Candidate Gene Approach - 2 Lecture 6 BSE

Complementation analysis

Which genes were missed and why? Genes required for cleavage and cellularization Development 104, (1988) Large deficiencies uncovered SEVEN genes essential for cellularization (14 th cell cycle). Three of these seven genes are cytoskeleton related genes.

Genes regulating morphological changes during gastrulation These genes were mostly missed in the Heidelberg screen because the defects in gastrulation produced by them did not have a cuticular phenotype that could be observed

Enhancer screen The art and design of genetic screens: drosophila melanogaster Daniel St. Johnston VOLUME 3 | MARCH 2002 Compulsory reading Enhancer screen for sevenless Genes are named after such screens e.g. suppressor of fused or enhancer of split etc. Some of the most successful screens of this type were carried out to find the components in the signal-transduction pathway downstream of Sevenless (Sev), which is a receptor tyrosine kinase (RTK) that controls cell-fate choice between one of the eight photoreceptors in the eye, the R7 cell, and non-neuronal cone cells30.A weak allele of sev provides just enough signalling activity for most of the R7 cells to form, but these cells are transformed to cone cells if there is any further reduction in the efficiency of signal transduction. Under these conditions, halving the dose of some of the downstream components in the pathway causes most of the R7 cells to become cone cells,without affecting signalling through the other RTKs in the fly. This sensitized genetic background therefore allowed Simon et al.30 to identify mutants in components of the RTK signalling pathway as dominant enhancers of a visible eye phenotype.

1.Homeotic genes, co-linearity and establishment of the body plan  Ed Lewis and Walter Gehring, Denis Duboule (vertebrates) Homeotic genes confer identity to a body segment. Homeotic mutations transform certain parts or an entire body segment into the corresponding structures of another body segment, thereby changing the architecture of the organism. What have we learned from the drosophila, C. elegans and zebrafish genetic screens

Systematic analysis by Ed Lewis ( helped Dennis Duboule (Trends Genet. 1994;10:358–364) propose the idea of “posterior prevalence”. Elimination of entire bithorax complex converts all the segments into T2 -> thus second thoracic (T2) segment is proposed to be the “ground state”. The three BX-C complex genes are expressed in a nested pattern - Ubx from T3 to A8, abd-A from A2 to A8 and Abd-B from A5 to A8. Mutation in extra sex combs (esc), a transcriptional repressor gene that lies outside the BX-C locus, can apparently activate all of the BX-C genes in all segments T2-A8, generating a mutant fly in which all segments resemble A8. Deletion of Ubx => A1 and T3 develop as T2; deletion of abd-A => T2-A1 develop more-or less normally but A2-A4 develop as A1; Abd-B - T2-A4 develop normally but A5-A8 develop as A4 (reference - Based on genetics alone, Ed Lewis thought there were genes in the BX-C complex but now it is known that of the different mutant loci characterized by Ed Lewis only three, Ubx, abd-A and Abd- B code for proteins. It is now known that iab-2 to iab-8 are non-coding regulatory elements, possibly encoding miRNAs - Nat Rev Genet. Oct 2008; 9(10): 789–796.Nat Rev Genet. Oct 2008; 9(10): 789–796. The concept of colinearity and posterior prevalence

Comparative anatomy and Hox gene expression There is a correlation between the span of specific Hox genes in a given species and the identity of the vertebral element.

Gene targeting data also support the notion that Hox genes assign regional identity HoxB6 is expressed throughout the axial skeleton Unlike in flies in vertebrates there are four copies of Hox clusters and thus to realize phenotype all the copies have to be deleted in mouse.

First set of morphogens were identified Morphogen gradient model - According to the gradient model, pattern is established by one or a few multifunctional regulatory substances, called morphogens, that are distributed in monotonic gradients. These morphogens specify cell fate in a concentration- dependent fashion such that cells acquire different fates based on the concentration of morphogen that they contain. First set of morphogens were identified – Bicoid, Dorsal, Nanos and Dpp

The entire pathway for determination of dorso- ventral polarity was uncovered from this screen

The concept of lateral specification/inductive signaling and Notch/Glp-1/Lin-12 gene Lateral specification Inductive signaling

Juxtacrine signaling The concept of lateral specification/inductive signaling and Notch/Glp-1/Lin-12 gene