Download Developmental Biology, 9e

BIOL 370 – Developmental Biology
Topic #8
The Genetics of Axis Specification in Drosophila
Lange
While we know a large amount about fruit fly genetics due to decades of
research beginning most notably with Thomas Hunt Morgan (Nobel
Prize in 1933) and his students, fruit fly development is not as well
understood in some ways, due to its complexity.
Laser confocal micrographs of stained chromatin showing superficial cleavage in a Drosophila
embryo
We see here syncytial eggs. Synctyial cells are defined as having a
multinucleated mass of cytoplasm that is not separated into
individual cells.
From what we do know, however, especially interesting for
Drosophila development is:
• CELL MEMBRANES do not form until after the 13th nuclear
division.
• Prior to the 13th nuclear division, the nuclei share the same
“common” cytoplasm
• This allows information from each nuclei to diffuse through
the entire embryo until that time. (Potential value?)
• Cell specification occurs by the interactions of the
components within a SINGLE multinucleated cell.
Fertilization in Drosphila:
• Sperm enters an ACTIVATED EGG.
• Activation of the egg occurs during ovulation
• When the female oviposits (lays) her eggs, the pressure of going
through the ovipositor opens calcium channels in the membrane
of the oocyte.
• The open calcium channels allows calcium to flood into the
oocyte.
• Due to this influx of calcium, the nucleus of the oocyte initiates
meiotic processes associated with nuclear division
For the male in fertilization, there is only one site on the egg
where sperm can enter…
The micropyle:
This structure is actually a channel/tunnel in the chorion
of the egg (chorion is the name given to the shell of the
egg).
Drosophila sperm have
extremely long tails. In
D. melanogaster, the
length of a single sperm
can be up to 1.8mm in
length.
This is almost as long
as the adult fly, is
longer than the egg,
and is about 300X
longer than a human
sperm.
Formation of the cellular blastoderm in Drosophila
From the syncytial blastoderm at
roughly division 10, microtubules
begin to surround the nuclei creating
a nucleus/cytoplasm/microtubular
“island” called an ENERGID.
Following division 13, the egg cell
membrane begins to fold inward,
creating the cellular blastoderm.
Nuclear and cell division in Drosophila
A = embryo in the syncytial stage
B = cellular blastoderm
Fruit Fly Developmental Stages & Life Cycle
Gastrulation in Drosophila:
• While they appear vastly different, the general body plan of the
multicellular embryo, the larva, and the adult are the same.
• Head region
• Multilple (11) segmental units
• Tail region
Each of these 11 segments has a unique identity as well. For example in
the first three thoracic segments:
1 = legs
2 = legs and wings
3 = legs and halteres
Halteres are small knobbed structures modified from the hind wings in some twowinged insects (like Drosophila). They are moved rapidly in a countercurrent pattern
to the wins and function as gyroscopes. They help with balance in flight and
informing the insect about rotation of the body during flight.
Gastrulation in Drosophila
Gastrulation in Drosophila (Part 4)
This is the 1st instar
larvae that is readily
visible in a fruit fly
culture.
1st Instar
Schematic representation of gastrulation in Drosophila
(Late in B & Early into C):
Start of gastrulation
Left = what will become ventral
body surface
Right = dorsal surface
Comparison of larval (left) and adult (right) segmentation in Drosophila
H segment = head segment
T segments = thorax segments
A segments = abdominal segments
(A) Early cleavage (15 minutes–1.5 hours).
cleave in the central region.
Nuclei
(B) Migration of cleavage nuclei (1.5 hours). Nuclei
migrate to the periphery.
(C) Formation of syncytial blastoderm (2 hours). Pole
cells form posteriorly.
(D) Cellular blastoderm (2.5 hours). Cell membranes
form between the nuclei.
(E) Early gastrulation (3.5 hours). The ventral furrow
forms. Thickening of the posterior plate below the pole
cells.
(F) Midgut invagination (3.5–4 hours). The midgut
invagination can be seen ventrally, as can the cephalic
furrow.
(G) Germ band extension (4–5 hours). Invagination of the
hindgut can be seen dorsally.
(H) Stomodeal invagination (5–7 hours). Invagination of
the stomodeum can be seen ventrally.
