Chapter 6 - The genetics of axis specification in drosophila

Question 1

Drosophila as a model organism:

Answer 1

• Easy to breed
• Hardy
• Prolific
• Tolerant of diverse conditions
• The polytene chromosomes of its larvae are readily identified
• Completely sequenced genome
• Ability to generate transgenic flies at high frequency

Question 2

Early Drosophila development

Answer 2

Cell membranes do not form until after the thirteenth nuclear division. Prior to this time, the dividing nuclei all share a common cytoplasm and material can diffuse throughout the whole embryo.

The specification of cell types along the AP and DV axis is accomplished by the interactions of components within the single multinucleated cell.

These axial differences are initiated at an earlier developmental stage by the position of the egg within the mother’s egg chamber.

The axes are specified by interactions between the egg and its surrounding follicle cells prior to fertilization.

Question 3

Fertilization - different!

Answer 3

• The sperm enters an egg that is already activated.
• There is only one site where the sperm can enter the egg (micropyle).
• By the time the sperm enters the egg, the egg has already begun to specify the body axes; the sperm enters an egg that is already organizing itself as an embryo.

Question 4

Drosophila egg activation, details:

Answer 4

Egg activation accomplished at ovulation, a few minutes before fertilization begins.
As the drosophila oocyte squeezes through a narrow orifice, calcium channels open and calcium flows in, restarting the meiotic division and mRNA translation without fertilization.

Question 5

Micropyle?

Answer 5

Site of sperm entry.
A tunnel in the chorion (egg-shell) located at the future DA region of embryo.
Allows sperm to pass through one at a time, preventing polyspermy.

Question 6

Blockage of polyspermy?

Answer 6

Micropyle only allows passage of one sperm at a time = no polyspermy.
No cortical granules to block polyspermy, although cortical changes are seen.

Question 7

Most insect undergo superficial cleavage

Answer 7

wherein a large mass of centrally located yolk confines cleavage to the cytoplasmic rim of the egg.
Cells do not form until after the nuclei have divided several times.

Question 8

Syncytium

Answer 8

a single cell with many nuclei residing in a common cytoplasm, created when karyokinesis (nuclear division) occurs without cytokinesis (cell division).

Question 9

Early nuclear division

Answer 9

occur centrally within a syncytium. Very rapid rate of division, accomplished by S and M phases in absence of G phases of cell cycle.

Question 10

Later division

Answer 10

nuclei and their cytoplasmic islands (energids) migrate to the periphery of the cell in cycle 10 with a progressively slower mitosis rate. => the syncytial blastoderm (no other cell membranes than the egg itself).

Question 11

Formation of cellular blastoderm (individual cell membranes)

Answer 11

After cycle 13, the cellular blastoderm forms by ingression of cell membranes between nuclei.

Question 12

Pole cell formation

Answer 12

The pole cells (germ cell precursors) form in the posterior during the 9th cycle when they reach the surface and become enclosed by cell membranes.

When the nuclei reach the periphery of the egg, each nucleus becomes surrounded by microtubules and microfilaments.

Question 13

The nuclei divide within a common cytoplasm, but this does not mean the cytoplasm is itself uniform.

Answer 13

Each nucleus within the syncytial blastoderm is contained within its own little territory of cytoskeletal proteins (collectively energids).

Question 14

Cellular blastoderm

Answer 14

all the cells are arranged in a single-layered jacket around the yolky core of the egg.

• Involves a delicate interplay between microtubules and microfilaments.
• Membrane movements, nuclear elongation, and actin polymerization all appear to be coordinated by the microtubules.
• Consists of ca 6000 cells and is formed within 4 hrs of fertilization.

Question 15

Phases of cellularization:

Answer 15

1. Characterized by the invagination of cell membranes between the nuclei to form furrow canals.
2. Begins when furrow canals have passed the level of the nuclei. Rate of invagination increases, and actin-membrane complex begins to constrict at what will be the basal end of the cell.

