Molecular & Developmental Biology (BIOL3530)

With Dr. Brian E. Staveley
Department of Biology
Memorial University of Newfoundland

Model Organism: Drosophila melanogaster

Drosophila melanogaster: early embryogenesis
The Drosophila egg is the shape of a sausage.
It has a micropyle at the anterior end (site of sperm entry).
With fertilization, the fusion of nuclei is followed by rapid mitotic divisions (90 minutes) and no cleavage.
A syncytium is formed (many nuclei/common cytoplasm).
After nine divisions, nuclei move to the periphery to form the syncytial blastoderm (2 hours).

Drosophila melanogaster: embryogenesis
By 13 mitoses the membranes sprout to surround the nuclei to form cells (cellular blastoderm).
~15 cells at posterior (pole cells) are sequestered and become the germline.
During first ~3 hrs. large molecules such as proteins can move between nuclei until the cellularization occurs.
Single layer of cells give rise to all tissues.
Gastrulation starts at ~3 hrs.
1) Mesoderm forms from ventral tissue.
2) Midgut from endoderm at the anterior and posterior ends.
3) Ectoderm remains on outside.

Drosophila melanogaster: gastrulation
The mesodermal tube forms from ventral tissue then cells separate & move to internal locations under the ectoderm.
The mesoderm becomes muscle and connections tissues.

In insects, the nerve cord lies ventrally (vertebrates: dorsal).
Neuroblasts form a layer between mesoderm and outer ectoderm.
The midgut (anterior & posterior) grow from threads and fuse.
= anterior and posterior midgut
Ectoderm becomes epidermis.
No cell division occurs during gastrulation but division restarts afterward.

Drosophila melanogaster: segmentation
The germband (ventral blastoderm) is main trunk region.
The process of germ band extension pushes the posterior end over dorsal side.
The first signs of segmentation grooves appear to outline parasegments which give rise to segments.
Segments are formed from the posterior of one parasegment and the anterior of the next.
There are14 parasegments:  3 mouth, 3 thorax, 8 abdominal.

Drosophila melanogaster: larvae
The larvae hatch at 24 hrs post-fertilization.
The larval structures of note include ...
The anterior end is the acron.
The posterior end is the telson.
Along with the head, the larvae has 3 thoractic segments and 8 abdominal segments.
The ventral side of the larvae has denticle belts, alternating patches of denticle hairs and cuticle on each segment, used for locomotion.

Drosophila melanogaster: metamorphosis
Three instar stages of larval life are separated by molts.

1st instar   -(Molt)->    2nd instar -(Molt)->  3rd instar

Third instar larvae forms pupae (pupation) to undergo metamorphosis.
The adult tissues arise from imaginal discs and histoblasts.
The imaginal discs are small sheets of epidermis (~40 cells each of cellular blastoderm) which grow throughout larval life.
6 leg, 2 wing, 2 haltere, 2 eye-antenna, plus genital, head discs and ~10 histoblasts (nest of cells in the abdomen which give rise to the abdominal segments).

Drosophila development: the body plan
Genes that control development in Drosophila are very similar to those that control development in vertebrates.
Drosophila is the best understood developmental system with great impact upon our knowledge of all development.  (for example, Hox genes were first found in Drosophila.)
Bilateral symmetry is established by the A/P and D/V axes.
The larvae has an anterior acron, three thoracic and eight abdominal segments and a posterior telson.
Early patterning occurs in the syncytial blastoderm and it becomes multicellular at the beginning of segmentation.
Concentration gradients of proteins (transcription factors) can diffuse, enter nuclei & provide positional information.

Technique: Mutagenesis and genetic screening
Although mutants can arise spontaneously, induced mutation and screening has become the standard way to identify developmentally important genes.
To generate mutants in a specific gene, a chemical mutagen, such as ethyl methane sulfonate (EMS), is fed to a large number of male flies.
The sperm cells of these males are exposed to the mutagen.
The males are bred to females that carry a balancer chromosome of the gene of interest.
Individuals carrying a mutagenized chromosome and a balancer are isolated.
These are crossed to individuals carrying the balancer chromosome.
In the next generation, offspring that carry both the mutagenized chromosome and the the balancer chromosome (balanced heterozygotes) are crossed.
Homozygous progeny are examined and balanced heterozygous siblings are selected to maintain the line.

Drosophila development: maternal and zygotic genes
Maternal genes establish the body axes.
Maternal gene products, mRNAs and proteins are expressed in the ovary.
Zygotic genes are expressed by an embryo.
About fifty maternal genes set up the A/P and D/V axes: the framework of positional information (spatial distributions of RNA and proteins).
Zygotic genes respond to maternal gene expression.
First broad regions are established, then smaller domains (with a unique set of zygotic gene activities) in a hierarchy of gene activity.

