The effect of Hedgehog signaling on in vivo neuronal morphogenesis

Neurons exhibit complex and diverse morphologies. The shape of a neuron has important functional implications, as it determines what signals a neuron receives and how these signals are integrated into neuronal circuits. To understand how the Hedgehog signaling pathway regulates neuronal morphogenesis in vivo, we are comparing dendritic arborization (branching) in Drosophila melanogaster (fruit fly) larvae with reduced, increased, and control (wild type) levels of Hedgehog signaling. Preliminary data suggests both increases and decreases in Hedgehog signaling affect the number of branches produced by epidermal sensory neurons. ___________________________________________________________________________________________________________________


Introduction
The Hedgehog (Hh) pathway is an evolutionarily conserved signal transduction cascade which mediates how cells sense and process external information. While it is repeatedly used throughout development, our research specifically focuses on its role in neuronal outgrowth and circuit formation.
The proteins involved in the Hh pathway determine whether signaling occurs through a canonical or noncanonical mechanism [1,2] Our goal is to understand how Hh signaling regulates neuronal morphogenesis in vivo. To accomplish this, we are studying how changes in Hh signaling activity affect the multi-dendritic epidermal sensory neurons of the fruit fly Drosophila melanogaster, a model organism, which is amenable to genetic analysis.

GAL4/UAS system:
The GAL4-upstream activating sequence (UAS) system was used to regulate gene expression in Drosophila [3]. To perform this technique, flies are crossed to produce progeny that carry both a GAL4 gene and a UAS-gene of interest. The GAL4 protein is a transcriptional activator which binds DNA at UAS sites and activates transcription. A genomic enhancer sequence is positioned upstream of the GAL4 gene so that GAL4 is expressed in a tissue specific manner. In our study, the pickpocket (ppk) enhancer sequence was used to express GAL4 in class IV multidendritic neurons. UAS sites were located upstream of DNA sequences encoding Patched; Dicer, which is a protein that mediates the cellular short interfering (siRNA) response; and siRNAs targeting Hh and Ptc mRNA. siRNA gene silencing: siRNAs, are short RNA sequences, ranging from 20-25 base pairs in length, which are used to reduce or silence gene expression [4]. siRNAs are designed to bind to complementary mRNA sequences of interest, such as Hh and Ptc mRNA in our study. When the siRNA binds its target mRNA, a protein complex containing the Dicer protein binds and cleaves the double-stranded siRNA-mRNA molecule. Hence, the targeted mRNA is not translated into protein.
Visualization of neurons and quantitative analysis of dendrite branching: The GAL4/UAS system was used to drive Green fluorescent protein (GFP) expression in class IV multidendritic neurons. Fly larvae were bred to carry ppkGAL4, UAS-tandem GFP, UAS-Dicer 2, and either a UAS-siRNA molecule targeting Hh or Ptc, or UAS-ptc, which encodes for the Ptc protein. Neurons were examined in live larvae 140 hours after egg laying (AEL) using a Zeiss fluorescence microscope and camera, and Axiovision software. Dendrites were traced, and Sholl analyses were performed with Image J software.

Results and Discussion
Preliminary data suggests Hedgehog signaling regulates dendritic arborization in class IV multidendritic sensory neurons (Fig. 1). An increase in dendritic branching was observed in larvae with reduced Hh signaling: hh siRNA and ptc siRNA. Conversely, a decrease in dendritic arborization was observed in larvae with increased Hh signaling, UAS-ptc. While these results appear promising, a larger sample size must be analyzed to determine whether the data can be replicated and the differences are statistically significant, as the number of neurons and larvae currently examined are in the single digits. Furthermore, experiments are underway to study the effect of reducing other Hh signaling pathway proteins, including Smoothened and Fused.