True anophthalmia is the most severe congenital eye malformation. With absence of the eye, optic nerve, chiasm and optic tracts. Identifying the genes that cause genetic true anophthalmia should improve our understanding of the critical processes required for development of the eye. Recessive loss-of-function mutations in SMOC1 have been identified as the cause of Ophthalmo-acromelic syndrome (OAS), a multisystem disorder which has true anophthalmia as a prominent feature with characteristic limb and facial malformations. In order to establish the function of SMOC1 in development I used the zebrafish as a model organism to support a link between SMOC-1 and BMP signalling. As a first step I characterised the genomic structure of zebrafish smoc1 gene. I was able to correct an error in the zebrafish genome (Zv8) that annotated zsmoc1 as two fragmented and rearranged orthologous loci. However, using RTPCR I could show that there is in fact a single intact zsmoc1 transcript. In addition, I was able to identify an un-annotated 5’ coding exon using 5' RACE which showed that the full open reading frame includes a signalling peptide. RT-PCR was also used to identify several novel zsmoc1 splice isoforms. To explore the link between zsmoc1 and bmp signalling I used injection of antisense morpholino oligonucleotide and capped mRNA to examine the effects of loss-of-function and overexpression respectively of smoc1 and genes functioning in the bmp signalling pathway. The resulting embryos were analysed using morphometric analysis (Kishimoto scale), a quantitative assay of dorsalisation/ventralisation and live imaging of reporter transgenic fish. I developed a quantitative RT-PCR assay for expression of dorsal (otx2 and runx3) and ventral (eve1 and gata2) marker genes. I established a reliable system for live imaging of zebrafish development between 8 hpf and 24 hpf. By combining this system with fluorescent transgenic reporters marking the eye field (rx3:gfp reporter) and BMP-signaling (BRE:gfp reporter) I was able to accurately quantitate the effect of smoc1 depletion on eye size and SMAD1/5/8 signalling in the eye. These results support the predictions from the Drosophila homologue Pent that zsmoc1 functions as an antagonist of bmp signalling. Finally, I describe my attempt to produce a zebrafish model for OAS using genome editing technology. I designed, produced and validated transcription activator like effectors nucleases (TALENs) targeted to the zsmoc1 open reading frame using the Voytas Goldengate method. I designed and optimised a novel strategy to demonstrate targeted cutting activity for in vitro validation. Following injections of the in vitro validated TALEN into zebrafish embryos I used Ion Torrent sequencing to assess the in vivo activity of the engineered TALEN pairs. Unfortunately these TALENs were not able to cut the targeted locus in vivo.

Pattern recognition receptors coordinate innate immune responses by sensing infection or injury. Nucleotide-binding, leucine rich repeat, and pyrin domain-containing protein 3 (NLRP3) is a cytosolic PRR which perceives diverse pathogenic and sterile insults. NLRP3 orchestrates inflammatory signaling responses by forming inflammasomes with the adaptor protein apoptosis-associated speck like protein with a caspase recruitment domain (ASC) and the cysteine protease caspase-1. Assembly of the intracellular macromolecular inflammasome complex culminates in proximity-induced autocatalysis of caspase-1. Caspase-1 activation promotes cell death by pyroptosis and activation and secretion of proinflammatory cytokines interleukin (IL)-1β and IL-18. While NLRP3-mediated inflammation protects against bacterial, fungal, viral, and parasitic infections, aberrant NLRP3 activation is implicated in numerous inflammatory diseases and heritable syndromes. Mechanistically, inflammasome activation requires a preliminary NF-κB-activating priming step (signal 1) and a subsequent NLRP3-specific stimulus (signal 2). While there is enormous molecular diversity among NLRP3-specific agonists, this second signal appears to engage a common pathway involving cation flux. Furthermore, NLRP3 associates with mitochondria and mitochondrial damage is implicated in NLRP3 activation, although the precise role for mitochondria in inflammasome assembly remains controversial. We previously demonstrated that the mitochondrial phospholipid cardiolipin binds to NLRP3 and is critical for NLRP3 inflammasome activation. Here, we further investigated how mitochondria contribute to NLRP3 activation. We found that liposomes containing molar concentrations of cardiolipin that resemble mitochondrial cardiolipin levels can induce NLRP3-dependent caspase-1 autoactivation. Unexpectedly, we discovered that caspase-1 binds directly to cardiolipin, causing inflammasome-independent caspase-1 complex formation and autocatalysis at higher cardiolipin densities. Finding that caspase-1 and NLRP3 are independently capable of binding to cardiolipin, we more thoroughly examined the association of inflammasome components with mitochondria. Normally confined within mitochondrial inner membranes, cardiolipin relocates to outer membranes of stressed mitochondria. We found that reactive oxygen species (ROS) produced in response to signal 1 facilitate cardiolipin externalization to the outer membrane during priming. We also determined that this coincides with ROS-dependent recruitment of NLRP3 and caspase-1 to the outer membrane of mitochondria at priming. In contrast, we found that NLRP3 activation by the signal 2 agonist nigericin induces calcium-dependent recruitment of the adaptor ASC to mitochondria and caspase-1 activation. Finally, to determine what type of mitochondrial damage was necessary to promote NLRP3 inflammasome activation, we examined how different NLRP3 agonists affect mitochondria. We found substantial variability in the extent of mitochondrial damage induced among different NLRP3 agonists. Collectively, our findings illustrate that mitochondria serve as innate immune signaling platforms through multiple stages of NLRP3 inflammasome activation. Further, paralleling lipid A interactions with caspase-11, we have demonstrated that caspase-1 is capable of binding to the phospholipid cardiolipin.