We are always interested in recruiting enthusiastic scientists and welcome new ideas and techniques. Please contact Sean to enquire about Honours, Masters, PhD or Postdoctoral positions in the lab. Below is a list of ongoing projects.

Regulation of cell-specific alternative splicing

Dscam2 produces two protein isoforms (A and B) that differ at a single immunoglobulin domain. Isoform expression is highly regulated with most cells expressing either A or B, but not both. This is unusual as most alternatively spliced genes studied to date express different ratios of isoforms rather than one isoform exclusively. We are interested in identifying factors that regulate Dscam2 alternative splicing and Josh Li is the PhD student spearheading this project. Our hypothesis is that cell-specific splicing programs regulate many alternatively spliced genes involved in the development of a specific neurons, and thus represent “hubs” for neurodevelopment.

Isoform-specific homophilic binding
Isoform-specific homophilic binding of Dscam2.
Isoform Dscam2A contains one variable immunoglobulin domain (blue), nine invariable immunoglobulin domains (black), six fibronection domains (grey) and a transmembrane domain (dark blue).
(B) Dscam2B differs with Dscam2A at a single variable immunoglobulin domain. 
(C) Homophilic binding of Dscam2 is isoform-specific and can lead to repulsion. 
(D) No binding occurs between Dscam2A and Dscam2B.  ​
Isoform-specific expression of Dscam2 in lamina neurons
  Isoform-specific expression of Dscam2 in lamina neurons. Isoform A is expressed in L2, L3 and L5 neurons (left) and isoform B is expressed in L1 and L4 neurons (right). This is a composite image from two different isoform reporter lines where random subpopulations of lamina neurons were labelled using the  Flpout technique . Photoreceptors (red).


The role of regulated isoform expression in synapse formation

We demonstrated in 2014 that regulated alternative splicing of Dscam2 is required for attaining the proper morphology of neurons and we have expanded these studies to synapses. Photoreceptor synapses contain multiple postsynaptic elements that express different isoforms of Dscam2. We generated knock-in animals that only express a single isoform from the endogenous Dscam2 locus and have been analysing them for synaptic defects. Our hypothesis is that when the postsynaptic cells express the same isoform, they will be unable to pair as Dscam2 acts repulsively in these cells.

Construction of the Dscam2 single isoform mutant lines
Construction of the Dscam2 single isoform lines
(A) The variable region of the endogenous Dscam2 locus has been removed via recombination mediated cassette exchange. A cDNA containing exon 9 and each of the alternative exon 10s was exchanged for the variable region to create single isoform lines that express one isoform of Dscam2 from all Dscam2 positive cells.
Adapted from (Lah et al. 2014)

Synaptic functions of Dscam2 at the neuromuscular junction

Dscam2 produces two alternative isoforms that mediate isoform-specific homophilic binding and are expressed in different subsets of cells. Thus, the two Dscam2 isoforms could function as distinct homophilic recognition molecules in different neurons or alternatively, each isoform could have unique functional properties. To address this, we are investigating how Dscam2 and regulated isoform expression affects synaptic physiology at the larval neuromuscular junction (NMJ). We hypothesize that Dscam2 has synaptic functions that may be independent of homophilic binding.  

Dscam2 isoform B, but not isoform A is expressed in motor neurons. Shown are larval brain/ventral nerve cord preparations from isoform reporter lines. Motor neurons that extend from the nerve cord to the muscles express isoform B.

Isoform B
Isoform B
Isoform A
Isoform A

A role for Dscam2 in the mushroom body

We have found that in the learning and memory centre of the Drosophila brain, the mushroom body, Dscam2 protein gets trafficked to the dendritic compartment. We are investigating whether this is due to protein or mRNA localisation and the role that Dscam2 plays in the formation of mushroom body synapses. We hypothesise that Dscam2 plays a role in the formation of mushroom body claws, the postsynaptic structures that receive input from the antennal lobe.

Mushroom body axons
Mushroom body axons (green) in the central brain of the fly. Magenta staining is a presynaptic marker.


Dscam2 and Down Syndrome

Vertebrate DSCAM resides on chromosome 21 in humans within a region that has been deemed as critical for Down syndrome. Since Down syndrome is caused by trisomy at chromosome 21, we have generated flies that contain an extra copy of the Dscam2 gene. Dscam2 can function as both an adhesive and a repulsive cue and we are investigating whether trisomy for Dscam2 can disrupt this balance and lead to wiring phenotypes in the fly.

BAC rescue and trisomic
Generation of flies trisomic for Dscam2. (A) Schematic of the modified Dscam2 region. Exon5 was flanked with B3 recombinase sites (green) using recombineering. (B) A control bacterial artificial chromosome (BAC) containing a non-functional Dscam2; exon5 has been replaced with a selection marker (Kana Rpsl). (C) Wild-type photoreceptor array. (D) Dscam2 (Ds2) mutant photoreceptor array. (E) Rescue of the Dscam2 mutant phenotype with the BAC. (F) No rescue with the non-functional BAC. 


Studying motor neuron disease genes in the fly

We are collaborating with Naomi Wray’s lab (IMB/QBI) to investigate genes associated with sporadic Amyotrophic Lateral Sclerosis (ALS, also called motor neuron disease) through GWAS studies in the Wray lab. We are generating mutations in fly homologues of these genes using CRISPR and then analysing the morphology, synaptic composition and neurophysiology of the larval neuromuscular junction (NMJ). The goal is to not only validate the GWAS associations, but also to understand how these proteins function at the NMJ.

Millard lab Honours projects – August 2018

1.      Mapping binding sites for the factor that regulates Dscam2 alternative splicing using CRISPR-Cas9 in Drosophila.

2.      Validating sporadic Motor Neuron Disease candidate genes in the fruit fly.

3.      Investigating whether the splicing factor, Rbfox1, represses Dscam2.10B isoform expression.

4.      Investigating whether Dscam2 and Presenilin function in the same signaling pathway in motor neurons.

5.      Assessing whether different cytoplasmic isoforms of Dscam2 confer dendritic localization.