Question 16

Mid-blastula transition

Answer 16

slowdown of nuclear division, cellularization, and concomitant increase in new RNA transcription
- Maternally provided mRNAs are degraded and control of development is handed over to the zygotic genome

Question 17

Coordination of mid-blastula transition and maternal-to-zygotic transition is controlled by several factors:

Answer 17

• The ratio of chromatin to cytoplasm; consequence of increasing DNA amount while cytoplasm remains constant
• Smaug protein; RNA-binding protein with known roles in translational repression, also targets maternal mRNAs for destruction during this transition. Smaug accumulation appears to regulate the progression from maternal to zygotic developmental control.
• The Zelda transcription factor; regulates activation of zygotic genes, encoded by a maternal mRNA. Many of the genes that initiate the pathways of sex determination, DV polarization and AP polarity are initiated by Zelda. Possible that genes with highest affinity activated first, lower affinities later.

Question 18

General Drosophila body plan ?

Answer 18

same in embryo, larva and adult: distinct head and tail ends with repeating segmental units between. Top three segments = thorax, bottom eight = abdomen.
Each segment of the adult fly has its own identity, e.g. only wings.

Question 19

Thoracic segments

Answer 19

T1 = Prothorax, only legs
T2 = Mesothorax, legs and wings
T3 = Metathorax, legs and balancing organs (halteres)

Question 20

Fates of tissues immediately before gastrulation begins

Answer 20

Endoderm in each end, pole cells posterior, dorsal ectoderm (internal), side ectoderm (neuroectoderm) and mesoderm at ventral. Amnioserosa extraembryonic layer surrounding the embryo.

Question 21

Drosophila gastrulation step 1-3

Answer 21

1. Segregation of presumptive mesoderm, endoderm and ectoderm.
   a. Prospective mesoderm cells constitutes the ventral midline of the embryo
2. Mesoderm folds inward to produce ventral furrow
   a. Eventually pinches off from the surface to become a ventral tube within the embryo
3. Prospective endoderm invaginates to form two pockets at the anterior and posterior ends of the ventral furrow.
   a. Pole cells are internalized along with endoderm

Question 22

Drosophila gastrulation step 4-6

Answer 22

4. Embryo bends to form cephalic furrow – separates the future head region (procephalon) from the germ band, which will form the thorax and abdomen.
5. The ectodermal cells on the surface and the mesoderm undergo convergence and extension, migrating toward the ventral midline to form the germ band, a collection of cells along the ventral midline that includes all the cells that will form the trunk of the embryo.
   a. Germ band extends posteriorly and wraps around the tip (dorsal) surface of the embryo
   b. By end of germ band formation, cells destined to form the most posterior larval structures are located immediately behind the future head region.
6. Body segments begin to appear, dividing ectoderm and mesoderm.

Question 23

Drosophila gastrulation step 7-10

Answer 23

7. Germ band retracts, placing the presumptive posterior segments at the posterior tip of the embryo.
8. Dorsal closure – the two sides of the epidermis are brought together.
   a. Amnioserosa (previously most dorsal structure) interacts with the epidermal cells to stimulate their migration.
9. While the germ band is in its extended position, several key morphogenetic processes occur: organogenesis, segmentation, and segregation of the imaginal discs (cells set aside to produce the adult structures).
10. The nervous system forms from two regions of ventral ectoderm
    a. Neuroblasts differentiate from this neurogenic ectoderm within each segment (and also from the non-segmented region of the head ectoderm).
    b. => In insects the nervous system is located ventrally, rather than being derived from a dorsal neural tube as in vertebrates.

Question 24

”Forward genetics” approach

Answer 24

randomly mutagenize flies and then screen for mutations that disrupted the normal formation of the body plan. Genes involved in mutant phenotypes are cloned and then characterized with respect to their expression patterns and their functions.

Question 25

The oogonium

Answer 25

Each oocyte is descended from a single female germ cell

Question 26

Formation of nurse cells

Answer 26

Before oogenesis begins, the oogonium divides four times with incomplete cytokinesis, giving rise to 16 interconnected cells: 15 nurse cells and the single oocyte precursor.

Question 27

Egg chamber

Answer 27

the 16 germline cells plus a surrounding epithelial layer of somatic follicle cells, where the oocyte develops.

As the oocyte precursor develops at the posterior end of the egg chamber, numerous mRNAs made in the nurse cells are transported along microtubules through the cellular interconnections into the enlarging oocyte.

