CHAPTER 11 – Drosophila - Micropyle

o Hole in the anterior end of chorion

o Where sperm enters the oocyte
Insects
- Anteroposterior sequence of segments
o Head – 6 segments CLEAVAGE AND BLASTULA
▪ Procephalic – 3
▪ Gnathal – 3 Superficial cleavage
o Thorax – 3 segments • Rapid synchronous nuclear divisions without cellular
▪ Prothorax (T1) cleavage
▪ Mesothorax (T2)
▪ Metathorax (T3) Syncytium
▪ T1,T2,T3 – pair of legs (ventral side) • Large cytoplasmic mass with several nuclei
▪ T2,T3 – pair of wings (dorsal side)
o Abdomen – 8-11 segments (varies/sp) 8th nuclei division
• pole cells are formed at the posterior end
Drosophila melanogaster (Fruit fly)
- First organism whose development was understood • incorporating the nuclei at the posterior end of pole plasm,
- Life cycle – 2 weeks becoming the drosophila's germ line
- Small size
- No procephalic segments 9th nuclei division
- Gnathal segments appear transiently • most nuclei migrate to the periphery to form the syncytial
- Only T2 bears wings blastoderm
- T3 – Halteres (balancing structures)
- 8 abdominal segments (A1-A8) • those nuclei remaining internally are later incorporated into
- Principal nerve cord – ventral side vitellophages which end up in the gut lumen
- Heart – dorsal side
- No specialized vascular system 3rd Hour
o Oxygen is brought to the tissues by diffusion • cell membranes grow inwards to form plasma membrane to
through trachea separate the nuclei forming the cellular blastoderm
- Holometabolous
o Undergoes abrupt and complete metamorphosis **the columnar epithelium of blastoderm is quite thick, destined to
o Egg->larva (2molts)->pupa->adult fly become part of embryo. Only a think dorsal strip becomes an
extraembryonic antisera
GAMETOGENESIS

a. Spermatogenesis GASTRULATION
b. Oogenesis
- One germ cell divides 4x =16 cells - Commences at about 3 hours with the formation of a deep
o 1 cell = Oocyte furrow along much of the embryo length that consists of a
o 15 cells = Nurse cells mesodermal invagination along the ventral midline (ventral
*par1 gene – oocyte formation furrow VF)
(absence = 16 nurse cells) - Anterior and posterior midgut (PMG) : invaginations at
- Egg Chamber respective ends.
o Ovarian follicle cells surrounds oocyte and - Cephalic furrow – appears laterally at 65%EL
nurse cells
- Follicle cells Concurrent with gastrulation:
o From gonads, Somatic in origin - Germ bands elongate, driving posterior end with pole cells
o Become divided into three populations round to dorsal side of the egg
1. Squamous (nurse cells)
2. Columnar (oocyte) 4 hours: The first neuroblasts appear in neurogenic ectoderm
3. Border Cells (both ends of oocyte)
o Secrete vitelline membrane and chorion Segmentation: Initially appears at the extended germ-band stage
(tough outer coat surrounding egg)
- Border Cells Parasegments:
o Important for determination of - Initial repeating pattern
anteroposterior pattern - Definitive segments each form from the posterior 2/3 of 1
- Nurse Cells parasegment combined with the anterior 3rd of the next
o Become polyploidy - important for body plan construction
o Export large amounts of RNA and protein into
oocyte 7.5 hours:
- Oocyte 1. germ band retracts
o Becomes visibly polarized 2. epidermal grooves rearrange themselves to separate
▪ Dorsoventral axis definitive segments
▪ Anteroposterior axis 3. anterior and posterior midgut fuse in the middle
4. ventral nerve becomes segregated
- Pole plasm
o Forms at the posterior end of oocyte 10-11 hours:
o Contains determinant for germ cells 1. Displace amnioserosa into the anterior = Dorsal Closure
2. Head involutes into the anterior = scarcely represented of
the larva
FERTILIZATION
- In uterus
 o localized UV irradiation to produce small defects
o injection of labeled horseradish peroxidase
Later stages: o Principal features of fate map (prospective regions):
1. Malphigian tubes (excretory organs) are formed at the ➢ exist for all the larval segments = no region of
junction of posterior midgut and hindgut, muscles, fat body, indeterminacy
and the gonads arise from mesoderm ➢ arranged in anteroposterior sequence from 75 to
2. Central nervous system is formed by the ganglia of the 15% of the egg length
