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 have recently identified Muscleblind (Mbl), a conserved splicing factor, as a regulator of cell-specific Dscam2 isoform expression (Li and Millard 2019). Mbl promotes expression of isoform B while inhibiting the expression of isoform A. Future studies will interrogate the mechanisms through which Mbl regulates Dscam2 cell-specific isoform expression. 

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).
Isoform
Mbl represses Dscam2 isoform A. (C1-C3) Isoform B is expressed in photoreceptors (R cells). A marker specific to R cells is shown in green and isoform B reporter expression (tdTom) is in red. Isoform B is expressed in both R cells (carrots) and in deeper regions of the optic lob (arrowhead). (D1-D3) Isoform A is not expressed in R cells (carrots), but is expressed in deeper regions of the optic lobe (arrowhead). (E1-E3) When mbl is knocked down in photoreceptors, isoform A turns on in these cells (carrots). From Li and Millard (2019) Sci Adv.

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. When these postsynaptic cells express the same Dscam2 isoform, fewer synapses and morphological defects in dendrites are observed (Kerwin et al. 2018). We interpret this as evidence for inappropriate repulsion between the postsynaptic cells when they express the same Dscam2 isoform.  

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)
Decreased synapse number in Dscam2 single isoform lines. (A) A schematic of a photoreceptor synapse indicating the pre and post-synaptic markers used in this study. L1 and L2 normally express different isoforms of Dscam2. (B) Confocal images of Brp (presynaptic) puncta in WT (B) and single isoform lines (C, D). (E) Quantification.

 

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.

 

Identifying components of the Dscam2 signalling pathway

Dscam2 has different functions in different neurons, but the molecular pathways that lead to these different outputs have not been identified. We are currently trying to identify proteins that interact with Dscam2 to better understand its functional diversity. One approach is to tag the endogenous Dscam2 gene with GFP, use this tag to pull it down from fly brain lysates and then identify associated proteins using mass spectrometry.

Dscam2::GFP is functional in the fly visual system (A) Schematic of an endogenous GFP-tagged Dscam2 that we generated using CRISPR. (B-C) Animals heterozygous (B) and homozygous (C) for Dscam2::GFP have normal photoreceptor projections, in contrast to Dscam2 mutant animals which have highly disorganised photoreceptors. Dscam2::GFP (green), photoreceptors (24B10 antibody, magenta). 

 

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.

Mutations in the fly homologue of the sALS candidate gene, GGNBP2, lead to smaller motor neuron terminals and fewer synapses. (A) Schematic of the CRISPR strategy used to delete the fly Ggnbp2 gene. (B-B’) WT larval motor neurons labelled with a membrane marker (magenta) and antibodies against the presynaptic marker, brp (cyan). (C-C’) Ggnbp2 mutant larval motor neurons labelled as in B.  

 

Millard lab Honours projects – 2019

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.

Group Head

Staff

Students

​2019

Li, JS and Millard SS (2019). Deterministic splicing of Dscam2 is regulated by Muscleblind. Sci Adv 5(1): eaav1678.

2018

Millard SS, Pecot MY (2018). Strategies for assembling columns and layers in the Drosophila visual system. Neural Development 13(1):11.

Kerwin SK, Li JS, Noakes PG, Shin GJ, Millard SS (2018). Regulated alternative splicing of Drosophila Dscam2 is necessary for attaining the appropriate number of photoreceptor synapses. Genetics 208 (2): 717-28.

2017

Lim NR, Shohayeb B, Zaytseva O, Mitchell N, Millard SS, Ng DCH, and Quinn LM (2017). Glial-specific functions of microcephaly protein WDR62 and interaction with the mitotic kinase AURKA are essential for Drosophila brain growth. Stem Cell Reports 9: 1-10.

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

Li Q, Ha TS, Okuwa S, Wang Y, Wang Q Millard SS, Smith DP and Volkan-Cayirlioglu P (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)
  • Kevin Mutemi (former MSc student) - enrolled in the EMBL PhD program, Germany
  • Josh Li (former PhD student) - postdoc Harvard 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.