Question 28

Anterior-posterior polarity in the oocyte

Follicular epithelium surrounding the developing oocyte is initially uniform with respect to cell fate, but this is broken by two signals organized by the oocyte nucleus. Both involve the gene gurken.

Answer 28

• Gurken message synthesized in nurse cells, but transported specifically to the oocyte cytoplasm.
• Gurken signal is received by the nearby posterior follicle cells through a torpedo encoded R protein, which results in ”posteriorization”.
• Posterior follicle cells send a signal back into the oocyte, recruiting Par-1 protein to the posterior edge of the oocyte cytoplasm.
• Par-1 organizes microtubules specifically with their minus (cap) and plus (growing) ends at the AP ends of the oocyte, respectively.
  o Orientation of microtubules is critical; different motor proteins will transport their mRNA / protein cargoes in different directions.
• Oskar mRNA transported to posterior end.
  o Oskar protein recruits more Par-1, stabilizing the microtubule orientation and allowing more material to be recruited to the posterior pole of the oocyte.
  o Posterior pole ends up with its own distinctive cytoplasm, pole plasm, containing the determinants for producing the abdomen and germ cells.
• Cytoskeletal rearrangement in oocyte accompanied by increase in oocyte volume (transfer of cytoplasmic components from nurse cells)
  o Including maternal messengers such as bicoid and nanos mRNAs, critical for establishing AP polarity of embryo.

Question 29

Dorsal-ventral patterning in the oocyte

Answer 29

• As oocyte volume increases, the oocyte nucleus is pushed by the growing microtubules to an anterior dorsal position, where a second major signalling event takes place.
  o Gurken message becomes localized in a crescent between the oocyte nucleus and cell membrane.
  o Gurken forms an AP gradient along the dorsal surface of the oocyte. Short diffusion distance.
• Follicle cells exposed to Gurken protein, those closest to nucleus, induced to become the more columnar dorsal follicle cells. Establishing the DV polarity in the follicle cell layer that surrounds the growing oocyte.

The Gurken-Torpedo signal initiates a cascade of gene activities that create the DV axis of the embryo.
Through a bunch of middle reactions a R on ventral side of egg are transducing a signal into the egg, whereas dorsal side does not.

Question 30

Maternal deficiencies of either the gurken or the torpedo gene cause ?

Answer 30

ventralization of the embryo.

Gurken only active in oocyte. Torpedo only in somatic follicle cells.

Question 31

Dorsal, the ventral morphogen

Answer 31

Dorsal protein – distinguishes dorsal from ventral.

• The maternal mRNA is deposited in the oocyte by the nurse cells.
• Translated Dorsal is found throughout the syncytial blastoderm, synthesized about 90 minutes after fertilization
• Dorsal is only translocated into the nuclei in the ventral part of the embryo.
• Acts as TF in nucleus, repressing dorsal genes, activating ventral.

Question 32

Establishing a nuclear Dorsal gradient

Answer 32

• Cactus protein keeps Dorsal in a complex in cytoplasm.
• Separation from cactus initiated by ventral activation of the Toll R by Spätzle.
  o Spätzle binds Toll
  o Activated Toll activates Pelle (protein kinase)
  o Active Pelle phosphorylates Cactus (prob through intermediate)
  o Phosphorylated Cactus is degraded => Dorsal enters nucleus.
• Spätzle gradient -> Toll activation gradient -> Dorsal translocation gradient
  => highest Dorsal concentrations in ventral most cell nuclei.

Question 33

Effects of the Dorsal protein gradient

Answer 33

Large amounts of Dorsal instruct cells to become mesoderm, whereas lesser amounts instruct the cells to become glial or ectodermal tissue.
Dorsal -> Lateral: amnioserosa, dorsal ectoderm, lateral ectoderm, neurogenic ectoderm, mesoderm.

Question 34

Dorsal protein specifies the cells to become mesoderm in two ways:

Answer 34

1. Activates specific genes that create the mesodermal phenotype; twist, snail, fgf8, fgf8 R, rhomboid. (Enhancers have low affinity for Dorsal => only transcribed in cells with high concentrations of Dorsal).
2. Also determines mesoderm indirectly, by directly inhibiting dorsalizing genes.