ventral nerve cord. ➢ ANTERIOR: prospective procephalic head structures
occupy the 25%
ORGANOGENESIS : prospective anterior midgut
Larval stages ➢ POSTERIOR: posterior midgut, Malpighian tubules,
o hatch at 24 hrs post-fertilization. proctodeum, & germ cells
o Has no legs REGIONAL SPECIFICATION
o Head tucked away in the interior
o 3 thoracic and 8 visible abdominal segments Dorsoventral Axis
o Specializations on the epidermal cuticle (during late - Responsible for the formation of
development) o Mesoderm
- Used to assess the phenotypes of late embryo o Neurogenic region
lethal mutations o Epidermis
o DORSAL side: region from T2 to A8 is covered with fine hairs - Arise from gradient of protein product of the maternal gene
o VENTRAL side: denticle belts on each of the thoracic and dorsal controlling the ventral half and zygotic gradient of
abdominal segments Dpp controlling the ventral half
• Occupies the anterior part
• Straddles the segment boundary Maternal gene dorsal
• Extends slightly into the posterior of the next - Becomes distributed in the nuclei of the blastoderm in
segment ventral-dorsal gradient
• Thoracic & abdominal segments can be - Regulates zygotic genes twist, rhomboid and zerknüllt
distinguished by the shapes and sizes of the which control formation of the various bands of tissue from
denticle belts ventral to dorsal
o POSTERIOR end: telson (unsegmented)
o ANTERIOR end: acron Territories formed during dorsoventral specification:
o Larval head is complex - Amnioserosa
o Horny cephalopharyngeal skeleton - the most prominent - Epidermis
component of head secreted by stomodeal part - Neurogenic zone
o Imaginal discs – small nest of cells in the 1st-instar larva - Mesoderm
• The imaginal discs are small sheets of epidermis • Disposition of territories is controlled by Dorsal protein, which is
(~40 cells each of cellular blastoderm) which grow the gene product of dorsal gene
throughout larval life.
o Abdominal segments are formed from abdominal histoblasts DORSAL REGION
(grow only in pupal stage) gurken
o Third instar larvae forms pupae (pupation) to undergo - Required in the oocyte
metamorphosis. - Encodes a growth factor related to vertebrate TGFα
o The adult tissues arise from imaginal discs and histoblasts. - Present just in the vicinity of the nucleus
o 6 leg, 2 wing, 2 haltere, 2 eye-antenna, plus genital, head - Positioned on the dorsal side of the oocyte
discs and ~10 histoblasts (nest of cells in the abdomen
which give rise to the abdominal segments). Egfr/torpedo
o - Expressed all over the follicle cells
Time after fertilization • Protein product of the gurken stimulates the Egfr product,
thereby activating the ERK signaling pathway and repressing pipe
Hours Days Developmental event

24 1 Hatching from egg; 1st instar larva begins VENTRAL REGION

pipe
49 2 1st molt; 2nd instar begins - Functions to create an active extracellular ligand localized on
the ventral side of the oocyte
72 3 2nd molt; 3rd instar begins - Codes for an enzyme responsible for adding sulfate groups
to extracellular glycosaminoglycans (GAGs)
120 5 Puparium formation; puparium white • Local sulfation of GAGs sequestrates proteases produced by
genes snake and easter which activate gene product of spätzle
122 5.1 Puparium fully colored gene

124 5.2 “prepupal molt” spätzle

- Synthesized as an inactive precursor requiring proteolytic
Pupation; cephalic complex, wings, legs
132 5.5 cleaveage for activation
everted
- Activates a receptor encoded by the Toll gene
169 7 Eye pigmentation
Toll Activation on the ventral side commences after fertilization
189 7.9 Bristle pigmentation biegins
Dorsal protein
216 9 Adult ready to emerge from pupa case - Transcription factor homologous to the vertebrate factor
NFκB
Fate map
- Its mRNA is uniformly distributed in the oocyte
o for the cellular blastoderm-stage Drosophila embryo:
 - Enters nuclei on the ventral side during syncytial blastoderm : synthesized during the syncytial stage at 1–3 hours of embryonic
forming a ventral-dorsal gradient of nuclear protein development and forms an exponential concentration gradient from
anterior to posterior.
