Axis Specification and Patterning II Segmentation and Anterior posterior axis specification in the Drosophila embryo

Segmentation and anterior-posterior body plan The hierarchy of genes identified in the genetic screens pioneered by Christiane Nϋsslein-Volhard 1)Genes that establish anterior posterior polarity 2)Genes that divide the embryo into specific number of segments each with a different identity Zygotic genes

Classical experiments: Klaus Sanders (1975) Lligated the egg early in development from the middle. One half became an anterior end of the embryo and other the posterior half the middle segments were missing. Destruction of RNA in the anterior of insect eggs with Ultraviolet light or RNAse Embryos lacked a head and thorax and developed into embryos with two abdomens and two telson (tails) Normal Irradiated Postulate: Morphogen gradients emanating from the two poles interacting to produce positional information.

What were these morphogens present in gradients in the early embryo? The genetic screen carried out by Christiane Nϋsslein-Volhard and colleagues.

Maternal gradients: polarity regulation by oocyte cytoplasm

Bicoid as the anterior morphogen was established by the strategy of “find it, lose it, move it” “find it” Bicoid protein and bicoid mRNA were both found to be present in a gradient Highest in the anterior (head-forming region)

“lose it” “move it”

Anterior determinant: Localization of the bicoid mRNA to the anterior of the oocyte  3’ UTR of bicoid mRNA is critical for its localization.  bicoid mRNA synthesized in nurse cells interacts with Exuperantia and Swallow proteins.  This complex is transported out of nurse cells on microtubule rails riding a Kinesin.  In the oocyte bicoid mRNA associates with the dynein proteins present at the minus end of microtubules in the anteriror.

Posterior determinant: nanos mRNA is localized to the posterior pole of the oocyte  nanos mRNA seems to be “trapped” in the posterior end by passive diffusion.  nanos mRNA interacts with products of oskar, valois, staufen, vasa and tudor.  The oskar mRNA and Staufen protein are transported first to the posterior of the oocyte via Kinesin motors.  Staufen permits the translation of oskar mRNA. The Oskar protein then binds nanos mRNA and traps it in the posterior.

Gradients of specific translational inhibitors: Localized translation of hunchback and caudal transcripts Bicoid Caudal 2/devo_02.html

How is it that different concentrations of Bcd at different points along the A/P axis of the embryo lead to transcription of different target genes?  The Bcd gradient provides positional information along the axis in a dose- dependent manner and efforts have been made to understand how this could be achieved. As the Bcd protein encodes a DNA-binding transcription factor, it was initially proposed that the thresholds of Bcd concentration required for the expression of its target genes depend on the number and on the affinity of Bcd binding sites found in their regulatory regions.  However, it is now clear that other elements in target gene promoters and the integration of positive and negative transcriptional inputs from proteins bound to these elements are major determinants for the interpretation of positional information along the anterior-posterior axis.

Segmentation genes

Gap genes The gap genes are activated or repressed by maternal effect genes and are expressed in one or two broad domains along the anterior posterior axis. The transcription pattern of different gap genes are initiated by different concentrations of Hunchback and Bicoid.

Figure 6.29 Architecture of the gap gene network Interactions between AP polarity genes and gap genes  High levels of Bicoid and Hunchback induce the expression of giant  Kruppel transcripts appear where Hunchback transcripts decline.  The Caudal protein which is highest in the posterior activates the giant and knirps genes in the posterior.

Figure 6.30 Messenger RNA expression patterns of two pair-rule genes, even-skipped (red) and fushi tarazu (black) in the Drosophila blastoderm Pair rule genes: The first indication of segmentation Primary pair rule genes: hairy, even-skipped and runt Expressed in 7 stripes each. Secondary pair rule genes: fushi tarazu, odd-skipped, odd-paired, sloppy-paired and paired

Figure 6.31 Specific promoter regions of the even-skipped (eve) gene control specific transcription bands in the embryo Each stripe pattern is the result of distinct enhancers which are modular in nature. Genetic studies: Deletion of each enhancer leads to disappearance of a particular stripe. Biochemical studies: LacZ reporter placed under an enhancer is expressed in a single stripe

Segment polarity genes:  They reinforce the paprasegment boundaries established by earlier transcription factors.  They establish cell fates within each parasegment through cell-cell signaling.  Encode members of the Wingless and Hedgehog signaling pathways.  engrailed is expressed in those cells with high levels of Eve, Paired or Ftz and inhibited in cells with high levels of Odd–skipped, Runt or Sloppy-paired.  Thus engrailed marks the anterior part of each parasegment.  wingless is transcribed in cells that see little or no Eve or Ftz but which have Sloppy-paired.  Thus wingless is transcribed in a row of cells just anterior to those making Engrailed.

The key to maintaining the pattern of engrailed and wingless is in activation of engrailed (en) in the cells that will express Hedgehog.

The diffusion of the Wingless and Hedgehog proteins provide the morphogen gradients by which cells acquire distinct identities with a parasegment.

 At Stage 9-10 the Wg protein gradient is symmetric and present anterior as well as posterior to the cells expressing Wg.  At Stage 11 the Wg protein gradient becomes asymmetric such that Wg protein is only present in a gradient anterior to the wg expressing cell.  This asymmetry in Wg distribution and correspondingly signaling activity is a result of its rapid endocytosis and degradation in cells posterior from which it is secreted, in a process that is promoted by Hh signaling.  Wg signaling, in turn, attenuates Hh signaling anterior to the En/Hh-expressing cells, thereby allowing its activity in only the posterior direction. Cold Spring Harbour Perspectives Wnt/Wingless Signaling in Drosophila Sharan Swarup and Esther M. Verheyen Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia,V5A1S6, Canada

EMBO reports vol. 2 | no. 12 | pp 1083–1088 | 2001 Generating patterns from fields of cells Examples from Drosophila segmentation Bénédicte Sanson University of Cambridge, Department of Genetics, Downing Site, Cambridge CB2 3EH, UK  Wg in the cells anterior to it inhibits ser expression while Hh also inhibits ser expression in cells posterior to the Hh expressing cells thus restricting Ser to the middle of the parasegment. Rho expression is repressed by Wg signaling and is positively reinforced through a combination of Hh and Ser signaling.  Thus during stage 12, each parasegment is divided into four domains that express specific genes that are responsible for the intra-parasegmental patterning of the embryo.  Within each segment the binary decision between specification of naked cuticle or denticle cell fates is dependent on the expression of a transcription factor encoded by the shaven baby (svb) gene. Wg signaling specifies naked cuticle by repressing the expression of svb.

Homeotic selector genes After the segmental boundaries are set the pair- rule and gap genes interact to regulate the homeotic selector genes.

Ultrabithorax Antennapedia Loss of expression of Ubx. T3 acquires identitiy of T2. Antennapedia misexpresed in the head and the thorax

Initiation and maintenance of homeotic gene expression patterns  pair-rule and gap genes interact to regulate the homeotic selector genes  abdA and AbdB are inhibited by Krϋppel and Hunchback which prevent their expression in the head and thorax region.  Antennapedia is activated by particular levels of Hunchback thus only in parasegment 4 which becomes T2.  The expression domains of homeotic genes are dynamic.  There is also reciprocal interactions between homeotic genes such as bithorax complex genes repress the expression of Antennapedia.