(I) Shortening of the germ band (9–10 hours). Foregut
and hindgut invaginations are deep.
(J) Shortened embryo (10–11 hours). Hindgut is now fully
posterior.
(K) Dorsal closure (13–15 hours). The ectoderm closes
dorsally. The midgut broadens. The head involutes.
(L) Condensation of ventral nervous system (15 hours–
hatching). The gut regions are joined. The nervous
system forms ventrally.
The Value of Cell Polarity:
• Organ development is a carefully scripted process of cellular
proliferation, differentiation, and apoptosis.
• Persistently delineating cell polarity by identifying the apical (top of
the cell) and basolateral (bottom and sides of the cell) surfaces is
crucial for the proper development of organs.
• Defects in this system can result in severe developmental
abnormalities and embryo death.
• Several of the key proteins defining cell polarity have been identified,
but the role these proteins play during development is still often
unclear.
(The above based upon information from G. Brennan (accessed 04/15/13).)
The anterior-posterior axis is specified during oogenesis (Part 1)
Gurken proteins (green spheres (aka a “pickle”) when produced
by the oocyte will bind with the Torpedo receptors in the terminal
follicle cells driving what will become the posterior axis.
The anterior-posterior axis is specified during oogenesis (Part 2)
The anterior-posterior axis is specified during oogenesis (Part 3)
Germline chimeras made by interchanging pole cells between wild-type embryos and embryos from
mothers homozygous for a mutation of the torpedo gene
The exchange of the pole cells affects further differentiation of the embryos.
• The follicle cells that were made deficient for torpedo guided abnormal
development in genetically normal embryos.
•
• In mutant strains, follicle cells were made “wild” for torpedo guided
NORMAL development in genetically abnormal embryos.
Generating dorsal-ventral polarity in Drosophila
Effect of mutations affecting distribution of the Dorsal protein, as seen in the exoskeleton patterns
of larvae
These images represent
another mutation this time
guiding development of only
dorsal cells.
• (A) shows typical
development of this
mutation in the larval
form
• (B) shows how normal
appearing development
can be induced via
injection of wild-type
mRNA.
Gastrulation in Drosophila
Dorsal-ventral patterning in Drosophila
Left-right axis formation in Drosophila involves the microfilament cytoskeleton
The Drosophila also has a distinct right/left division and is not simply a
mirror image bilaterally. Areas that most readily show this asymmetry
include the hindgut (HG), and the male (MG) and female (FG) gonads.
Normal and irradiated embryos of the midge Smittia
Effects of radiation (UV light) on the early development of the midge
can be profound if they alter ventral/dorsal polarity. Here the bottom
(irradiated) midge displays NO HEAD and two abdominal/tail regions.
Syncytial specification in Drosophila
Syncytial – a multinucleated mass of cytoplasm that is not
separated into individual cells.
Schematic representation of experiments demonstrating that the bicoid gene encodes the
morphogen responsible for head structures in Drosophila
In this experiment by Driever et. al. (1990), bicoid mRNA from wild type
Drosophila are injected into mutant strains or abnormal locations in
other wild strains. Note the results in terms of head development.
Bicoid mRNA and protein gradients shown by in situ hybridization and confocal microscopy
Caudal protein gradient in the syncytial blastoderm of a wild-type Drosophila embryo
***Note that the gradient is complementary to that of bicoid.
Model of anterior-posterior pattern generation by Drosophila maternal effect genes
Bicoid protein gradient in the early Drosophila embryo
Three types of segmentation gene mutations
In normal, unmutated Drosophila, each segment
produces bristles called denticles in a band
arranged on the side of the segment closer to the
head.
Gap Segmentation Mutation where the active gene
differentiates the whole thorax region uniformly, and
the mutant form lacks these segments.
Pair-rule mutation where alternate segments do not
develop normally in the mutant form.
Segment Polairty mutation where the portion of the
posterior segment does not develop in the mutant
Homeotic gene expression in Drosophila
Homeotic genes cause the development of specific structures in plants and animals.
A four-winged fruit fly constructed by putting together three mutations in cis-regulators of the
Ultrabithorax gene
End.