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Bicoid morphogen is among the earliest triggers of differential spatial pattern of gene expression and subsequent cell fate determination in the embryonic development of Drosophila melanogaster. This maternally deposited morphogen, diffusing along the anterior-posterior axis of the embryo, establishes a concentration gradient which is sensed by target genes. In most computational model based analyses of this process, the translation of the bicoid mRNA is thought to take place at a fixed rate in the anterior pole of the embryo. Is this process of morphogen generation a passive one as assumed in the modelling literature so far, or would available data support an alternate hypothesis that the stability of the mRNA is regulated by active processes? This thesis demonstrates a Bicoid spatio-temporal model in which the stability of the maternal mRNA is regulated by being held constant for a length of time, followed by rapid exponential degradation. With the mRNA regulation, three computational models of spatial morphogen propagation along the anterior-posterior axis are analysed: (a) passive diffusion with a deterministic differential equation, (b) diffusion enhanced by a cytoplasmic flow term and (c) stochastic diffusion modelled by Gillespie simulation. Comparison of the parameter estimation in these models by matching to the publicly available data, FlyEx, suggests strong support for mRNA regulated stability. With a non-parametric Bayesian setting, we have applied Gaussian process regression to infer the mRNA regulation function as a posterior density. With synthetic data obtained from a linear spatio-temporal dynamical system and the experimental measurements (FlyEx), this approach is capable of inferring the driving input. Apart from confirming the validity of a regulated mRNA source, this work also demonstrates the applicability of a powerful non-parametric model of Gaussian processes in a spatio-temporal inference problem. In line with recent experimental works, we have also analysed this model with a spatial gradient of maternal mRNA, rather than being fixed at the anterior pole. Our final work is to analyse the dynamical topology of the gap gene network, which is the major developmental activity, taking place after the establishment and interpretation.

I have analysed the regulation and function of the Him gene to gain new insights into Drosophila heart development and its controlling factors. My results show that Him is important during the early specification of pericardial cells and cardioblasts. Loss of Him leads to a reduced number in both of these cell types by the end of embryogenesis. Over-expression of Him throughout the heart results in supernumerary pericardial cells. Him is expressed in all embryonic pericardial cells from embryonic stage 12 to approximately stage 15. I have identified an enhancer fragment that reproduces this expression pattern. Phylogenetic footprinting revealed three highly conserved regions within this sequence. I undertook an extensive mutational analysis of this enhancer to identify regulatory elements within it. I identified Tinman as a direct activator of Him expression. My data indicate that Him is activated in a widespread area of the dorsal mesoderm and the amnioserosa and is actively limited to the pericardial cells. A 5 bp mutation within the enhancer sequence allows for expression within the cardioblasts. Both heart cell types develop from the dorsal mesoderm and some share immediate progenitors. By stage 13, Him is pericardial cell specific and Mef2 is cardioblast specific. This is essential for normal heart development. If Him is not excluded from the cardioblasts, expression of the muscle-cell specific differentiation gene myosin is disrupted, similar to what has been described for Mef2 null mutants. If Mef2 is expressed in pericardial cells, the larval development of the pericardial cells is severely disturbed. A possible explanation for these data is that Him is part of a genetic program that prevents the premature differentiation of heart cells and its down-regulation permits the pericardial cells to undergo their correct development.

fruitless (fru) is a pleiotropic gene which produces an array of variant transcription factor isoforms to fulfil a range of developmental and behavioural roles, from the regulation of axonal pathfinding during embryogenesis, all the way to the precise orchestration of individual steps of the Drosophila melanogaster male courtship ritual. Much of the transcriptional differentiation is achieved through the use of multiple promoters, sex-specific splicing and variant C-termini. Alternative splicing at the 3′ end enables generation of transcripts containing one of at least three zinc finger (Zn-F) domains (A, B or C). Due to the close proximity of these Zn-F–encoding exons at the fru locus, almost all extant fru mutants reduce or eliminate expression of all isoforms from a given promoter(s). This has prevented genetic dissection of their individual roles, and limited functional assignment to the zinc finger triumvirate as a whole. Using gene targeting by homologous recombination, this project set out to generate precise null mutations for type-A and -B Fru isoforms in an attempt to determine which aspects of fru function are conferred by these isoforms. Isoform-specifc antibodies were also generated to confirm the loss of individual isoforms within the generated mutants, and to investigate the expression of different isoforms throughout development. Generation of such an antibody to FruB proteins enabled the developmental expression pattern of this isoform to be assessed for the first time, and expression in the male-specific serotonergic neurons of the abdominal ganglion suggested a possible role for male-specifc FruB isoforms in male fertility. Investigation of the developmental expression patterns of FruC revealed a novel immunostaining pattern for this isoform in a group of coalescing cells which appear towards the end of embryonic development. Isolation of specific-isoform mutants was achieved, giving rise to multiple mutant phenotypes, varying in severity between mutant lines. Analysis of mutants lacking type-A and -B Fru isoforms demonstrated the importance male-specific type-A and/or type-B isoforms in establishing male fertility, and suggested essential roles for sex-non-specifc type-A and/or type-B isoforms in the viability and morphology of both sexes.