1. 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

2. Waardenburg’s syndrome
Mutation in PAX3 gene on chromosome 2q35
Paired-box transcription factor gene
Deafness, white forelock, iris heterochromia

3. 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

4. Developmental genes discovered through mutations
Frog
Fruitfly (Drosophila)
Zebrafish
Worm (C. elegans)
Mouse
spontaneous
ENU-induced
transgenics
knock-outs

5. Ways to study genes in development
In situ hybridization
Whole mount
Sections

6. 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
anterior
posterior
ventral
early
embryo

7. 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

8. 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

9. 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
Cleavage and implantation
Etc…..

10. 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

11. 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

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

13. 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)

14. 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)

15. 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

16. 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 or
OFF
Downstream regulatory factors induced
Default regulatory factors operate
New development pathway induced
Default developmental pathway maintained

17. Early Drosophila embryo
Example: Sex Determination
Early Drosophila embryo
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
X:A = X:A = 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

18. Example: Development of male 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 dpc
PGCs colonizing genital ridge differentiate into precursor cells of either male or female gametes under control of cell interactions in developing gonad

19. 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

20. 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

21. Ectodermally derived eye imaginal disc
Morphogenetic furrow moves from posterior to anterior
Progress of furrow driven by wave of ommatidial differentiation
Drosophila

22. Genetic pathway controlling eye development
BMP4/BMP7
toy ey
dac
Pax6
lens placode
Dach
dpp
so eya
Eya Six3/Optx2
Drosophila Mouse/Human

23. 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 eye phenotype in BOR
Eya1-/-
Holopresencephaly
microphthalmia
Anophthalmia

24. The Human Genome Project
As of June 26, 2000
Finished sequence
24% of genome
Draft sequence
85% of genome
38,000 predicted genes

25. 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
The ability to undertake DNA sequencing on a large scale started a revolution in biology.
Became possible to determine complete sequence of any organism and obtain full description of genes and other important biological information stored in genomes.
1. Possible to define complete set of proteins required for life form
2. Possible to make full comparisons of protein sets between 2 species, to discover basis of similarities and differences, and to explore evolutionary relationship between them.
S. cerevisiae genes encode all functions required for a eukaryotic cell.
C. elegans: Complete set of genes for multicellular organism.
Encoding proteins involved in basic eukaryotic cell functions, development, cell-cell interactions, motility, feeding and reproduction.
Sequencing of the Mouse Genome
Finished sequence 20.3 Mb
0.65 % of genome
Draft sequence
180 Mb
5.8 % of genome

26. 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) CF
Inheritance - autosomal recessive
Location - chromosome 7q31
Mutation - deletion of 3 bp at codon 508 HD
Inheritance - autosomal dominant
Location - 4p16.3
Mutation - cytosine/adenine/guanine repeat >35 times
RB1
Childhood tumors of the retina are associated with inactivation of the retinoblastoma gene
ATP7B
In Wilson’s disease, toxic levels of copper accumulate and damage many tissues and organs, including the basal ganglia of the brain accounts for 70% of mutations