Genetics of Axis Specification in Drosophila: Anterior-Posterior Axis Determination Gilbert - Chapter 9

Axis Specification 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!!!

distinctive structures The genetic screen looked 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 Nanos 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