Autism spectrum disorders (ASDs) are neuropsychiatric disorders with symptoms including impairment in social communication and interaction, repetitive or stereotypic behaviours, restricted interests and abnormal response to stimuli from the environment. A large proportion of the genetic basis of ASDs remains unaccounted for and is referred to as the ‘missing heritability’. Some of this missing heritability appears to be due to gene-gene interactions. While mutations in a single gene may be without functional consequence, the presence of mutations in two interacting genes in the same genome can increase the likelihood of phenotypic effects. ASDs, intellectual disability and several other neuropsychiatric disorders, share overlapping susceptibility genes, suggesting that gene-gene interactions may contribute to shared phenotypes across syndromes. This thesis aims to examine the interactions between two key synaptic genes, nrxn1 (neurexin1, a susceptibility gene for ASDs) and lrrtm2 (leucine-rich repeat transmembrane protein 2, a candidate gene for intellectual disability), in the development of locomotor behaviours and spinal cord cytoarchitecture in zebrafish. The NRXN1 and LRRTM2 proteins are known to bind to each other and regulate synapse development in vitro. A loss-of-function strategy in zebrafish was applied to assess the roles of nrxn1 and lrrtm2 in development of locomotor function and spinal cord cytoarchitecture in vivo.
In Chapter 2, I investigated the role and interaction of nrxn1 and lrrtm2 in development of somatosensory reflex behaviours of zebrafish embryos. Individual loss of function of β-nrxn1b or lrrtm2 using anti-sense translational blocking morpholinos (MOs) perturbed the somatosensory touch reflex of 28-30 hpf embryos and 2 dpf larvae. Dose-response analyses revealed subthreshold MO levels for β-nrxn1b and lrrtm2. Subsequent simultaneous subthreshold knock down of both genes revealed significant behavioural deficits while separate injections of subthreshold levels of either β-nrxn1b or lrrtm2 MO failed to elicit phenotypes, indicating a synergistic interaction between β-nrxn1b and lrrtm2 in the emergence of behavioural phenotypes.
In Chapter 3, spontaneous slow swimming pattern of 6 dpf larvae was assessed to investigate the role and interaction of β-nrxn1b and lrrtm2 in complex behaviours. Larvae with loss of function of either β-nrxn1b or lrrtm2 displayed aberrant swimming behaviours involving out-of-phase tail and fin movements and restricted tail undulation. Simultaneous subthreshold knock down of both genes showed greater than additive penetrance of phenotypes indicating that β-nrxn1b and lrrtm2 continued to genetically interact to control complex behaviours of zebrafish larvae.
In Chapter 4, I investigated whether loss of β-nrxn1b and lrrtm2 function perturbed the neural cytoarchitecture in the spinal cord. Co-injection of subthreshold doses of β-nrxn1b and lrrtm2 MOs resulted in aberrant branching of motor neuron axons, reduced density of interneurons and motor neurons in the embryonic spinal cord. Subthreshold dose of either MO alone was without consequence. These results suggested that genetic interactions between β-nrxn1b and lrrtm2 were controlling the development of the basic components of the neural circuitry underlying locomotor activity. These results may provide the anatomical substrate for behavioural phenotypes.
The results in this study found that two key synaptic cell-adhesion genes cooperated synergistically to regulate locomotor behaviours and neural cytoarchitecture during neurodevelopment. Considering that β-NRXN1 is a susceptibility gene for ASDs and LRRTM2 is a candidate gene for intellectual disability, the interaction of these two genes at both behavioural and cellular levels strengthens the argument that ASDs and intellectual disability share a common functional mechanism, and also has implications for the understanding of gene-gene interactions in the etiology of ASDs, intellectual disability, as well as other neuropsychiatric disorders.