dissection-immunostaining-imaginal-discs-from-drosophila

Dissection and Immunostaining of Imaginal Discs from <em>Drosophila melanogaster</em>

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Indiana University

The compound eye of Drosophila melanogaster is configured by a differentiating wave, the morphogenetic furrow, that sweeps across the eye imaginal disc and transforms thousands of undifferentiated cells into a precisely ordered repetitive array of 800 ommatidia. The initiation of the furrow at the posterior margin of the epithelium and its subsequent movement across the eye field is controlled by the activity of the Hedgehog (Hh) signaling pathway. Differentiating photoreceptors that lie behind the furrow produce and secrete the Hh morphogen, which is captured by cells within the furrow itself. This leads to the stabilization of the full-length form of the zinc-finger transcription factor Cubitus interruptus (Ci(155)), the main effector of Hh signaling. Ci(155) functions as a transcriptional activator of a number of downstream targets, including decapentaplegic (dpp), a TGFβ homolog. In this report, we describe a mechanism that is in place within the fly retina to limit Hh pathway activity within and ahead of the furrow. We demonstrate that the helix-loop-helix (HLH) protein Extramacrochaetae (Emc) regulates Ci(155) levels. Loss of emc leads to an increase in Ci(155) levels, nuclear migration, apical cell constriction and an acceleration of the furrow. We find that these roles are distinct from the bHLH protein Hairy (H), which we show restricts atonal (ato) expression ahead of the furrow. Secondary furrow initiation along the dorsal and ventral margins is blocked by the activity of the Wingless (Wg) pathway. We also show that Emc regulates and cooperates with Wg signaling to inhibit lateral furrow initiation.

Extramacrochaetae imposes order on the Drosophila eye by refining the activity of the Hedgehog signaling gradient.

During the third and final larval instar stage, thousands of pluripotent cells within the Drosophila eye imaginal disc are transformed into a near perfect neurocrystalline lattice of 800 unit eyes called ommatidia. This transformation begins with the initiation of the morphogenetic furrow at the posterior margin of the eye field. The furrow, which marks the leading edge of a wave of differentiation, passes across the epithelium transforming unpatterned and undifferentiated cells into rows of periodically spaced clusters of photoreceptor neurons. As cells enter and exit the furrow they undergo dramatic alterations in cellular architecture and gene expression, many of which are required to propel the furrow forward and for proper cell fate specification. The Decapentaplegic (Dpp) and Hedgehog (Hh) signaling pathways are required for the initiation and progression of the furrow, respectively. Consistent with a role in furrow progression, the loss of Hh pathway activity results in a "furrow stop" phenotype. In contrast, reductions in levels of the helix-loop-helix transcription factor, Extramacrochaetae (Emc), lead to the polar opposite phenotype--the furrow accelerates. Recently, we demonstrated that the furrow stop and furrow acceleration phenotypes are molecularly connected. Emc appears to serve as a brake on the furrow by dampening the activity of the Hh pathway. Loss of Emc leads to an upsurge in Hh pathway activity and a faster moving furrow. The acceleration of the furrow appears to be due to an increase in levels of the full-length isoform of Cubitus Interruptus (Ci (155)) and Suppressor of Fused [Su(fu)]. Here we will briefly review the mechanisms by which Hh drives and Emc impedes the progression of the furrow across the developing retina.

Article	Year
Extramacrochaetae imposes order on the Drosophila eye by refining the activity of the Hedgehog signaling gradient. Development (Cambridge, England)\| PubMed ID: 23536565	2013
Hedgehog and extramacrochaetae in the Drosophila eye: an irresistible force meets an immovable object. Fly\| PubMed ID: 24406336	2014

Carrie M. Spratford

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