Drosophila Development

Why should we care about how fruit flies are patterned and establish their axis?

Conserved mechanisms between invertebrates and vertebrates in how our bodies were patterned as embryos

What happens 0 to ~1 hour after fertilization?

Egg undergoes rapid nuclear divisions without cell division (syncytial stage)

What happens 2-3 hours after fertilization?

Cellularization occurs, and the embryo begins to form distinct tissue layers

What happens 3-10 hours after fertilization?

Major body segments start to form, and the germ band extends (posterior end that curves around itself)

What happens 10-13 hours after fertilization?

The germ band retracts, and organogenesis proceeds

What happens roughly 24 hours after fertilization?

Embryo completes development, hatches and emerges as a first-instar larva

When looking at early cleavage divisions of a fly embryo, what is characteristic of the syncytial blastoderm?

Cells have migrated to the periphery

• pole cells form

When looking at early cleavage divisions of a fly embryo, what is characteristic of MBT?

Cell membrane has formed and grows inward (cellular blastoderm)

What drives cellularization?

Microtubules and microfilaments

What does cellularization form?

Epithelium of cells, facilitating gastrulation (so cells can move together)

Apical adherens junctions between cells

What features mark gastrulation?

• Ventral furrow: invagination on ventral surface that internalizes ~1000 cells then closes up (precursors of mesoderm)

• Anterior/Posterior midgut invaginations = how endoderm gets inside

  • Cephalic furrow is boundary between head and rest of body

• Pole cells in posterior midgut invagination = germ cell precursors (oocytes or sperm)

When does the germ band extend and retract?

Extends at end of gastrulation and retracts (~12 hours) when organogenesis starts

What is germ band extension driven by?

Converging and intercalating at the ventral midline

• cells inserting in between each other allows extension along perpendicular plane

What does the germband eventually develop into?

Future thoracic and abdominal segments

What gives oocyte anterior-posterior polarity and pre-pattern the early embryo?

Maternal Effect Genes (MEGs)

What are the anterior (head) morphogens?

Bicoid and hunchback

What are the posterior morphogens?

Nanos and caudal

What is step 1 in the anterior-posterior patterning of Drosophila?

1. Maternal Effect Genes (MEGs)

• mRNAs deposited in oocyte

• Set up early polarity

• Translated at fertilization

• Creates cytoplasmic polarity

What do MEGs activate?

2. Gap Genes

• Expressed from zygotic genome

• Broad expression domains

• 1-2 broad stripes created

What do gap gene products activate?

3. Pair-rule genes

• 7 stripes

• define boundaries of segments

• Most important

What do pair-rule gene products activate after cellularization?

4. Segment polarity genes

• Give each segment polarity

What are MEGs expressed by?

Nurse cells of the egg chamber

• Oocyte precursor cell (oogonium) undergoes 4 incomplete cell divisions to produce 16 interconnected cells → 15 nurse cells, 1 oocyte

• Nurse cells supply proteins and MEGs (bicoid, hunchback, nanos, caudal mRNAs)

How are MEGs localized to set up early anterior-posterior axis during oogenesis?

Nurse cells are localized to anterior end with polarized microtubules on posterior half

• Positive towards posterior, negative towards anterior

• Bicoid mRNA localized on negative end

• Nanos mRNA localized to positive posterior end

  • mRNA will produce proteins that diffuse

What mRNA is associated with kinesin I?

Oskar mRNA associated with kinesin I; Oskar protein associated with nanos mRNA

What establishes the dorsal-ventral axis?

Organization of nucleus on dorsal side

What is the order of Syncytial specification?

1. MEG mRNAs (bicoid and nanos) are distributed assymetrically in oocyte

2. mRNAs translated at fertilization → proteins diffuse

3. Proteins form morphogen gradients (syncytium) and are incorporated into cells at cellularization

What does bicoid encode?

Morphogen responsible for head structures

What will a bicoid-deficient mutant have?

Two tails (one at each end)

If bicoid is added to mutant anterior, mutant middle or WT posterior end what will happen?

Anterior: WT; head at anterior

Middle: Head in middle

Posterior: Two heads (one at each end)

**head develops wherever bicoid is placed (bicoid dominant over nanos)

What mRNA is asymmetrically distributed? Symmetrically distributed?

• Asymmetric: bicoid and nanos

• Symmetric: hunchback and caudal

If the mRNA is symmetric, then how is hunchback protein higher on anterior side and caudal protein higher on the posterior side?

Bicoid protein halts the translation of caudal

• High [Bicoid] binds BRE in caudal mRNA 3’ UTR, and eIF4E that is bound to the 5’ cap

• Low [Bicoid]: eIF4E and elF4G can interact normally

Nanos halts the translation of hunchback

• High [Nanos] binds pumillo on NRE and causes deadenylation of PolyA tail

• Low [Nanos]: Only Pumilio bound to NRE → translation

What is the WT gap gene?

