Birt-Hogg-Dubé syndrome (BHD) is caused by mutations in the gene encoding folliculin (FLCN). How this leads to the BHD clinical manifestations is not yet clear. Since homozygous mutations of FLCN are lethal in mice, rats and dogs at early embryonic stage (Hasumi et al., 2009), zebrafish is a valuable alternative model to study the developmental functions of FLCN. Newly published research from Kenyon et al. (2016) examines the role of FLCN in zebrafish development using morpholino oligonucleotides to generate a zebrafish BHD model and reconcile the expression of FLCN transcripts in the developing embryo with the phenotype associated with the morpholino knock-down of FLCN.
Using genome level analysis of the zebrafish transcriptome authors analysed FLCN expression at different stages of development. In situ hybridization was used to identify FLCN expression during embryo development. Raised FLCN expression was seen at different developmental stages in fin bud, somites, eye and regions of the brain. Overlap in the expression of FLCN and the proliferating cell nuclear antigen (PCNA) gene was observed in brain regions suggesting that FLCN may play a role regulating proliferation in the embryo brain. To test this and other FLCN functions the authors used morpholino oligonucleotides to block FLCN expression. Efficiency of morpholino gene knockdown was assessed by PCR, however, the authors were unable to assess protein levels. Knockdown of the FLCN gene by microinjection of FLCN morpholino resulted in clear phenotypic effects at different development stages. Embryos injected with FLCN morpholino showed developmental arrest, increased cell death in the brain, brain oedema, problems with tail circulation and larger and thinner yolk extension when compared with mismatch control morpholino injected embryos. This phenotype was rescued by injection of FLCN RNA in embryos, suggesting that the was caused by reduced expression of FLCN and that FLCN may be required for the development of the zebrafish embryo in the brain.
As BHD patients have an increased risk of developing kidney tumours (Menko et al., 2009) the authors studied the effect of FLCN on kidney development in zebrafish embryos using a pronephros and exocrine pancreas transgenic cell line. Injecting FLCN morpholino into the cell line did not cause defects in pronephric development when compared with controls. BHD was also recently described as a novel ciliopathy (Luijten et al., 2013), however, in this study, there was no difference in cilia expression in the pronephros and central canal cilia in embryos injected with FLCN morpholino. Author discuss that it is possible that although cilia seem to be present in the FLCN knockdown embryos, they may not be functional.
Since there were no obvious defects in motile cilia morphology and in the developing kidney, the authors examined other processes that might be affected by FLCN deficiency. FLCN has been shown to affect cell cycle progression (Nahorski et al., 2012; Laviolette et al., 2013; Kawai et al., 2013; Lu et al., 2014). Authors used an in vivo labelling tool to monitor cell cycle regulation in zebrafish embryos after FLCN knockdown. The zFucci system is composed of two transgenes that fluorescently label G1 and S-M phase nuclei in the living zebrafish embryo. zFucci transgenic embryos were injected with FLCN morpholino or mismatch control and S-M and G1 phases were monitored. Results showed a significant drop in the number of cells in S-M phases and a corresponding increase in G1 cells in FLCN morpholino injected embryos particularly in the retina and brain suggesting a disruption of the cell cycle in the brain as a result of FLCN knockdown. The authors did not try to rescue the phenotype. To determine if the change in cell cycle behaviour could be attributed to a specific development stage authors used time-lapse confocal microscopy in FLCN morpholino injected and mismatch control embryos. Results showed that initially embryos showed comparable levels of cells in G1 phase but as time progressed there was an increase in cells in G1 phase in FLCN morpholino injected embryos suggesting that there is no specific stage with a dramatic change in cell cycle behaviour but instead a gradual loss of proliferation of the cells. These results show that FLCN plays a role in cell cycle, however, the exact nature of the role remains to be determined and authors discuss that the cell cycle effects might be secondary to other consequences of FLCN knockdown like the regulation of AMPK signalling.
In summary, the study shows that FLCN is transcribed during embryonic development with elevated transcription levels in proliferating tissues of the zebrafish embryo, that FLCN is probably required for embryonic brain morphogenesis and that it affects the cell cycle particularly in the brain. BHD patients show no defects in the brain, however, all known BHD patients only show a mutation in one copy of the FLCN gene (Wei et al., 2009). This study provides previously undescribed and informative insights into the role of FLCN in vertebrates.
- Kenyon EJ, Luijten MN, Gill H, Li N, Rawlings M, Bull JC, Hadzhiev Y, van Steensel MA, Maher E, & Mueller F (2016). Expression and knockdown of zebrafish folliculin suggests requirement for embryonic brain morphogenesis. BMC developmental biology, 16 (1) PMID: 27391801