Craniofacial Research Group

Head of Lab: Dr Timothy Cox

His research is primarily aimed at elucidating the molecular defects and pathological consequences underlying the presentation of human developmental syndromes, particularly those affecting the craniofacial region.

His Lab group has expertise in many advanced molecular and cell biology techniques, including mutation detection, prokaryotic and eukaryotic expression methodologies, immunohistochemistry and the yeast two-hybrid system. The green fluorescent protein (GFP) is also used in a variety of contexts to monitor proteins and gene activity in live cells and tissues by immunofluorescence. We are also using the latest technologies (for example, the Cre / loxP recombination system) for manipulation of the mouse genome in order to generate accurate models of each of the disorders we are interested in studying.

The research undertaken in the Laboratory includes:

Research Title:

Mechanisms of tissue morphogenesis: the role of genetic, epigenetic and environmental factors.

Research Aims:

The overall objectives of the lab are to understand the role of various genetic pathways and environmental factors in regulating the precise morphogenesis of complex tissues and organs. In particular the lab is interested in morphogenesis of the midface and the contribution of both genetics and environmental factors to the incidence of common clefts of the face, such as cleft lip and palate. We utilize both the mouse and chick as model systems, employing a variety of advanced genetic manipulation techniques. For the latter system, we have invested considerable effort in developing new technologies that can capitalize on the many advantages of the chick system. However, novel approaches with regard to genetic modification of the mouse are also opening up avenues by which to investigate the impact of maternal and environmental factors that influence the susceptibility of the fetus to clefting.

Research Undertaken

Craniofacial Projects

Novel gene delivery and gene knockdown technologies to transiently manipulate embryological mechanisms.

The chick system offers many advantages for the understanding of human developmental mechanisms but has been under-utilized over the years because of the inability to undertake genetic manipulations. Recently, some advances have been made on this front with the advent of micro-electroporation of embryos and the development and utilization of siRNAs and morpholinos.

• We have been establishing new methods by which to deliver antisense oligonucleotides and expression vectors to the face, an organ traditionally difficult to access and manipulate in ovo, as well as more convenient methods by which to assess and monitor the impact on facial development.
• To date, we have established an ex ovo facial explant culture system that enables the process of fusion of the facial processes to be observed (potentially in real time). This system is also being optimized for the delivery of morpholinos and expression vectors using a novel biodegradable industrial nanogel.
• As an alternative, we are exploring the feasibility of using a variation on the micro-electroporation procedure to also introduce DNAs into the facial epithelia.
• Both procedures are showing encouraging results and promise to significantly speed up the analysis of gene function in facial development. The techniques should also be applicable to other developmental systems.

The contribution of genetic and environmental factors on the success of fusion of the facial primordia.

The A strain mouse is the only inbred mouse strain that shows a susceptibility to classic cleft lip and palate, although the phenotype is only partially penetrant and dependent on both the maternal genotype and nutritional factors. Recent work has determined that two gene loci are solely responsible for the genetic contribution in this strain.

• We are beginning to utilize this strain to investigate the role of other genes and genetic pathways on influencing the incidence of clefting.
• The strain will also allow further studies on identifying the maternal factors and the mechanism of action of other environmental factors (ie. epigenetic mechanisms) on disease presentation.

The role of specific genes and gene products, implicated by mutational analysis in human cleft lip and palate, in modulating behaviour of the facial epithelia.

The facial epithelia destined to contact and fuse with those on the apposing prominences undergo a dramatic series of cellular changes immediately prior to contact indicating that these epithelia are being genetically reprogrammed.

We are investigating the impact on such changes in knockout mice that show facial dysmorphism (and/or are knockouts of gene that in humans are associated with clefting), as well as in the chick embryos in which gene expression has been transiently up or down regulated.

The regulation of gene expression in the craniofacial primordia.

We are also interested in understanding the in vivo factors that regulate the expression of genes in specific craniofacial tissue, since in many cases gene expression appears to be coordinated by a complex series of factors in a temporal and spatial manner that is unique to the developing embryo.

To this end, we are undertaking in ovo promoter dissection of select craniofacially expressed genes using micro-electroporation of promoter-reporter fusion constructs.

Heart Projects

The role of X chromosome inactivation in presentation of a cardiomyopathy.

X-linked diseases comprise around one fifth of all hereditary diseases because even recessive phenotypes present in the hemizygous males. However, presentation of disease phenotypes in females is highly variable, which has been attributed to the random or clonal nature of X chromosome inactivation.

• We have generated a heart-specific knockout of a candidate disease gene for an X-linked cardiomyopathy.
• Using complex breeding strategies and novel X-linked reporter genes, we are assessing whether the proportion of cells harbouring the mutant X chromosome or their position within the heart contribute more to disease presentation.
• The genetic contribution from different strains of mice is being used to influence the proportion of cells harbouring the mutant chromosome.

This model is providing new insight into epigenetic factors that influence disease presentation but also hints at providing insight into the capacity for early remodeling of damaged or diseased cardiac