Animal model systemDrosophila melanogaster

Graduate Institute of Biomedical Sciences, Department of Biochemistry

Dr. Li-Mei Pai

Why???

醫學一 8F, 5520pai@mail.cgu.edu.tw

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 evolutionPax6 and Eyeless

Homologous genes initiate the development program for the same organ in animals separate by 500 million years of evolution

• Genes & DevelopmentMutants in the EGFR signaling pathwayLittle gene redundancy

Receptor (torpedo)

Wild type

Ligand (signal)

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

Easily to be cultured , large population

Imaginal disc

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

More than 100 genes!!

How to know who are they??tomorrow

b:balancerDTS; dominant Temp. sensitive

Superficial Cleavage in a Drosophila early EmbryoSyncytial blastoderm

Gastrulation in Drosophila

Model of Drosophila Anterior-Posterior Pattern Formation

Maternal effect genes

Zygotic genesSyncytial blastoderm

Cellular blastoderm

Egg development in Drosophila

each egg chamber: 3 types of cellsOocyte with nucleus (germinal vesicle-GV)Connected to 15 nurse cells }---germ-lineSurrounded by a monolayer of about 1000 somatic follicle cells

Egg shell

Fig. 5-10

Signals from older to younger egg chambers

Red arrow: Delta-Notch induces anterior polar follicle cellsJAK-STAT: form the stalk cellsYellow arrow: signals induce E-cadherins expression

The oocyte move towards one end in contact with follicle cellsBoth 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.

A/P Determination during oogenesis

Axis Determination during oogenesis

Gurken—TGF-aTorpedo--EGFR

1. posterior

Fig. 5-12

mRNA localization in the oocyte

Dynein-gurken and bicoid to the plus endKinesin—oskar to the minus end

The sequential expression of different sets of genes establishes the body plan along the anterior-

posterior axis

Localized mRNA and ProteinsTranslated after fertilization—

Temporal sequence

The effects of mutations in the maternal gene system

Three classesAnteriorPosteriorterminal

head and thoracic

abdominal

acron and telson

Independent Genetic Pathways Interact to Form the Anterior-Posterior Axis

Bicoid--fertilized—translatedProtein diffuses and forms morphogen gradient

No head and thoracicPrick at the anterior of normal egg

Partial rescued

Approach I: transplantationThe bicoid gene is necessary for

the establishment of the anterior structure

Approach II: expression patternThe distribution of the maternal mRNA and protein

of bicoid

Short Half life

Transcription factor---Activates zygotic gene

In situ RNAhybridization

ImmunostainingAntibody interaction

Approach III: relationship between genesPosterior determination

9 maternal genesAbnormal abdominal DevelopmentOskar localizes nanos mRNA

Nanos suppresses the translationof 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 regulationP-element mediated transformation

-hunchback expression

Thresholds of Transcription factorkrÜppel 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-numberFusi tarazu—even number

Syncytium just before cellularizationEach stripe is specified independently

Transcription networkThe specification of the second even-skipped (eve)

stripe by gap gene proteins

Bicoid and Hb activate eveKruppel 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 patternWingless=Wnt hedgehog

The cuticle of each segment in the abdomen of the adult Drosophila

Different bristles, pigmentation, and gene expressionen- clone—anterior type cuticle

Segment polarity genes and compartment

The expression of the engrailed geneAnterior margin of each parasegment

Mutations upset the A/P polarity of the segmentThey are activated in response to pair-rule genesEngrailed (en) —cell lineage boundary, defines a compartmentEn: homeodomain transcription factor

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 nucleusWith signal---full length Ci acts as an activator in the nucleus

SHH mutation-50% reduction in gene expressionholoprosencephaly,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 syndactylyabnormalities 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 withMutation in APC

C-myc target gene

Metamorphosis

Homeotic selector genes

Each segment unique identity—master regulator genesHomeotic 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 specifiersBithorax-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—10Combinatorial manner

Mutation in HoxD13—synpolydactylyExtra digits & interphalangeal webbing (hetero)Similar but more severe & bony malformation of hands, wrists (Homo)

Axis Determination during oogenesis

Gurken—TGF-aTorpedo--EGFR

1. posterior2. dorsal

Fig. 5-12

http://www.youtube.com/watch?v=GntFBUa6nvs

The EGFR signal establishes the D-V axial pattern of the egg chamber

Fig. 5-11

Gurken—TGF- (a green)Actin-cell outline (red)

Blue-dorsal anteriorFollicle cells

Torpedo--EGFR

The Key determinant in D/V polarity is pipe mRNA in follicle cells

windbeutel—ER protein pipe—heparansulfate 2-o-sulfotransferase (Golgi) nudel—serine protease

The activation of Toll

Toll protein activation results in a gradient of intranuclear dorsal protein

Spatzle is processed in the perivitelline space after fertilization

Fig. 5-8

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 activationDorsal + cactusToll activation –tube (adaptor) and pelle (kinase)Phosphorylate cactus and promote its degradation

B cell gene expressionDorsal=NF-kBCactus=I-kB

The mechanism of localization of dorsal protein to the nucleus

Fig. 5-9

Dorsalized embryo—Dorsal protein is not in nucleiDpp is everywhereTwist and snail are not expressed

Threshold effect—integrating Function of regulatory binding sites

Regulatory element=developmental switches

Nuclear gradient in dorsal protein

Fig. 5-14

Zygotic genes pattern the early embryoDorsal protein activates twist and snail represses dpp, zen, tolloid

Rhomboid----neuroectodermRepressed by snail (not most ventral)

Binding sites for dorsal protein in their regulatory regions

Model for the subdivision of the dorso-ventral axis into different regions by the gradient in nuclear dorsal protein

Fig. 5-13

Dpp protein gradient

Cellularization---signal through transmembrane proteinsDpp=BMP-4(TGF-b)Dpp protein levels high, increase dorsal cellsshort of gastrulation (sog) prevent the dpp spreading into neuroectodermSog is degraded by Tolloid (most dorsal)

References:1. Principles of Development2nd edition, by Lewis Wolpert (P48-52)

2. The genetics of axis specification in DrosophilaThe Chapter 9 of Developmental Biology by Scott Gilbert, 9th edition