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Animal model system
Drosophila melanogaster
Why???
Graduate Institute of Biomedical Sciences,
Department of Biochemistry
Dr. Li-Mei Pai
醫學一8F, 5520
[email protected]
My exploration in Science
• 東吳大學 微生物 (Bachalor)
• 陽明大學 微免所 (Master-EBV)
• 美國 北卡州立大學 教堂山分校 ( The University North
Carolina, Chapel Hill—Ph.D)
• Thesis: The function of Drosophila armadillo gene
• (Development, 1997)
•
美國 普林斯敦 大學 (Princeton University—Postdoctoral
fellow)
• Study: Identify Cbl oncogene in Drosophila body
patterning (Cell, 2000)
Functional homologous genes during evolution
Pax6 and Eyeless
Homologous genes initiate the development program for
the same organ in animals separate by 500 million years of evolution
•
Genes & Development
Mutants in the EGFR signaling pathway
Little gene redundancy
Wild type
Ligand (signal)
Receptor (torpedo)
GAP (negative regulator)
Fewer genes
4 pairs of chromosomes
Functions of 13,600 genes???
Development of the Drosophila body plan
Axis determination
Signaling pathway
Transcriptional and translational regulationfunctions
Life cycle of Drosophila (very short)
4 stages: embryo, larva, pupa, adult
Imaginal disc
Easily to be cultured , large population
Edward B.
Lewis
Christiane
Nüsslein-Volhard
Eric F.
Wieschaus
Body patterning of fly
One cell to an organism
Genetic screening strategy for identifying
developmental mutants
b:balancer
DTS; dominant Temp. sensitive
More than 100 genes!!
How to know who are they??
tomorrow
Superficial Cleavage in a Drosophila early Embryo
Syncytial blastoderm
Gastrulation in Drosophila
Model of Drosophila Anterior-Posterior
Pattern Formation
Maternal effect genes
Zygotic genes
Syncytial blastoderm
Cellular blastoderm
Egg development in Drosophila
Egg shell
Fig. 5-10
each egg chamber: 3 types of cells
Oocyte with nucleus (germinal vesicle-GV)
Connected to 15 nurse cells }---germ-line
Surrounded by a monolayer of about 1000 somatic follicle cells
Signals from older to younger egg chambers
Red arrow: Delta-Notch induces anterior polar follicle cells
JAK-STAT: form the stalk cells
Yellow arrow: signals induce E-cadherins expression
A/P Determination during oogenesis
The oocyte move towards one
end in contact with follicle cells
Both the oocyte and the posterior
follicle cells express high levels
of the E-cadherin
If E-cadherin is removed,
the oocyte is randomly positioned.
Then the oocyte induces surrounding
follicle cell to adopt posterior fate.
Axis Determination during oogenesis
Gurken—TGF-a
Torpedo--EGFR
1. posterior
Fig. 5-12
mRNA localization in the oocyte
Dynein-gurken and bicoid to the plus end
Kinesin—oskar to the minus end
The sequential expression of different sets of genes
establishes the body plan along the anteriorposterior axis
Localized mRNA and Proteins
Translated after fertilization—
Temporal sequence
The effects of mutations in
the maternal gene system
Three classes
Anterior
Posterior
terminal
head and thoracic
abdominal
acron and telson
Independent Genetic Pathways Interact to Form the
Anterior-Posterior Axis
Approach I: transplantation
The bicoid gene is necessary for
the establishment of the anterior structure
Bicoid--fertilized—translated
Protein diffuses and forms
morphogen gradient
No head and thoracic
Prick at the anterior of normal egg
Partial rescued
Approach II: expression pattern
The distribution of the maternal mRNA and protein
of bicoid
Short Half life
In situ RNA
hybridization
Transcription factor--Activates zygotic gene
Immunostaining
Antibody
interaction
Approach III: relationship between genes
Posterior determination
9 maternal genes
Abnormal abdominal Development
Oskar localizes nanos mRNA
Nanos suppresses
the translation
of the maternal mRNA
of Hunchback(hb)
The expression of Gap genes
First zygotic genes—transcription factors
Mutant –large section of the body is missing
Blastoderm—proteins diffuse away
but with short half life
Approach IV: the effects of gene
copies
Maternal bicoid protein controls
zygotic hunchback expression
Dosages of maternal bicoid genes
Bicoid = homeodomain transcription factor
Approach V transcriptional regulation
P-element mediated transformation
-hunchback expression
Thresholds of Transcription factor
krÜppel gene activity is
specified by Hunchback protein
kruppel is the target genes of hunchback
Increase dose of hunchback –
kruppel shift posteriorly
The striped patterns of activity of pair-rule genes
Pair-rule genes in 14 segments
Even-skipped—odd-number
Fusi tarazu—even number
Syncytium just before cellularization
Each stripe is specified independently
