Cystic Fibrosis Zebrafish Model

General Characteristics

Lifespan: 2-3 years

Age to sexual maturity: ~10-12 weeks

Rat

The Cystic Fibrosis Model

Zebrafish (Danio rerio) are freshwater fish belonging to the Cyprinidae family, order Cypriniformes (same as carp). Zebrafish are a commonly used model for biomedical research. The development of the CF zebrafish model in 2010 provided the community with a useful model system for understanding some of the effects of CFTR function on organogenesis.

In spite of genome duplication, there is only one cftr gene in zebrafish. Human CFTR and zebrafish cftr only share 55% sequence identity, but the structures are essentially identical in the dephosphorylated, ATP-free conformation. In addition, many CF-causing missense mutational sites are identical between the two species. Importantly, Cftr is also activated by agents that elevate cAMP such as forskolin and IBMX1 and by direct PKA phosphorylation.2 The first 3D structures of the channel were obtained for zebrafish Cftr, including the dephosphorylated, ATP-free form and the phosphorylated, ATP-bound form.3

The first indication of a functional role for Cftr in zebrafish came from a forward genetic screen that identified Cse1l as a negative regulator of intestinal fluid secretion.1 It was shown that loss of Cse1l leads to a secretory diarrhea-like phenotype that could be prevented by pharmacological inhibition of Cftr.1 Interestingly, a proteomic study of human CFTR showed that CSE1L is bound to the channel.4

Production of zebrafish with mutant cftr

Zebrafish mutants for cftr were generated at Duke University using TAL effector nucleases (TALENs) mediated gene editing.5 Two alleles were published, a null mutant cftrpd1049, which produces no protein, and a hypomorph cftrpd1048 that has a two-amino-acid deletion within a transmembrane helix of the channel that impairs surface delivery of the protein.5 The mutants are maintained as heterozygotes, containing one mutated cftr allele. Heterozygotic in-crosses result in the expected Mendelian ratios (1:2:1), producing 25% mutant offspring.

The same study by Navis et al. that generated mutants also produced a functional cftr-GFP transgenic using BAC recombineering. Live microscopy revealed cftr-GFP is first expressed in Kupffer’s vesicle (KV), a transient, fluid-filled structure that is the organ of laterality in zebrafish. It was shown that loss of CFTR-mediated fluid secretion impairs KV lumen expansion, leading to defects in organ laterality.5 Although  CFTR is not required for L/R asymmetry in mammals, its role in lumen formation was later shown in the mouse salivary gland,6 demonstrating a core conserved function in tube formation.

Disease Manifestations

 

In addition to KV, cftr-GFP is expressed in the intestine and the pancreas. In the intestine it is expressed at low levels in most enterocytes and at very high levels in a subset of intestinal cells in which the protein localizes subapically (Navis and Bagnat, unpublished). In the pancreas, cftr-GFP lines the pancreatic duct. Although cftr mutants show normal intestinal development, they present a severe pancreatic defect (Navis and Bagnat, 2015).

 

Pancreatic Disease

Initial development of the pancreas appears to be normal in mutant cftr zebrafish. Once the pancreas is formed, the mutants exhibit severe exocrine pancreatic destruction. Loss of cftr function causes the zebrafish pancreatic ducts to become filled with mucus, leading to the pancreatic acinar tissue being rapidly destroyed. In addition, the cftr mutant pancreas exhibits increased neutrophil recruitment to the affected tissue and later replacement of the affected tissue with fibrosis. The onset of the pancreatitis phenotype is around 14-16 days post fertilization.7 Interestingly, while homozygous cftr mutants exhibit high mortality, a variable fraction survives to adulthood and shows normal growth rates, suggesting the presence of modifier genes in the background. This phenomenon may allow the implementation of forward suppressor genetic screens to identify modifier genes.

Available Strains

 

CF zebrafish strains Note Source
Cftrpd1048 hypomorphic mutant [A]
Cftrpd1049 null mutant [A]
TgBAC(cftr-GFP)pd1041   [A]
TgBAC(cftr-RFP)pd1042   [A]
TgBAC(cftr:Gal4)pd1101   [A]
[A] = Bagnat Lab, Duke University    

Contact for Information or Questions about this Model

e-mail: michel.bagnat@duke.edu

References

1. M. Bagnat, A. Navis, S. Herbstreith, K. Brand-Arzamendi, S. Curado, S. Gabriel, K.E. Mostov, J. Huisken, D.Y.R. Stainier (2010). Cse1l is a negative regulator of CFTR-dependent fluid secretion. Curr. Biol., 20:1840-1845.
2. Z. Zhang, J. Chen (2016). Atomic structure of the cystic fibrosis transmembrane conductance regulator. Cell, 167: 1586-1597.
3. Z. Zhang, J. Chen (2017). Conformational Changes of CFTR upon Phosphorylation and ATP Binding Cell, 170 (2017).
4. Wang X, Venable J, LaPointe P, Hutt DM, Koulov AV, Coppinger J, Gurkan C, Kellner W, Matteson J, Plutner H, et al. (2006). Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. Cell, 127:803-15.
5. Navis, L. Marjoram, M. Bagnat Cftr controls lumen expansion and function of Kupffer’s vesicle in zebrafish (2013). Development, 1703-1712.
6. Nedvetsky PI, Emmerson E, Finley JK, Ettinger A, Cruz-Pacheco N, Prochazka J, Haddox CL, Northrup E, Hodges C, Mostov KE, Hoffman MP, Knox SM. (2014). Parasympathetic innervation regulates tubulogenesis in the developing salivary gland. Dev Cell. 30:449-62.
7. Adam Navis and Michel Bagnat. (2013). Loss of cftr function leads to pancreatic destruction in larval zebrafish. Dev Biol. 2015 399: 237-48.