Developmental biology: Fishy fingers
Published online 25 April 2012
Mutant zebrafish provide a model for an emerging class of human diseases
Adult zebrafish (top). Cilia are present in the kidneys of a normal zebrafish embryo (middle) but absent from those of mutant embryos lacking the talpid3 gene (bottom).
© iStockphoto.com/isoft and © Philip Ingham
A team of researchers led by Phillip Ingham of the A*STAR Institute of Molecular and Cell Biology has created mutant zebrafish that highlight the unusual mechanism underlying how cells respond to a key molecular signal controlling vertebrate development1.
The talpid3 gene was originally discovered through a mutation in chickens that causes a characteristic limb deformity, called polydactyly, as well as craniofacial defects. These abnormalities arise through disruption of the primary cilium, a finger-like structure that protrudes from the cell surface and plays a crucial role in the cellular response to the Hedgehog signal.
Animals with mutations in the Hedgehog gene are also characterized by severe malformations of the limbs, brain and face. Related defects are found amongst the broad range of abnormalities associated with ciliopathies, an emerging class of human genetic disease that share dysfunction of the primary cilium as their underlying cause.
To analyze talpid3 function in a system more amenable to genetic manipulation, Ingham and his colleagues identified the zebrafish version of the gene and fused it to the gene encoding green fluorescent protein. This allowed them to visualize the Talpid3 protein in live zebrafish embryos, revealing that it is localized to a structure called the centriole, which is critical for development and positioning of the primary cilium.
They then mutated the gene using a DNA cutting enzyme called an endonuclease that they genetically engineered to target specific DNA sequences found only in the talpid3 gene. Surprisingly, the resulting mutant fish developed more or less normally, apart from abnormal kidneys, but died about one month after hatching.
Ingham reasoned that the talpid3 gene product supplied to the egg by the mother might suffice to support normal embryonic development. To test this, the team transplanted mutant egg progenitors into normal 'surrogate' females — as predicted, embryos derived from the mutant eggs failed to make primary cilia, disrupting the cellular response to Hedgehog and causing the same abnormalities associated with the chicken mutant.
These findings underline the crucial role of the primary cilium in Hedgehog signaling in all vertebrates. They provide a new model for investigating the interaction between Talpid3 and other proteins implicated in ciliopathies, as well as the role of the primary cilium in the cellular response to Hedgehog.
“It is becoming clear that components of the Hedgehog signaling pathway interact at the primary cilium, but exactly why is unclear,” says Ingham. “We are now generating mutations in other genes that affect primary cilia and in genes encoding Hedgehog pathway components. This will give insights into human ciliopathies.”
The A*STAR-affiliated researchers contributing to this research are from the Institute of Molecular and Cell Biology
- Ben, J. et al. Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates. Development 138, 4649–4978 (2011). | article