Drosophila development: the A/P axis
Three classes of maternal genes set up the A/P axis
Maternally expressed genes distinguish the anterior from the posterior.
Maternal effect mutants result in females that can not produce normal progeny.
Three mutant classes are 1) anterior,  2) posterior and 3) terminal classes.
Anterior class: loss of head and thorax (sometimes replaced with posterior).
Posterior class: loss abdominal segments.
Terminal class: missing acron and telson.
bicoid, hunchback, nanos and caudal are key to A/P axis.

Drosophila development: maternal genes
bicoid is sequestered in the oocyte during oogensis.
bicoid sets a A/P morphogenic gradient and controls the first steps in embryo development and, thus, is essential to the developing organism.
bicoid mRNA is localized to the anterior end of the unfertilized egg.
After fertilization, the mRNA is translated and a concentration gradient is formed along the A/P axis.
bicoid was the first evidence of a morphogen gradient.

Drosophila development: clues to the role of bicoid
1) bicoid (bcd) females lay eggs that give rise to embryos missing the head and thorax (and have an anterior telson).
2) Embryos missing anterior cytoplasm resemble above.
3) bcd embryos rescued by anterior cytoplasm injections.
4) Anterior cytoplasm can induce ectopic head & thoracic segments by injection in the middle of a bicoid egg.
5) in situ hybridization shows bicoid RNA is at the anterior part of the unfertilized egg (attached to cytoskeleton).
6) Protein not in egg, forms A/P gradient after fertilization.
7) bicoid: transcription factor & morphogen.
8) other anterior-group (group 1) maternal genes are involved in bicoid localization and translational control.

The posterior pattern is controlled by nanos & caudal protein gradients (group 2)
nanos mRNA is localized to the posterior pole of the egg.
nanos is NOT a morphogen like bicoid but acts to suppress translation of another maternal gene, hunchback (hb).
hunchback is maternal (present at low levels in embryo) AND zygotic (the latter is activated by high bicoid levels).
nanos (and pumilio) bind hb mRNA to prevent translation.
caudal mRNA is distributed evenly.
The P-A gradient of caudal is established by inhibition of caudal protein synthesis by bicoid.
[bcd and hb run in A to P gradients & caudal runs P to A.]
Anterior and posterior extremes are specified by cell-surface receptor activation

Group 3 maternal genes specify the acron and telson regions.
torso mutants develop neither acron nor telson regions.
torso encodes a uniformly distributed receptor protein which is activated by ligand present only at the anterior and posterior parts of the vitelline membrane.
The ligand is released after fertilization.
torso (a receptor tyrosine kinase) signals to direct terminal zygotic gene expression.
D/V polarity is due to vitelline membrane proteins.
At fertilization, a protein deposited on the ventral vitelline membrane initiates a series of reactions which, in part, activates (cuts)  spatzle: the ligand for the uniformly distributed receptor Toll.

dorsal provides positional information along the D/V axis
dorsal provides positional information along the D/V axis
In the syncytial blastoderm, dorsal (a transcription factor) is activated and enters nearby nuclei.
Dorsal is in highest concentration in ventral nuclei (little or none is present in the dorsal nuclei).
Toll signals the degradation of the maternal protein cactus.
Without Toll signal, cactus binds dorsal to keep it in the cytoplasm.
Dorsal and cactus are homologues of vertebrate NF-kappa-B and I-kappa-B.

Polarization of the body axes during oogenesis
Polarization of the body axes during oogenesis
In the germarium, a stem cell gives rise to 16 cells by four mitotic divisions which become the oocyte and 15 nurse cells, all which are connected by cytoplasmic bridges.
A sheath of somatic follicle cells surround the nurse cells and oocyte to form the egg chamber which secrete the vitelline membrane and egg shell.
Signals from older egg chambers act to polarize younger ones.
Some common signals are here.

A/P and D/V axes of the oocyte are specified by interactions with follicle cells
The oocyte induces follicle cells to adopt posterior fate & the anterior follicle cells are not in contact with the oocyte.
The signal from the oocyte to the follicle cells is the gurken protein, a member of the TGF-alpha family.
gurkin binds to torpedo, a receptor tyrosine kinase similar to the EGF receptor.
Follicle cells signal back to reorganize the oocytes cytoskeleton which direct bicoid mRNA to the anterior and oskar mRNA (which specifies germ plasm) and nanos mRNA to the posterior.
Later the D/V axis is set up by gurken (again) which signals to establish dorsal follicle cells (which do not produce the ventral follicle cell proteins needed for establishing ventral embryo fates).