By the cellular responses to the Dorsal gradient, the embryo becomes subdivided from the VD regions into mesoderm, neurogenic ectoderm, epidermis (lateral and dorsal ectoderm), and amnioserosa.

Question 35

LR and inside-out axes

Answer 35

Whereas vertebrate asymmetry appears to be regulated by microtubules, asymmetry in Drosophila appears to be regulated by microfilaments.
Very little is known about the formation of the LR axis

Because the insect embryo is built along the cortex of a yolk-filled cytoplasm, it also has an ”inside-out” axis. The outer (apical) part of each blastoderm cell is made from the egg cell membrane, while its inner (basal) membrane contacts the yolk.

Question 36

Segmentation and the AP body plan

Gene hierarchy:

Answer 36

1. Establish AP polarity
2. Divide embryo into a specific number of segments, each with a different identity

Hierarchy initiated by maternal effect genes that produce mRNAs localized to different regions of the egg.

Question 37

Segmentation and the AP body plan

Zygotic genes expressed:

Answer 37

1. Gap genes
2. Pair-rule genes
3. Segment polarity genes
4. Homeotic selector genes

Question 38

Gap genes:

Answer 38

Activated / repressed by the maternal effect genes.
Expressed in certain broad (ca 3 segments wide), partially overlapping domains
Encode TFs
Regulate transcription of pair-rule genes
Mutations here cause gaps in the segmentation pattern.

Question 39

Pair-rule genes

Answer 39

Divide the embryo into periodic units
Regulated by gap gene TFs
Encode TFs that activate the segment polarity genes

Transcription of different pair-rule genes results in a striped pattern of seven transverse bands perpendicular to the AP axis.

Question 40

Segment polarity genes

Answer 40

Their mRNA and protein products divide the embryo into 14 segment wide units, reinforcing the periodicity of the embryo.

Question 41

Homeotic selector genes

Answer 41

Regulated by protein products of gap, pair-rule and segments polarity genes
Encodes TFs that determines the characteristic structure of each segment.

Question 42

Maternal gradients: Polarity regulation by oocyte cytoplasm

Answer 42

Two ”organizing centres” in the insect egg; one in anterior and one in posterior

• Morphogenetic gradients emanate from the two poles during cleavage, and these interact to produce the positional information determining the identity of each segment.
• Egg sequesters an mRNA that generates a gradient of anterior-forming material

Question 43

The molecular model: Protein gradients in the early embryo, Morphogenic genes:

Answer 43

• One set for anterior
• One set for posterior
• One set for the terminal regions at both ends

Question 44

Two maternal mRNAs are most critical to formation of the AP axis:

Answer 44

• Bicoid: located near anterior tip of the unfertilized egg, translated after ovulation and fertilization, protein product diffuses, forming gradients critical for AP patterning
• Nanos: located near the posterior, same

Distributions occur as a result of the dramatic polarization of the microtubule networks in the developing oocyte

Bicoid and Nanos gradient forms a coordinate system based on their ratios. Each position along the axis is distinguished from any other position.
These are established by diffusion as well as by active degradation

Question 45

Anterior can be recognised by ?

Answer 45

the micropyle on the shell (lille dut ud); this structure permits sperm to enter.

Question 46

At completion of oogenesis:

Answer 46

• Bicoid message is anchored at anterior
• Nanos message at posterior

Both are dormant until ovulation and fertilization, at which time they are translated.

Question 47

Two other maternally provided mRNAs (hunchback and caudal) are critical for patterning the anterior and the posterior regions of the body plan respectively.

Answer 47

• Synthesized by the nurse cells of the ovary and transported to the oocyte, where they are distributed ubiquitously throughout the syncytial blastoderm
• Translation of their mRNAs (hunchback and caudal) is repressed by the diffusion gradients of Nanos and Bicoid proteins, respectively.

