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Drosophila melanogaster Genetic studies Microsurgical manipulation One of the best understood developmental systems 13,600 genes Axis determination PowerPoint PPT Presentation


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Drosophila melanogaster Genetic studies Microsurgical manipulation One of the best understood developmental systems 13,600 genes Axis determination Signaling pathway Transcriptional regulation. P48-52. 4 stages: embryo, larva, pupa, adult . Rapid division 9 mins/division 9 divisions.

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Drosophila melanogaster Genetic studies Microsurgical manipulation One of the best understood developmental systems 13,600 genes Axis determination

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Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Drosophila melanogaster

Genetic studies

Microsurgical manipulation

One of the best understood developmental systems

13,600 genes

Axis determination

Signaling pathway

Transcriptional regulation

P48-52


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

4 stages: embryo, larva, pupa, adult


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Rapid division

9 mins/division

9 divisions

13 divisions

Single cell


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Transgenic flies


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

One single epithelial layer –all tissues

Mesoderm—muscle, connective tissues

Endoderm---midgut (foregut and hidgut-

Ectoderm)

Ectoderm---nervous tissue and epidermis


Gastrulation

gastrulation


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Larva hatch 24 hrs

Acron: associated with head

Telson:posterior terminal structure

3 thorcic and 8 abdominal segments—specialization in cuticle

(denticle belts and cuticular structure)


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Genitalia

Sex comb

Pigmentation

Small wing

p. 421-431


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Sex determining signal--- Sex-lethal (X chromosome)

Transformer-spliced + transformer 2


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Repression by autosome

2X higher numerator


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Dosage compensation

Barr body

Xist-non-coding RNA

Male specific gene

Repressed by Sxl


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Primordial germ cell -special cytoplasm

Germ plasm-polar granules, pole plasm


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Oskar—organization and assembly of

the pole plasm

mRNA-posterior pole—3’ untranslated region


Polarization of the body axes during oogenesis

Polarization of the body axes during oogenesis


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Cyst formation:

16 cell cyst enter a long S phase

only one (Oocyte) continues meiosis

Oocyte—4 ring canals

15 cells become nurse cells

after germarium nurse cells left meiotic cycle, grow rapidly without division, and form polytene chromosomes


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

A/P 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.


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Microtubule cytoskeleton reorganization

is essential for localization of

bicoid and oskar mRNA


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Maternal effect mutations---reguired for

pole plasm assembly

Lack polar granules: grandchildless mutation (homozygote female—

Progeny—sterile)

Central role: Oskar, Vasa, and Tudor

Pole cell number = amount of oskar RNA

Ectopic pole cells: oskar RNA at the anterior pole

MtlrRNA (mitochondrically encoded large

ribosomal RNA)

+ gcl RNA for pole cell formation

antisense reduce pole cells

mtlrRNA rescue UV-irradation

Vasa: DEAD-box RNA helicase—translational regulator


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Germ cell migration

Germ cell—extragonadal origin, migrate to

reach the somatic gonad

a. posterior end

b. gastrulation

c. migrate dorsally through the wall of the posterior midgut

d. associate with the somatic gonadal precursors

e. GC align with somatic gonadal mesoderm

f. coalesce to form the embryonic gonad


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

PGC migration----Genes and mechanisms

Genetic screen—somatically expressed genes

Guidance (cues):

Wunen: repulsive signal (exclude migrating pole cells from wrong places)

Misexpression wunen: transform a tissue permissive to PGC to repulsive one

Phosphatidic acid phosphatase 2 (transmembrane protein)

Columbus: factor (gonadal mesoderm) attracts pole cells

Misexpression Columbus—attract PGCs to tissues other than gonadal mesoderm

3-hydroxy-3-methylglutaryl coenzymeA reductase (cholesterol biosynthesis in human, but fly does not make cholesterol)

nanos, pumilio mutants stall at the outer gut surface

differentiate prematurely---act as complete migration to the somatic gonads

nanos target: RNA binding protein Sex lethal (Sxl)---splicing and translational regulation

also depend on specific germ plasm components, e.g polar granule component (Pgc)


Patterning of the fly embryo

Patterning of the fly embryo


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Localized mRNA and Proteins

Translated after fertilization—

Positional information to activate zygotic genes

Temporal sequence

parasegment

Pattern in the segment

Segment identities


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

p.350-358

Appendages:imaginal discs—pattern formation

Ectoderm invagination-epithelium(20-40 cells—larva 1000X)