A/P axis is divided into broad regions by gap genes
The gap genes, the first genes expressed along the A/P axis are transcription factors.
The gap genes are initiated by bicoid in the synctial blastoderm.
hunchback acts to help switch on the other gap genes (giant, Kruppel and knirps).
Mutants of gap genes have large sections of the body pattern missing.
Gap gene proteins are short lived (half-life of minutes) and extend only slightly outside of where the gene is expressed (bell-shaped concentration distribution.)

bicoid protein signals anterior hunchback expression
Zygotic hunchback expression is in the anterior half of the embryo.
Suppression in the posterior half, produces a gradient running A to P.
Anterior expression is switched on by high levels of bicoid.
Increased anterior bicoid expression will result in extending the hunchback gradient toward the posterior half of the embryo.
bicoid (homeodomain transcription factor) directly binds the hunchback promoter in several places.

hunchback activates and represses other gap genes
Kruppel is activated by a combination of bicoid and low levels of hunchback but is repressed by high levels of hunchback.
This locates Kruppel expression to the centre of the embryo.
knirps is repressed by high levels of hunchback .
In this way the initial gradients of morphogens can lead to the establishment of regions within the syncytial blastoderm which themselves lead to the beginning of segmentation.

Technique: Transgenic Drosophila
P element transformation is accomplished by cloning a sequence of interest (genomic region, cDNA or control region/reporter gene fusion) and a marker gene (often the white gene)  into a cloned transposable P element.
The cloned DNA along with a source of transposase (helper plasmid) are injected into the pole plasm of an early embryo.
If incorporated into the germline, progeny of the injected individual that express the marker gene can be selected.
These will also carry the transgene.
Transgenesis allows manipulation of developmental processes segmentation: pair-rule gene activation

Technique: Targeted Gene Expression
One way to control gene expression is by fusing the heat-shock promoter to a given gene.
Another method involves the two-part Gal4/UAS system.
Gal4, a yeast transcription factor, is fused to Drosophila control sequences by
1) recombinant DNA cloning (and making transgenic Drosophila)
and 2) enhancer trap (random integration/selection)
to produce the transcription factor in a desired developmental pattern
and generate a driver line.
The responsive line is generated by cloning a coding region downstream of several copies of the UAS (up-stream activating sequence) and transgenesis.
By mating individuals of these lines, the targeted gene is expressed in the selected times and places.

Zygotic gene expression along D/V axis is controlled by dorsal protein
dorsal drives gene expression to activate and inactivate a number of genes by binding to the regulatory genes of many of genes it controls.
dorsal specifies the most ventral cells as prospective mesoderm.
High levels of dorsal activates twist and snail (required for mesoderm and gastrulation).
Low levels of dorsal activate rhomboid (which is suppressed by snail) to give rise to the neuroectoderm.
decapentaplegic (dpp), tolloid and zerknult are suppressed by dorsal and are restricted to the most dorsal regions.
zerknult specifies the amnioserosa.

Zygotic genes pattern the early embryo
The most ventral region become mesoderm (muscle and connective tissue).
Ventral ectoderm becomes neurectoderm (some epidermis and all nervous tissue).
Dorsal ectoderm becomes dorsal epidermis and the amnioserosa (an extra-embryonic membrane).
The endoderm from the terminal regions, give rise to the midgut.

dpp protein patterns the dorsal region
dpp is a member of the TGF-beta family of secreted growth factors.
After cellularization, dpp is expressed in cells that do not have dorsal in the nucleus.
It produces a gradient of activity by binding an inhibitory protein sog (short gastrulation).
sog is very similar to the vertebrate protein chordin.

Parasegments (PS) are the basic module of fly development
Parasegments arise first & each segment is made from the posterior part of one PS and the anterior of the next.
Parasegments are delimited by  periodic expression pair-rule (PR) genes.
Transient grooves on embryo surface (after gastrulation) define the 14 PS.
Parasegments act as developmental units: "piece-meal fly".

Pair-rule genes delimit the parasegments
Pair-rule genes delimit the parasegments and are expressed in 7 transverse stripes (every 2nd parasegment).
Pair-rule expression determined by gap gene activity to interpret a series of broad expression patterns to make a repeated series of stripes.

Gap gene activity positions stripes of pair-rule expression
Pair-rule genes are expressed in  alternate parasegments.
even-skipped defines odd parasegments.
fushi-tarazu define even parasegments.
Striped expression pattern of pair-rule genes begins just before cellularization.
After cellularization, each pair-rule gene is restricted to a few cells in seven stripes.
Some pair-rule genes define segment boundaries.
Stripes appear slowly, first fuzzy then later become sharply defined.
even-skipped is first expressed at low level in all nuclei but then redefines into stripes.

Each stripe is independently specified
The 2nd stripe of even-skipped (eve) requires bicoid & hunchback.
giant represses eve to form a sharp anterior border.
Kruppel represses eve to form a sharp posterior border.
Since each stripe is independently controlled by combinations of transcription factors (gap genes).
Each pair-rule gene has complex control regions with multiple binding sites for each of the different factors.
Some factors activate and other inactivate.
Some require the activity of the primary pair-rule genes (such as eve and hairy).

