Playing it by ear: Investigating the role of the inner ear in lymphatic development

Abstract

Proper development and patterning of the intricate lymphatic vascular network is crucial for its function in maintaining fluid homeostasis, immune surveillance and lipid absorption in the gut. Uncontrolled lymphatic development is associated with many pathological conditions including cancer metastasis and graft rejection. Conversely, loss of functional lymphatics can lead to lymphoedema – a build-up of lymph in tissues. Development of the lymphatic system requires guidance structures and molecular cues to direct growth, however, little is known about the molecular mechanisms involved in lymphatic guidance. Zebrafish are an ideal model organism to study lymphatic development as the molecular mechanisms are conserved between vertebrates, and imaging can be used to visualise in-vivo vessel growth in transparent larvae. This study focused on otolithic lymphatic vessel (OLV) development, which grows in a stereotypical fashion along the inner ear (otic vesicle), but its guidance is unknown. Live imaging studies revealed a potential OLV guidance structure within the otic vesicle, which was termed the superficial three-pronged structure (STPS). To investigate the role of the STPS, genetic and laser ablation strategies were employed. Two mutants with OLV-specific defects were identified in a forward genetic screen, one with severely reduced OLV growth called the OLV mutant, while conversely in a putative ptch1 mutant, the OLV was hyperbranched. Additionally, a foxi1 mutant with known otic vesicle development defects was also used. Loss of the STPS through either ablation or through mutations disrupting STPS development, correlated with impairment of OLV growth, while the hyperbranched OLV phenotype was associated with expansion of the STPS. This suggests the STPS is required for the guidance of lymphatic growth. Inhibitor studies revealed that OLV growth is dependent on VEGFR signalling. Finally, the STPS was known to express high levels of Collagen type-II (Col2), and immunofluorescence analysis suggests the presence of this ECM component is associated with OLV growth, suggesting Col2 may be acting as a potential lymphatic guidance molecule. In conclusion, this study identified the STPS as a potential lymphatic guidance structure. Future studies will investigate the molecular mechanism driving OLV growth along the STPS and further validate the OLV mutants used in this study.