor gene
Deafness, white forelock, iris heterochromia
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
Developmental genes discovered through mutations
Frog
Fruitfly (Drosophila)
Zebrafish
Worm (C. elegans)
Mouse
spontaneous
ENU-induced
transgenics
knock-outs
Ways to study genes in development
In situ hybridization
Whole mount
Sections
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
anterior
late
embryo
posterior
adult
ventral
early
embryo
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
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
Every stage of human (and other) development
is controlled by genes
The cell cycle
Interphase
G 1, G 2, S
Cell division (mitosis)
prophase, metaphase,
anaphase, telophase
Apoptosis (cell death)
Sperm development
Ovum development
Germ cell formation (male and female)
Fertilization
Cleavage 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
In place of normal antennae, an Ante
causes antennal precursor cells to d
Homeobox gene clusters in humans
cluster
number of genes
Chromosome
Hox 1
Hox 2
Hox 3
11
9
9
9
7p
17q
12q
2q
Hox 4
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/ DenyssDrash syndrome (abnormal sexual
differentiation and disordered renal
development)
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
Regulatory cascades: complex network of genes
coordinate developmental pathways
Cells achieve •
different roles
through series of “onoff” 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
Downstream regulatory
factors induced
New development
pathway induced
or
OFF
Default regulatory
factors operate
Default developmental
pathway maintained
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
maintenance
X:A = 0.5
X:A = 1
Sx/OFF
Sx/ON
tra/OFF
tra/ON
dsx RNA
splice
dsx RNA
splice
dsx-M
protein
dsx-F
protein
Repression of
-specific
structural genes
Repression of
-specific
structural genes
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
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
Example: Vertebrate eye development
The mouse
Whole mount in situ
hybridization
Pax6 expression in developing
mouse eye
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
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
Pax6 BMP4/
BMP7
toy
ey
so
dpp
eya
dac
Drosophila
lens placode
Eya
Six3/Optx2
Dach
Mouse/Human
Vertebrate genes
Drosophila homolog
loss of function
Pax6
eyeless, twin of eyeless
Aniridia, small eye
Bmp4
Dpp
no lens placode
Bmp7
60A
no lens placode
Eya1
eyes absent
no eye phenotype in BOR
Eya1-/-
Six3
sine oculis
Holopresencephaly
microphthalmia
Optx2
Optix
Anophthalmia
Dach1
dachshund
The Human Genome Project
As of June 26, 2000
Finished sequence•
24% of genome
Draft sequence•
85% of genome
38,000 predicted •
genes
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 Mb
5.8 % 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)•