Transcription factors mediate many essential processes. The role of transcription factors in mediating neuronal growth, function, and regeneration is not well known. It is particularly poorly understood how the development, branching, and regrowth of motor neurons are regulated. There is also little known about how transcription factors regulate dendrite (projections of neurons) and how this works in conjunction with axonal outgrowth and targeting.
Regulation of dendrite and axon positioning is necessary to define a neuron’s connections and is controlled by a combination of guidance receptors, adhesion molecules, and cytoskeletal regulators. The lab of Dr. Greg Bashaw studies how axons in Drosophila successfully navigate to their direct targets during development, with the ultimate goal of developing therapeutics for nerve regeneration and brain abnormalities.
Dr. Celine Santiago, recent alumna from the Bashaw lab, took on the momentous task of elucidating how the transcription factor Islet (Isl) coordinates axon and dendrite positioning though the guidance receptor Frazzled (Fra)/DCC. Regulation of dendrite and axon positioning is necessary in defining a neuron’s connections and is controlled though a combination of guidance receptors, adhesion molecules, and cytoskeletal regulators. In 2014, Celine showed that the neuronal receptor Roundabout (Robo) regulates axonal guidance in ventrally projecting RP3 motor neurons in response to the activity of the transcription factor Hb9. Celine’s most recent publication describes a parallel pathway by which the transcription factor Isl regulates fra expression and its importance for muscle target selection.
The crux of the paper is that a single transcription factor (Isl) can control the position of both the input and output of a neuron, even when acting upon the same receptor (Fra). Celine used a plethora of complex genetic models to show that only Isl is required for fra expression in RP3 motor neurons, but is not required for general survival of Drosophila motor neurons. This suggests that Isl’s effect on Fra is novel and specific. By generating separate isl- and fra-mutant flies, Celine also demonstrated that Fra is an essential downstream effector of Isl during the guidance of the RP3 axon to its target muscles. Ectopic overexpression and rescue experiments further confirm this relationship. Celine went on to show that another transcription factor, Hb9, can work in parallel with Isl to regulate different downstream targeting events in RP3 neurons, further complexing the mechanism behind some axon trajectory programs.
At later stages during development, RP3 neurons have reached their targeted position of an ipsilateral projection branching from the soma (cell body) and a large dendritic tree forming off the contralateral primary neurite. Celine visualized this in vivo by utilizing a membrane-tethered GFP transgene. With this system, Celine determined that there are severe midline crossing defects in RP3 axons when Fra is genetically ablated; however, there is little defect in midline crossing when Isl is deleted. This suggests that Isl is not required for early stages of RP3 neuronal development and axonal guidance, but is required in later stages of motor neuron differentiation and ventral target selection by controlling fra expression.
Deleting Isl does have its consequences, as RP3 neurons fail to form contralateral dendrite extensions into the intermediate zone of the Drosophila nerve cord. While the length of the dendritic projections does not change compared to isl controls, isl mutant RP3 neurons appear to have their dendrites in a laterally shifted position within the CNS. Despite this shift, the axons still appear to reach their correct target muscle groups. In contrast, fra mutants have a more severe lateral shift defect, although their axon projections also still reach the correct target muscle groups and there was no significant reduction in dendrite projection length. Combined, these findings demonstrate that the Isl-Fra signaling pathway is not a major controller of the outgrowth of motor neuron dendrites in the nerve cord. Using the limb3b-GAL4 construct to overexpress a UAS-HA-Fra transgene and visualize single RP3 neuronal dendrites, Celine rescued laterally shifted dendrites in isl mutants without affecting dendritic projection length or number of projections, which confirms that Isl directly regulates RP3 dendrite positioning though fra expression.
The transcription factor Islet (Isl) is required for expression of fra in RP3 motor neurons. Isl and Fra are required for axonal and dendrite targeting in RP3 motor neurons. Overexpression of fra rescues RP3 motor axon and dendrite targeting in isl mutants. A single transcription factor, Isl, is able to control both input and output of a subset of motor neurons through regulation of fra expression.
The final question Celine asked was whether changes in dendrite positioning could alter axonal positioning and targeting. There appeared to be no correlation between axonal and dendrite defects in isl mutants, with the majority of isl mutants showing a defect in either axonal or dendrite positioning but not both. This was also the case with fra mutants. Therefore, central targeting defects (axon or dendrite positional shifting within the nerve cord) can occur separately from defects in muscle target innervation in an RP3 neuron.
In conclusion, Celine effectively demonstrates that Isl directs RP3 motor axon targeting. She shows that this is regulated through the single transcription factor Fra, and Isl is able to concomitantly regulate branching of dendrites in the CNS and axons in the periphery of Drosophila. Future experiments will identify other transcription factors that control motor neuron growth and development in the hopes of developing potential therapies for patients with movement and coordination disorders.
Celine is now moving on to begin a post-doctoral fellowship studying the development and homeostasis sensation of touch in David Ginty’s lab at Harvard University. She’s excited to be switching over to a new model organism - the mouse.
Santiago C, Bashaw GJ. Iset Coordinately Regulates Motor Axon Guidance and Dendrite Targeting through the Frazzled/DCC Receptor. Cell Rep. 2017 Feb 14;18(7):1646-1659.