TERRITORIES FORMED DURING DORSOVENTRAL
SPECIFICATION: *bicoid protein gradient: functions to regulate the zygotic expression
of several of the gap genes:
1. Mesoderm (1) orthodenticle and (2) hunchback both encode homeodomain
o twist transcription factors, and (3) Krüppel encodes a zinc-finger
▪ Encodes bHLH protein transcription factor.
▪ Required for correct mesoderm differentiation
o snail Posterior system
▪ Encodes a zinc-finger transcription factor *nanos : has a protein product whose action is responsible in lifting
▪ Needed for invagination of the mesoderm inhibition on the transcription of gap genes in the future abdomen;
• These are upregulated by Dorsal codes for an RNA binding protein
protein at high nuclear
concentration *A number of mutations in maternal effect genes that cause loss of the
2. Neuroectoderm abdomen: mutations in nanos, oskar, and pumilio.
o rhomboid
▪ Upregulated by dorsal protein and repressed *nanos must encode the functional end product of the pathway.
by snail
▪ Leads to the lateral stripe in the prospective *oskar is required for the formation of the pole plasm,
neuroectodermal region
▪ Codes for transmembrane protein involved in *sequestration of the nanos mRNA, and pumilio is required for
EGF signalling relaying nanos activity from pole plasm to prospective abdomen.
*Motor proteins: translocates mRNAs for bicoid and oskar along
3. Epidermis these microtubules so that they end up anterior or posterior,
o zerknüllt respectively.
▪ Defines dorsal ectoderm
▪ Repressed by Dorsal protein *The key genes required for the tubule polarization process have
already been encountered as genes required for dorsoventral
patterning, namely gurken and torpedo
4. Amnioserosa
o decapentaplegic (dpp)
*Anterior border cells: the specialized group of follicle cells that
▪ Also repressed by Dorsal protein
develops not just at the anterior but instead form at both ends of the
▪ Encodes signaling molecule homologous to
oocyte IF the females lack either gurken and torpedo
vertebrate BMPs
▪ Brings about patterning of the dorsal half of
Terminal system
the embryo circumference
*The key gene in the pathway is torso, which also has a gain-of-
▪ Can induce amnioserosa at high dose and
function allele causing substantial suppression of segmentation in the
dorsal hairs at lower dose
thorax and abdomen.
▪ Conducts induction of the dorsal gene
together with screw (BMP homolog)
:encoded a cell-surface receptor of the tyrosine kinase class. It
o short gastrulation (sog) gene
stimulates the ERK signal transduction pathway and the gain-of-
▪ Cause the graded effect of dpp and screw
function phenotype arises from a constitutively active form of the
▪ Expressed in the lateral belt because it is
receptor.
repressed by snail
▪ Inhibits screw leading to dorsal-ventral
*Activation of torso leads, via the ERK pathway, to activation of two
gradient pf BMP activity
zygotic gap genes: tailless and huckebein
▪ Homolog of vertebrate chordin
- tailless encodes a transcription factor of the nuclear
o tinman
receptor family
▪ Encodes a homeodomain transcription factor
necessary for heart formation
- huckebein encodes a zinc-finger transcription factor.
