Drosophila melanogaster
Why???
Graduate Institute of Biomedical Sciences,
Department of Biochemistry
Dr. Li-Mei Pai 醫學一 8F, 5520 pai@mail.cgu.edu.tw

• 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.

Fig. 5-12
Axis Determination during oogenesis
Gurken —TGFa
Torpedo--EGFR
1. posterior

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 kr Üppel gene activity is specified by Hunchback protein kruppel is t he 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 conservedreceptor on the target cells, intracellular effectors , changes in the activity of the target transcription factor

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

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)

Fig. 5-12
Axis Determination during oogenesis
Gurken —TGFa
Torpedo--EGFR
1. posterior
2. dorsal http://www.youtube.com/watch?v
=GntFBUa6nvs

The EGFR signal establishes the D-V axial pattern of the egg chamber
Gurken —TGFa ( 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

Cellularization---signal through transmembrane proteins
Dpp=BMP-4(TGFb
)
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