Kruppel (boundary between two segments)

What is the pattern of pair-rule gene expression?

Expressed in 7 stripes, where each stripe spans two fragments (control development of every other fragment)

• Even-skipped (eve) and Paired (prd) for example

What are examples of segment polarity genes?

Engrailed (en) and wingless (wg)

MEGs establish broad anterior-posterior identity of the embryo, what establishes the segments?

The hierarchy of zygotic genes! Gap, pair-rule, segment polarity, the homeotic selector (HOX) establish the identity of each individual segment

What happens if there is a mutation in any of the zygotic segmentation genes?

Embryonic lethal

What happens in a Gap mutant?

Missing several segments in a row

What happens in a pair-rule mutant?

Missing portions of every other segment (can delete boundaries and reduce the number of segments)

What happens in a segment polarity mutant?

Polarity disrupted in each segment (segment number does NOT change!)

When are pair-rule genes induced?

Onset of cellularization

How many gap genes are known?

9 (like Kruppel; parasegments 4-6)

When pair-rule genes are expressed at cellularization, what regulates them?

Gap genes transcriptionally activate and repress

How are parasegments positioned relative to segments?

Shifted one compartment forward

If we were looking at pair-rule genes eve and ftz (fushi tarazu), how would they be placed?

Each would be in their own parasegment, in an alternating pattern

[eve] [ftz] [eve] [ftz]……

What is the difference between parasegments and segments?

1. Eve and Ftz are expressed in alternating stripes, and these patterns help establish parasegment boundaries. There are 7 stripes of Eve, 7 stripes of Ftz = 14 parasegments

2. Segments form, containing half of two neighbouring parasegments (one segment will have expression of two pair-rule genes to form posterior/anterior ends) => these are what we can actually see on a bug!

What are all of the segments of a Drosopholia embryo?

Ma: Mandibular

Mx: Maxillary

Lb: Labial

T1-T3: Thoracic

A1-A8: Abdominal

When are segment polarity genes expressed? What are they activated by?

AFTER cellularization. They are activated by different combinations of pair-rule proteins acting as TFs

How many stripes of expression do the products of the pair-rule genes create?

14 alternating stripes; 7 of Eve and 7 of Ftz

How does the expression of pair-rule genes change when comparing mid cellularization to late cellularization?

Mid-cellularization: thin to no boundary between Ftz and Eve

Late-cellularization: thicker bands of no expression acting as a boundary between Ftz and Eve => boundaries have been established so now segment polarity genes will be induced, to reinforce these boundaries

What is the function of segment polarity genes in Drosophila development?

They establish and maintain parasegment boundaries and help establish anterior and posterior polarity, allowing different regions of each segment to develop distinct structures

Which two segment polarity genes are involved in reinforcing parasegment boundaries?

• Engrailed (en)

  • TF

  • Anterior compartment of each parasegment

  • Activated by Ftz and Eve

• Wingless (wg)

  • Wnt Ligand

  • Posterior compartment of each parasegment

  • Activated where there are no pair-rule genes

How do Engrailed and Wingless reinforce parasegment boundaries?

Engrailed-expressing cells produce Hedgehog, which maintains Wingless expression in neighbouring cells by activating Patched and allowing transcription of Wg. Wingless signals back (WNT ligand) to maintain Engrailed and Hedgehog expression, creating a positive feedback loop that reinforces the boundary

Is the positive feedback loop between cells at the parasegment boundary a morphogen affect?

Since the cells depend on each other to maintain their gene expression patterns, this is an example of reciprocal signalling that maintains two adjacent cell populations, rather than concentration dependent

By reinforcing parasegment boundaries, segment-polarity genes differenciate cells on the anterior and posterior halves of segments. This does what?

While maintaining Para segment boundaries, they are also simultaneously polarizing each segment so they can go onto produce different identities and structures (posterior wg of parasegment 1 is beside anterior en of parasegment 2 and they have established a positive feedback loop with eachother)

• like the alternative pattern of the denticles

If polarity genes are mutated, what happens to denticle patterning?

If Wg or En is mutated, polarity will be lost within the segment and it will be a lawn of denticles rather than an alternating pattern

• its like a handshake. Wg requires En and En requires Wg

How many homeotic complexes are formed in Drosophila by HOX genes?

2 homeotic complexes at 3R!

• Antennapedia complex (head and thorax); 5 genes

• Bithorax complex (Abdomen); 3 genes

How are the genes within both homeotic complexes arranged?

Genes are ordered in the same sequence as the body plan they make

• most anterior gene = most anterior body part

What does ultrabithorax (Ubx) knockout do?

When Ubx is knocked out, the fly will form a pair of wings at both T2 and T3

• Ubx usually inhibits wing development at T3, and instead develops a pair of small balancing wings

What happens if Antennapedia (antp) is misexpressed in the head?

GOF! Antp usually develops the middle pair of legs

• Misexpression in the head causes a homeotic transformation since antennae develop as legs instead (ectopic expression of Antp)