Transcription network
The specification of the second even-skipped (eve)
stripe by gap gene proteins
Bicoid and Hb activate eve
Kruppel and Giant repress eve
Sites of action of activating and repressing
transcription factors
Segment polarity
A/P axis within one segment
Ventral epidermis of the abdomen—ventral denticle belts (anterior)
Mutation—alter the denticle pattern
Wingless=Wnt
hedgehog
The cuticle of each segment in the abdomen of the
adult Drosophila
Different bristles, pigmentation, and gene expression
en- clone—anterior type cuticle
Segment polarity genes and compartment
Mutations upset the A/P polarity of the segment
They are activated in response to pair-rule genes
Engrailed (en) —cell lineage boundary, defines a compartment
En: homeodomain transcription factor
The expression of the engrailed gene
Anterior margin of each parasegment
Interactions between hedgehog, wingless,
and engrailed
hh turn on wg expression,
wg maintain en expression
The hedgehog signaling pathway
Without signal—Ci is processed as a repressor into nucleus
With signal---full length Ci acts as an activator in the nucleus
SHH mutation-50% reduction in gene expression
holoprosencephaly,or failure of the midface and
forebrain to develop
(cleft lip and palate, hypotelorism)
Signaling pathways are conserved-receptor on the target cells,
intracellular effectors, changes in the activity of the target
transcription factor
Malformation: Polydactyly and syndactyly
abnormalities in one or more genetic programs
Greig cephalopolysyndactyly (GCPS):
loss of function mutation in GLI3 (Ci) —transcription factor
The wingless signaling pathway
More than 50%
Colon cancr with
Mutation in APC
C-myc target gene
Metamorphosis
Homeotic selector genes
Each segment unique identity—master regulator genes
Homeotic selector genes—control other genes-required
throughout development
Vertebrate Hox gene complex
Homeotic transformation of the wing and haltere
Homeotic genes—mutated into
homeosis transformation
As positional identity specifiers
Bithorax-haltere into wing
The spatial pattern of expression of
genes of the bithorax complex
Bithorax—Ultrabithorax –5-12
Abdominal-A—7-13
Abdominal-B—10-13
Bithorax mutant –PS 4 default state
Bithorax mutant –PS 4 default state
+Ubx—5,6
+Abd-A—7,8,9
+Abd-B—10
Combinatorial manner
Mutation in HoxD13—synpolydactyly
Extra digits & interphalangeal webbing (hetero)
Similar but more severe & bony malformation
of hands, wrists (Homo)
Axis Determination during oogenesis
Gurken—TGF-a
Torpedo--EGFR
1. posterior
2. dorsal
Fig. 5-12
http://www.youtube.com/watch?v
=GntFBUa6nvs
The EGFR signal establishes the D-V axial pattern
of the egg chamber
Gurken—TGF-a (green)
Actin-cell outline (red)
Fig. 5-11
Blue-dorsal anterior
Follicle cells
Torpedo--EGFR
The Key determinant in D/V polarity is
pipe mRNA in follicle cells
The activation of Toll
windbeutel—ER protein
pipe—heparansulfate 2-o-sulfotransferase (Golgi)
nudel—serine protease
Toll protein activation results in a gradient of
intranuclear dorsal protein
Fig. 5-8
Spatzle is processed in the perivitelline space after fertilization
The mechanism of localization of
dorsal protein to the nucleus
1. Toll mutant – dorsalized
(no ventral structure)
2. Transfer wt cytoplasm into Toll mutant
specify a new dorsal-ventral axis
(injection site =ventral side)
Without Toll activation
Dorsal + cactus
Toll activation –
tube (adaptor) and pelle (kinase)
Phosphorylate cactus and promote
its degradation
B cell gene expression
Dorsal=NF-kB
Cactus=I-kB
Fig. 5-9
Nuclear gradient in dorsal protein
Fig. 5-14
Dorsalized embryo—
Dorsal protein is not in nuclei
Dpp is everywhere
Twist and snail are not expressed
Threshold effect—integrating
Function of regulatory
binding sites
Regulatory element
=developmental switches
Model for the subdivision of the dorso-ventral axis
into different regions by the gradient
in nuclear dorsal protein
Zygotic genes pattern the early embryo
Dorsal protein activates twist and snail
represses dpp, zen, tolloid
Rhomboid----neuroectoderm
Repressed by snail (not most ventral)
Binding sites for dorsal protein
in their regulatory regions
Fig. 5-13
Dpp protein gradient
Cellularization---signal through transmembrane proteins
Dpp=BMP-4(TGF-b)
Dpp protein levels high, increase dorsal cells
short of gastrulation (sog) prevent the dpp spreading into neuroectoderm
Sog is degraded by Tolloid (most dorsal)
References:
1. Principles of Development
2nd edition, by Lewis Wolpert (P48-52)
2. The genetics of axis specification in Drosophila
The Chapter 9 of Developmental Biology by Scott
Gilbert, 9th edition