Transcript Bicoid mRNA - bthsresearch

Genetics of Axis Specification
in Drosophila: AnteriorPosterior Axis Determination
Gilbert - Chapter 9
Axis Specification
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Controlled by a variety of genes
Maternal effect genes
Gap genes
Pair-rule genes
Segment polarity genes
Homeotic selector genes
Genetic Screen for Genes involved in
Drosophila Development
• Nusslien-Volhard, Wieschaus
• Fed mutagens to Drosophila
• Then breed until mutation is homozygous
recessive
• Examined embryos for patterning defects
• Used embryonic cuticles to do screens
– Looked at pattern of denticles, shapes of
segments
Goals of Genetic Screen
• Create small mutations in fruit fly genome
– Enough to mutate EVERY gene in the
genome at least once
– Identify EVERY mutation in the genome
that affects embryonic development in the
fruit fly
– How many genes are there in fruit flies?
– Note - At this time the D. melanogaster
genome was not sequenced
How was the genetic screen
performed?
• Feed adult fruit flies with a mutagen
– EMS - causes high mutation rate in offspring
– Point mutations, short deletions, rearrangements
– Can cause defective proteins, absence of proteins
• THEN - breed the flies until the mutation is
homozygous, and look for ANY embryo that
has abnormalities
• TENS OF THOUSANDS OF EMBRYOS ARE
EXAMINED!!!
The wild-type body
is segmented
and each segment
has a unique identity
and thus produces
distinctive structures
-The genetic screen looked
for changes in this body plan
The molecular genetics of
pattern formation in candy corn
Anterior
Borrowed from
Mark Peifer,
Ph.D.
One of my great
prof’s. at UNC!
Posterior
The wild-type pattern
They collected a series of mutants
affecting the body plan
Wild-type
Posterior
group
Anterior group
Morphogenesis
defects
Gap gene
Borrowed from Mark
Peifer, Ph.D.
One of my great prof’s.
at UNC!
Wieschaus and
Nüsslein-Volhard
removed single genes
and looked for effects
on the body plan
Genes involved in
embryogenesis
• Genes controlling embryonic
development are either
–maternal-effect genes
•mRNA or protein already deposited in the egg
– zygotic genes
•Transcribed from nucleus of zygote
Maternal effect genes:
Anterior - Posterior Polarity
• Placed into developing oocyte by
maternal cells
– Nurse cells that surround the egg
• Several genes were discovered
• What phenotype would you be looking
for in your embryos??
bicoid mutants have no head end!!
Wildtype larva
bicoid mutant
Gilbert Fig. 9.13
Bicoid Mutant
• Lacks anterior structures, posterior
structures are duplicated
• Leads to lots of Molecular Biology
questions!
– How can we find this gene and its
sequence?
– What does the phenotype tell us about the
function of bicoid?
– What is the bicoid protein like in this
mutant?
Bicoid and Nanos
• Bicoid mRNA
– Concentrated in the future anterior end of
the ovum by the nurse cells
– Nurse cells are ovary cells of the mother
– The mRNA is held in place by a network of
microtubules
• Nanos mRNA
– Tethered to the cytoskeleton at the future
posterior end of the egg
Bicoid and Specification of the
Anterior pole
• Bicoid appears to be essential for the
formation of anterior structures
• Further evidence
– Bicoid mRNA is localized to the anterior
end of the oocyte
– As bicoid mRNA gets translated, a gradient
of bicoid is created from A to P
• More concentrated at most Anterior end
Localization of bicoid mRNA - What technique
was used??
Bicoid Protein gradient: What technique is used?
Bicoid - More evidence
• Bicoid mRNA (wild-type) can rescue the
Bicoid mutant phenotype
– Inject bicoid mRNA into anterior end of
bicoid mutant embryo
• Injection of WT bicoid mRNA anywhere
into the early embryo turns that area
into a head end!
What does bicoid do, and
how?
• Bicoid protein is arranged in a gradient
• It represses the molecules that control
posterior identity
– Represses translation of a molecule that
helps specify the posterior end - Caudal
– Caudal mRNA is found in entire embryo
– Caudal protein is found at posterior end
Caudal protein localization
What does bicoid do, and
how?
• Bicoid protein is a transcription factor
• Enters nuclei, activates downstream
gene expression
– Hunchback - essential for anterior pole
formation
• Hunchback activates transcription of further
head-specific gene products (swallow,
exuperantia, buttonhead, orthodenticle)
A-P axis specification: a
combination of protein gradients
• Nanos - mRNA tethered to posterior pole
– Forms a gradient from posterior to anterior
– Inhibits translation of Hunchback mRNA in the
posterior region
– What would be the phenotype of a Nanos mutant?
Resulting gradients
A-P gradient of bicoid
A-P gradient of hunchback
P-A gradient of nanos
P-A gradient of caudal
Maternal effect genes and AP
polarity - summary
• Maternal mRNA’s are tethered to either
A or P ends of oocyte
• Proteins are translated to create a
gradient
• Activate or repress embryonic gene
expression (hunchback, caudal)
• Result is and anterior region and a
posterior region
Hunchback protein localization