Specification occurs –segment being patterned-according to it


A p and d v compartment

A/P and D/V compartment


Wing blade

Wing blade

Ventral fold under dorsal-double layers of epithelium


Signal region and the compartment

Signal region and the compartment

Maintain compartment boundaries—communication between compartments

Hh—10 cells, induces expression of Dpp through activation of Ci


The hedgehog signaling pathway

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


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Intercellular signaling set up PS boundary

Wg distributed asymmetrically—less in posterior (endocytosis and degradation)


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

TGFb , Activin: R-Smad 2,3

BMPs: R-Smad 1, 5, 8

Common Smad4

Inhibitory Smads: I-Smad6, 7—recruting

Smurf (ubiquitin ligase to

receptor)

Cell, 95,737,1998


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Smad= Sma + Mad

Sma-C. elegans

Mad-Fly


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Dpp-secreted into both compartment

Long range signal—expression of spalt


Patterning the a p axis of the wing disc

Patterning the A/P axis of the wing disc

  • Dpp-morphogen

  • Low level—omb

  • High level—spalt

  • Clones can’t respond to Dpp—

  • no spalt and omb

  • 2. Ectopic hh-Dpp—localized

  • activation of spalt and omb

  • around the hh clones

  • 3. Ts mutant of dpp—reduction in

  • the region with expression of low

  • Threshold genes—omb

  • 4. Clones expression low or high

  • Dpp—distinquish these two types

  • of genes


Ectopic expression of hh and dpp

Ectopic expression of Hh and Dpp

L4—compartment boundary

L3– Hh

L2 ---adjacent to cells expressing spalt

Ectopic Hh in posterior—no effects

In anterior—mirrow-symmetric repeated pattern

Hh--Dpp


Expression of wingless green and vestigial

Expression of Wingless (green) and vestigial

Homeotic selector gene—

apterous (Lmx-1)

induces fringe and Serrate,

then Notch receptor activation –

Leading to Wingless expression

Wg—achaete, distal-less, vestigial

Wingless (green)

Vestigial (red)

D/V boundary

Dpp, Wg morphogen

GFP-dpp active transportation—

Endocytosis

Regulate their receptors

Dpp inhibits receptor—thick veins

Dpp high--receptor low, and dpp low

Receptor high—1, prevent spreading

2,cells reach threshold at low Dpp


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

nervous system: selection of a single neuroblast (lateral inhibition)

Notch –transmembrane protein

DSL family—Delta, Serrate, Lag-2

Kuzbanian—cleave Notch ECD

Presenilin—cleave Notch ICD


Leg disc extension

Leg disc extension

Jointed tubes of epidermis—secrete the hard cuticle (exoskeleton), inside:

Muscles, nerves


Fate map of the leg imaginal disc

Fate map of the leg imaginal disc

Proximo-distal segment

Center—distal end


Signaling centers in a p compartment

Signaling centers in A/P compartment

Dpp, wg meets—Dll (distal end)

homothorax (proximal)


Regional subdivision

Regional subdivision

Dpp, and Wg induce Dll and

inhibit homothorax

Activates dachshund between Dll and hth


Butterfly wing pattern

Butterfly wing pattern

Eyespot center—distal-less


Segmental identity of imaginal disc

Segmental identity of imaginal disc

Homeotic selector genes

Similar signal into different structures—

Different interpretation—

controlled by Hox genes

Antennapedia—PS4 and 5– 2 pairs of legs

If in head, antennae into legs (clones) –

which part of the leg—depends on their

position along the P/D axis

(positional values are similar)

Hth (proximal) and Dll (distal)—in antennae and leg

In combination as selector to specify antenna

No Hth, antenna into leg

In leg: antennapedia prevents Hth and Dll acting together

Dominant antennapedia mutant (gene on)—

blocks Hth and Dll in antennae disc, so leg forms


Imaginal discs and adult thoracic appendages

Imaginal discs and adult thoracic appendages

Bithorax mutation—Ubx misexpressed

T3 into T2 –anterior haltere into

Anterior wing


Drosophila melanogaster genetic studies microsurgical manipulation one of the best understood developmental systems 13

Postbithorax muation (pbx)—

Regulatory region of the Ubx—

Posterior of the haltere into wing

If both mutations—effect is additive—

Four wings


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