Genetic Pathways: Development of Body Axes in the Fly Embryo • The fruit fly embryo develops in a single day, hatching from its eggshell as a worm-like larva • The larva grows through 3 stages called "instars", then secretes a hard casing and becomes a pupa • Inside the pupal case the larval tissues undergo metamorphosis, generating the body of the adult fly ◦ Minimum time fron oen generation to the next is only 10 days '• Different life stages possess the same dorsoventral (DV) and anteroposterior (AP) body axes ◦ AP axis is organized into segments • The embryo uses a fundamentally different mechanism to establish DV and AP• AP axis is first established by asymmetric localization of maternal gene products at the anterior and posterior poles of the embryo • During egg formation, maternal mRNA from the bicoid gene becomes localized at the future anterior end of the egg • Individuals homozygous for loss-of-function alleles of the bicoid gene completely lack anterior structures◦ Form a larva with 2 rear ends instead • AP axis is first established by asymmetric localization of maternal gene products at the anterior and posterior poles of the embryo • Unlike bicoid, maternal mRNA from the oskar gene becomes localizedat the future posterior end of the egg • After 2 hours of embryonic development, the Drosophila embryo consists of a continuous monolayer of somatic cells surrounding a central yolk mass and a small number of posterior pole cells that segregate from the monolayer• Both bicoid and oskar mRNAs are initially produced at the anterior end of the egg• As the egg develops, microtubules grow from the anterior to the posterior end • An adapter protein links bicoid mRNA to the (-) ends of the microtuulesso it remains anteriorly • oskar mRNA becomes associated with motor proteins that transport it to the (+) ends of the microtubules ◦ Oskar mRNA relocates to the posterior end• Bicoid and oskar mRNAs have localization sequences in their 3'-UTRs◦ A fusion gene with the oskar protein-coding sequence fused to the bicoid 3'-UTR◦ When injected into the egg, this fusion mRNA localized to the anterior pole of the egg where it induced a second set of pole cells to form • The fly's DV axis is initially established by an extracellular signal that acts asymmetrically on the developing egg/embryo • Even as an embryo, the fly develops distinctive dorsal and ventral structures◦ The ventral surface is marked by segmentally repeating denticle belts (bands of fine bristles the larva will use for traction when crawling)◦ The dorsal surface has posterior spiracles (tubes that supply the developing embryo with air • Inside its eggshell the fly embryo is exposed to a ventral-to-dorsal concentration gradient of diffusible protein called Spatzle • Spatzel is the ligand for a receptor protein named Toll ◦ Toll is distributed uniformly over the entire egg cell membrane • Due to the gradient of ligand, the Toll receptor is strongly activated on the ventral side of the embryo (100% bound to ligand) and not activated at all on the dorsal side of the embryo (0% bound) ◦ Toll is activated to intermediate levels at intermediate positions • When Toll binds to Spatzle, it initiates a signal transduction pathway that ultimatley activates gene expression in nearby nuclei• The transcription factor is a proteins called DORSAL◦ w/o signal transduction, Dorsal is tethered in the cytoplasm by the protein Cactus • When Toll is activated by its ligand, this causes Cactus to be (1) phosphorylated (by Pelle and Tube)and as a result (2) degraded • Loss of cactus allows Dorsal protein to enter the nucelus where it can bind to the enhancers of its target genes • Ventral-to-dorsal gradients:◦ A extracellular gradient of Spatzle concentration ◦ A gradient of Toll activation in the cell membrane ◦ A gradient of Dorsal concentration inside the nuclei• Mutations in the dorsal and cactus genes give opposite phenotypes: ◦ Loss of function mutations of dorsal yield a dorsalized phenotype(without Dorsal protein, all cells develop as if they were located on the extreme dorsal side where there is normally no relocalization of Dorsal into the nucleus ◦ Loss of function mutations of cactus yied a ventralized phenotype( without cactus protein tethering it in the cytoplasm, Dorsal relocates into the nuclei of all cells regardless of position • Every protein we have talked about is a maternal gene product ◦ Only a mutation in the mother's gene would influence the embryo's phenotype ◦ But when it enters the nucelus, the Dorsal protein activates transcription of the embryo's own genes◦ For the latter genes, it’s the embryo's own genotype that will determine its phenotype• The genes twist and snial are only activaated by high concentrations ofthe Dorsal transcription factor • They are only transcribed in the most ventral region of the embryo • Only the most ventral nuclei in the Drosophilia embryo activate transcription of the twist and snail genes • Concentration-dependent transcription of Dorsal target genes • Note that the Dorsal TF can serve as either an activator or a repressor when it binds to the enhancers of different genes • Chemical Affinity ◦ A transcription factor only influences transcription when itsbound to the DNA ◦ The probability that a DNA binding site will be occupied by its transcription factor is a function of their affinity ◦ At low protein concentrations , only high affinity binding sites willhave an effect on target gene transcription ◦ At high protein concentrations, both high and low affinity sites will have an effect on target gene transcription • Binding site affinity determines the concentration dependent transcription of Dorsal target genes • Dorso-ventral patterning in flies ◦ The axis of dorso-ventral polairty is determined by an extracellular signal, Spatzle protein, in the fluid surrounding the embryo ◦ A concentration gradient of Spatzle protein outside the embryo leads, via signal transduction, to a concentration gradient of Dorsal protein in nuclei of the embryo◦ Dorsal protein activates different target genes depending on its nuclear concentration ◦ These target genes specify the formation of different tissues at different positions along the axis • Sog and rho expression patterns are more complicated ◦ Eventually both become restricted to lateral bands of tissue that DON'T