Question 48

Four maternal protein gradients in the early embryo (relevant for AP):

Answer 48

• An anterior to posterior of Bicoid
• An anterior to posterior of Hunchback
• A posterior to anterior of Nanos
• A posterior to anterior of Caudal

Question 49

The anterior organizing centre: the Bicoid and Hunchback gradients

Answer 49

High concentrations of Bicoid = anterior head structures
Slightly less = jaws
Moderate = thorax
Lacking = abdomen

Bicoid and Hunchback act synergistically at the enhancers of the ”head genes” to promote their transcription in a feedforward manner (both needed)

Question 50

Caudal protein gradient activates a number of zygotic genes, critical for abdominal development

Answer 50

Caudal activates the genes responsible for the invagination of the hindgut and thus is critical in specifying the posterior domains of the embryo

Question 51

Acron

Answer 51

the terminal portion of the head that includes the brain

Question 52

Telson

Answer 52

tail

Question 53

Third set of maternal genes whose proteins generate the unsegmented extremities of the AP axis: the acron and the telson.

Answer 53

• Critical gene appears to be torso – gene encoding a R tyrosine kinase (RTK)
• No torso, no acron or telson => formed through the same pathway

The TM Torso protein is evenly distributed throughout the cell membrane, but normally only activated at the ends of the egg.

Question 54

The end products of the RTK cascade activated by Torso diffuse into the cytoplasm at both ends of the embryo

Answer 54

• Thought to inactivate the Capicua protein (transcriptional repressor of tailless and huckebein)
• These two gap genes specify the termini of the embryo. Distinction between depends on the presence of Bicoid. No Bicoid = telson, with Bicoid = acron.

Question 55

Summarizing early AP axis specification in Drosophila, specified by 3 sets of genes:

Answer 55

1. Genes that define the anterior organizing centre.
   Located at the anterior end of the embryo, the anterior organizing centre acts through a gradient of Bicoid protein. Bicoid functions both as a TF to activate anterior-specific gap genes and as a translational repressor to suppress posterior-specific gap genes.
2. Genes that define the posterior organizing centre.
   The posterior organizing centre is located at the posterior pole. This centre acts translationally through the Nanos protein to inhibit anterior formation, and transcriptionally through the Caudal protein to activate those genes that form the abdomen.
3. Genes that define the terminal boundary regions.
   The boundaries of the acron and telson are defined by the product of the torso gene, which is activated at the tips of the embryo.

Question 56

Segmentation genes

Answer 56

mediates the transition from specification to determination, divide the early embryo into a repeating series of segmental primordia along the AP axis.

Question 57

Parasegments

Answer 57

”Transsegmental” units
Posterior compartment of one segment and anterior compartment of the immediately posterior segment. Often affected by mutations in segmentation genes = ”functional units” of the embryonic gene expression.

The expression patterns in the early embryo are delineated by parasegmental boundaries not by the boundaries of the segments.

In adult, parasegmental organization only seen in the nerve cord, not in adult epidermis, prob required for coordinated movement (think spinal cord).

Question 58

Stripe placement is a result of:

Answer 58

1. The modular cis-regulatory enhancer elements of the pair-rule genes
2. The trans-regulatory gap gene and maternal gene proteins that bind to these enhancer sites.

Question 59

Two regions of chromosome III contain most of the homeotic genes, overall referred to as homeotic complex (Hom-C).

Answer 59

• The first, known as the Antennapedia complex, contain genes that specify the head segments and contributes to the thoracic segment identities.
• The second, is the bithorax complex, with genes required for the identity of the third thoracic segments and abdominal segment identities.

Transcripts from the major homeotic selector genes can be detected in specific regions of the embryo and are especially prominent in the central nervous system.

Question 60

The proteins encoded by the gap and pair-rule genes are transient, once transcription patterns have become stabilized, they are ”locked” into place by alteration of the chromatin conformation in these genes.

Answer 60

• The repression of homeotic genes is maintained by the Polycomb family
• The active chromatin conformation appears to be maintained by the Trithorax proteins.

Question 61

Homeotic genes don’t do the work alone

Answer 61

• They regulate the action from up in the ”executive” suite, while the actual business of making an organ is done by other genes on the ”factory floor”.
• The homeotic genes work by activating or repressing a group of ”realisator genes”, they function to form the specified tissue or organ primordia.