The overall goal of the Millard lab is to understand how specificity is generated in the brain. This problem is best exemplified by considering that 100 trillion synapses are generated and maintained in the human brain using a toolkit of only 20,000 genes. We have been approaching this problem using molecular genetics in the fruit fly, Drosophila melanogaster. Most projects in the lab revolve around how a broadly expressed cell surface protein, called Down syndrome cell adhesion molecule 2 (Dscam2), is able to perform specific functions in different neurons. We are also interested in mechanisms of neurological disease, particularly those that involve changes in synaptic function.

Clones of mushroom body neurons in the Drosophila central brain generated using mosaic analysis with a repressible cell marker (MARCM).
Clones of mushroom body neurons in the Drosophila central brain generated using mosaic analysis with a repressible cell marker (MARCM).

 

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.
(A)
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.

 

How Dscam2 regulates sleep

Why we sleep is still an enigma. Current theories suggest that sleep is required for memory consolidation, synaptic homeostasis and metabolic clearance in the brain. Dscam2 is expressed in most of the structures known to regulate sleep in flies. In collaboration with Bruno van Swinderen’s lab at QBI, we are trying to understand how Dscam2 regulates sleep. Dscam2 mutants have fragmented sleep with a particular tendency to sleep less at night compared to the day. In addition, the mutant flies are more easily aroused by a vibration stimulus compared to control animals. These data suggest that Dscam2 may be required for either the development or the maintenance of the sleep circuitry in flies and we are investigating both of these possibilities.

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.

Epilepsy project

Mutations in many synaptic ion channels are associated with severe forms of epilepsy that are insensitive to drug treatments. In collaboration with Steve Petrou’s lab (Florey Institute) we are modelling epilepsy in flies. Using a modified version of the Drosophila ARousal Tracking (DART) assay developed by the van Swinderen lab to study sleep, we are tracking spontaneous and evoked seizures in flies with mutations in various ion channels. The goal of this project is to develop a quantitative assay for measuring seizures that can be used as a drug-testing platform.

Lab head

Staff

Students

2016

Tadros W, Xu S, Akin O, Yi CH, Shin GJ, Millard SS and Zipursky SL (2016). Dscam proteins direct dendritic targeting through adhesion. Neuron 89 (3):480-93.

2015

Li JS, Shin GJ, Millard SS (2015). Neuronal cell-type-specific alternative splicing: A mechanism for specifying connections in the brain? Neurogenesis 2:1, e1122699: 1-5.

Bosch DS, van Swinderen B and Millard SS (2015). Dscam2 affects visual perception in Drosophila melanogaster. Front. Behav. Neurosci. 9:149. doi: 10.3389/fnbeh.2015.00149.

2014

Lah GJ, Li JS, Millard SS (2014).  Cell-specific alternative splicing of Drosophila Dscam2 is crucial for proper neuronal wiring.  Neuron 17;83(6):1376-88.

2013

Qingyun Li, Tal Soo Ha, Sumie Okuwa, Yiping Wang, Qian Wang, S. Sean Millard, Dean P. Smith and Pelin Volkan-Cayirlioglu. (2013) Combinatorial rules of precursor specification underlying olfactory neuron diversity. Current Biol. 23, 2481-2490.

Paulk A, Millard SS, van Swinderen B.  (2013).  Vision in Drosophila:  Seeing the World Through a Model's Eyes.  Ann. Rev. Entomol. 58:313-32. 

2010

Millard SS, Lu Z, Zipursky SL, Meinertzhagen IA. (2010) Drosophila Dscam proteins regulate postsynaptic specificity at multiple-contact synapses. Neuron 9;67(5):761-8.

2008

Millard SS, Zipursky SL. (2008) Dscam-mediated Repulsion Controls Tiling and Self-avoidance. Current Opinion in Neurobiology 18(1):84-9.

Hattori D, Millard SS, Wojtowicz W, Zipursky SL. (2008) Dscam-mediated Cell Recognition Regulates Neural Circuit Formation. Annual Reviews in Cell and Developmental Biology 24:597-620.

2007

Millard SS, Flanagan JJ, Pappu KS, Wu W, Zipursky SL.  (2007) Dscam2 mediates axonal tiling in the Drosophila visual system.  Nature 447(7145):720-4.

Former lab members

  • Grace Shin (former postdoc) – currently doing a second postdoc at Columbia University
  • Danny Bosch (former PhD student, whereabouts unknown)
  • Wei Tan (former Hons student - currently working as an RA in Singapore)
  • Alex Quirk (former Hons student - applying for medical school)

Our team

Sean Millard
Sean enjoys mountain biking and fishing in his free time.
Millard Lab team
The lab at the annual Australian Fly Meeting in Warburton, VIC
Nissa
Nissa is a caffeine-addict who is always looking for new places to drink a tasty brew. If you can’t find her at a coffee shop, she’ll be on a hiking trail somewhere in southeast Queensland or zipping around one of Brisbane’s many bike paths.
Kevin at Easter
Kevin’s drive to discover the unknown extends to Easter egg hunts...

Find out more about our research environment and how to apply to do a short or long-term research project with us.