▪ Initially upregulated by twist
▪ Turned off except in the lateral region that is
Gap genes
adjacent to the dpp-expressing epidermis
Gap genes: mutant embryos have patterns bearing gaps of up to eight
contiguous segments. All the gap genes code for transcription factors,
Anteroposterior Axis
and because the early embryo is a syncytium, these can diffuse from
*IN THE ANTERIOR, mRNA for bicoid is deposited in the egg and a
one nucleus to another and exert their effects directly, with no need
gradient of bicoid protein activates various genes to generate regional
for cell–cell signaling.
subdivisions.
*Some important members of this group are: orthodenticle,
*IN THE POSTERIOR, mRNA for nanos is deposited, and its protein
hunchback, Krüppel, knirps, and giant.
product lifts the inhibition on gene activation in the future abdomen.
The regulatory relationships have been deduced mainly by two types
Anterior system
of experiment:
*bicoid : a maternal effect gene coding for a homeodomain
1. Examining the expression pattern of one gene in the absence of
transcription factor.
another. Expansion of a domain indicates repression and reduction of a
domain indicates activation in normal development.
:transcription occurs during oogenesis in both oocyte and nurse cells,
2. Examining the effect of uniform over expression of another gene.
and the mRNA becomes localized at the anterior of the oocyte.
*caudal: encodes a homeodomain transcription factor and is
homologous to the cdx gene family in vertebrates.

*Pair-rule system: function as a layer in the developmental hierarchy
between the gap genes and the segment polarity genes
*Segment polarity system: function to create the parasegment
boundaries of the early embryo.
:Once activated, they maintain their repeating pattern through a
positive-feedback loop between the cells on either side of each
boundary, defined by activity of the transcription factor genes
engrailed and cubitus interruptus (ci).
Hox genes
The Drosophila Hox complex is split into two regions called
the Antennapedia and the Bithorax complexes.
The Antennapedia complex corresponds to vertebrate
paralog groups 1–6 and contains the genes labial, proboscipedia,
Deformed, Sex combs reduced, and Antennapedia.
The Bithorax complex corresponds to vertebrate paralog
groups 7–10 and contains the genes Ultrabithorax (Ubx), abdominal-A,
and Abdominal-B.
DROSOPHILA DEVELOPMENTAL GENETICS
A. Transgenesis
P-element
➢ a transposable element which is important for the creation of
transgenic lines
➢ has been specifically used for insertional mutagenesis
The process of transgenesis:
1. The gene of interest is cloned into a P-element vector
containing a marker gene, often the wild-type allele of the
white gene which produces the red pigment in the eyes.
2. The recombinant P-element is injected into the posterior
pole of early embryos of the white genotype together with
plasmid or mRNA encoding the tranposase enzyme.
3. The resulting flies are crossed to white flies.
4. If the P-element has been integrated, the flies from the
injected embryos will produce transgenic offspring,
which can be identified by their possession of red eyes.
Purpose of transgenesis:
• To introduce enhancer traps
• To create strains carrying reporter constructs
• To rescue endogenous mutations
• To express genes in an ectopic manner
Phage ΦC31 integrase
➢ catalyzes recombination between special attB sites in the
injected plasmid and attP sites in the host.
*attP sites need to be previously introduced with a P-element
▪ position of integration can be chosen to avoid position
effects + larger DNA sequences can be introduced
B. Identification of developmentally important genes
Drosophila genome
• 13,000 genes, 5000 can be mutated to lethality
Mutagenesis screening
possible to assemble a fairly complete collection of genes which, when
mutated, give rise to pattern alterations in the embryo.
The screen illustrated in Fig. 11.5 is for autosomal recessive mutations
and like most screens depends on the use of special balancer
chromosomes which have following features:
1. suppress recombination
2. carry some visible marker gene
3. lethal in the homozygous condition.