The 3rd and 4th stripes of eve are highly directed by the hunchback gradient.

Segment polarity (SP) (or "Segmentation"_ genes and compartments
Segment polarity/segementation genes are ...
1) a diverse group of genes (not just transcription factors),
2) are expressed in 14 stripes,
3) act after cellularization and
4) are activated by the pair-rule genes.
engrailed (a transcription factor) is expressed in the anterior of each parasegment to define a boundary of cell lineage restriction.
engrailed is a selector gene which confers identity by a duration of expression.

Technique: Genetic mosaics
A genetic mosaic is an individual that has some tissues that carry cells of different genetic constitutions.
Formally accomplished via X-rays.
Flies carrying a yeast recombinase (FLP) and target sequence (FRT), can be induced to form clones of mutant tissue in an otherwise normal individual.

Expression of engrailed delimits a cell lineage boundary and defines a compartment
engrailed is expressed throughout the life of the fly (not transient like gap and pair rule genes).
A parasegment is a compartment that cells do not move between (cell lineage restriction).
Compartments can be detected by marking cells and following the fates of the clones (cell's descendants).
engrailed defines anterior margin of parasegment and thus the posterior portion of the segment.
Compartment boundaries can be studied in the adult wing which is normally divided into anterior and posterior compartments.
In a mosaics, engrailed mutant cells do not respect the "A/P boundary" and lineages are not restricted .

Segment polarity genes pattern the segments and stabilize parasegment and segment boundaries
Each larval segment has an A/P pattern:  the anterior part has denticles while the posterior part has naked cuticle.
In wingless & hedgehog mutants, the naked cuticle is converted to a mirror image duplication of the anterior part to give the "lawn of denticles" phenotype.
Segment polarity genes are expressed in a restricted subset of the cells of each parasegment.
wingless and hedgehog encode highly conserved proteins and are part of a number of signaling systems.
Parasegment boundary depends on the intercellular signaling between cells on either side of the compartment boundary involving segment polarity genes.
The patterning process is also apparent on the abdominal segments of adults.

Different mechanisms used by other insects for the body plan
Long germ band development develops all segments at once (Drosophila).
Short germ band development (Tribolium, the flour beetle), the anterior segments are formed in the blastoderm and the more posterior segments are added by growth of the posterior.
The mature germ bands appear to be similar (phylotypic stage, common to insects).
Although different growth processes are involved the same genes (i.e. Kruppel, wingless and engrailed) have conserved functions.

Segment Identity: selector and homeotic genes
Each segment has an unique identity.
Homeotic selector genes specify each segment to control other genes and maintain segment identity.
Two complexes [or a split complex] (Bithorax and Antennapedia: the HOM genes), together are homologous to the HOX gene complexes of vertebrates.
First identified by homeotic genes, mutations in which cause homeosis, the transformation of one structure into another structure.
Antenna to leg (Antennapedia) or haltere to wing (Bithorax)

Homeotic genes of the bithorax complex (BX-C) are responsible for the posterior segments
Bithorax complex (BX-C) consists of three homeobox genes (Ubx, abd-A & Abd-B).
Ubx is expressed from PS 5 and posterior.
abd-A is expressed in PS 7 and posterior.
Abd-B is expressed in PS 10 and posterior (and suppresses Ubx).
Expression is controlled by gap & pair-rule genes.
Larvae missing the complete bithorax complex, develop PS 5-13 as PS4, thus BX-C diversifies PS5-13 and PS4 is the default state modified by the BX-C proteins.
BX-C genes impose a new identity to the segments (selector genes).

Experiment: Replace BX-C components into embryos missing the complex.
BX-C absence: PS1-4, plus ten more PS4 like segments.
BX-C components were replaced by targeted gene expression.
Ubx only (missing abd-A and Abd-B): PS1-6 followed by seven more PS6-like segments.
Ubx plus abd-A (no Abd-B): PS1-9 plus 4 PS9 segments.
Parasegments must be acting in a combinatorial manner.
While gap and pair-rule genes control the original pattern of HOM gene expression, the polycomb and trithorax gene groups maintain the correct expression of these genes after first four hours.
The polycomb group maintain transcriptional repression of homeotic genes.
The trithorax group maintain expression of homeotic genes.

Antennapedia complex controls specification of anterior regions
Antennapedia complex (Antp-C) consists of 5 homeobox genes.
Antp-C control expression anterior parasegments in a manner similar to BX-C in the posterior segments (described above).
deformed mutants affect PS0&1.
Sex combs reduced mutants affect reduced PS2&3.
Antennapedia mutants affect PS4&5.
As with HOX genes in mammals, HOM gene expression order corresponds to the order of genes on the chromosome.

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