Males carrying a visible marker (a – distinct from the
balancer marker) are mutagenized and are then mated to
females carrying the balancer. Each individual offspring fly
represents one mutagenized gamete, so individual males
from the F1 generation are isolated and crossed again.
For creation of the F2, females are used which carry a
dominant temperature-sensitive lethal mutation on the
chromosome opposite the balancer and this enables
selection against offspring not carrying the mutant
chromosome. In the F2 generation each tube of flies
represents one of the original mutagenized gametes.
The viable F2 flies are mated with each other to produce the
F3 generation. As the F2s are heterozygous for the
mutagenized chromosome and the balancer, 25% of the F3
will have a homozygosed mutant chromosome.
If these homozygous mutants are viable, they will display
the recessive marker phenotype (a/a) from the mutagenized
males. If this marker is not visible, then the dead embryos
or larvae are examined to see whether they have any
significant pattern abnormality.
In the case where the mutants are lethal the stock is
maintained by breeding from the heterozygotes that have
one mutant chromosome and one balancer.
**These will breed true because both the possible homozygous
chromosome combinations that can arise from mating are lethal. The
original mutagenesis will induce mutations on all the chromosomes,
but those that do not lie on the balanced chromosome are mostly lost
in the outcrosses, so the method enables the screen to be carried out
for one chromosome at a time.
Once obtained, mutations are sorted into groups of alleles of
the same gene by complementation tests, and are
genetically mapped.
C. Types of Mutation
RECESSIVE MUTATIONS (most mutation, REDUCED FUNCTION, LOSS-
OF-FUNCTION)
HYPOMORPHS – alleles
HYPOMORPHIC
- phenotypes
- can be arranged in a series of increasing severity with the
amorphic phenotype as the limit form
ALLELIC SERIES
- can give useful information about function, particularly where
the weaker alleles give something recognizable and stronger ones
do not.
“NULL” alleles – give amorphic phenotypes
• Some alleles may be TEMPERATURE SENSITIVE (because
mutation affects the thermal stability of the protein product,
can help establish the developmental stages at which gene
product is required.
1. Permissive temperature – protein active at low
temperature
2. Nonpermissive temperature – protein inactive at
high temperature
• Shifting between temperatures:
 1. Mutant -Shift to nonpermissive is before time of gene
function
2. Normal –shift to nonpermissive is afterwards
DOMINANT MUTATIONS
1. Gain-of-function(more often) = production of active gene
product in positions
2. Dominant negative = mutant version of gene product
interferes with function of the wild-type version
3. Loss-of-function when locus is haploinsufficient = means
reduction in the level of the product to 50% of wild-type
level is sufficient to cause a mutant type, homozygous
phenotype will be more severe than heterozygous
Names of drosophila genes usually derive from the
appearance of the mutant phenotype
✓ May indicate opposite to normal function
Example: dorsal gene forms ventral parts
*Some names are named after the adult phenotypes of viable alleles
for embryonic lethal mutations
Example: Antennapedia is so named because of gain-of-function
alleles that converts antenna to leg
D. Cloning of genes
▪ A mutation caused by P-element insertion can be used as a
starting point for cloning the gene.
▪ The resulting clones are tested in situ hybridization to polytene
chromosomes to find which particular P-element they
represent.
Gene function
- Is studied by how the mutation of one gene affects the
expression of others.
- Effects can be direct or indirect
1. Direct
- The protein product of gene A is actually interacting with gene B
to regulate it
- If gene A codes for a transcription factor, there are three
methods of establishing directness:
1. Demonstrate interactions between gene A-product and gene
B-regulatory region by means of band shift assays
2. Genes A and B can be transfected together into Drosophila
tissue culture cells
3. Do a domain swap: a new DNA recognition domain is put
onto A and the corresponding DNA sequence is inserted into
the regulatory region of B. If the effects are maintained,
then it must be due to a direct molecular interaction.
2. Indirect
- Gene A could be turning on something else that regulates
gene B
- If gene A codes for an inducing factor or receptor then the
effect is necessarily indirect.