Fgf3‐Fgf4‐cis: A new mouse line for studying Fgf functions during mouse development

The fibroblast growth factor (FGF) family consists of 22 ligands in mice and humans. FGF signaling is vital for embryogenesis and, when dysregulated, can cause disease. Loss‐of‐function genetic analysis in the mouse has been crucial for understanding FGF function. Such analysis has revealed that multiple Fgfs sometimes function redundantly. Exploring such redundancy between Fgf3 and Fgf4 is currently impossible because both genes are located on chromosome 7, about 18.5 kb apart, making the frequency of interallelic cross‐over between existing mutant alleles too infrequent to be practicable. Therefore, we retargeted Fgf3 and Fgf4 in cis, generating an Fgf3 null allele and a conditional Fgf4 allele, subject to Cre inactivation. To increase the frequency of cis targeting, we used an F1 embryonic stem cell line that contained 129/SvJae (129) and C57BL/6J (B6) chromosomes and targeting constructs isogenic to the 129 chromosome. We confirmed cis targeting by assaying for B6/129 allele‐specific single‐nucleotide polymorphisms. We demonstrated the utility of the Fgf3Δ‐Fgf4flox‐cis mouse line by showing that the caudal axis extension defects found in the Fgf3 mutants worsen when Fgf4 is also inactivated. This Fgf3Δ‐Fgf4flox‐cis line will be useful to study redundancy of these genes in a variety of tissues and stages in development. genesis 54:91–98, 2016. Published 2016. This article is a US Government work and is in the public domain in the USA.

[1]  N. Itoh,et al.  The Fibroblast Growth Factor signaling pathway , 2015, Wiley interdisciplinary reviews. Developmental biology.

[2]  René Bernards,et al.  Feedback and redundancy in receptor tyrosine kinase signaling: relevance to cancer therapies. , 2014, Trends in biochemical sciences.

[3]  B. Morrow,et al.  Endoderm‐specific deletion of Tbx1 reveals an FGF‐independent role for Tbx1 in pharyngeal apparatus morphogenesis , 2014, Developmental dynamics : an official publication of the American Association of Anatomists.

[4]  R. Jaenisch,et al.  Generating genetically modified mice using CRISPR/Cas-mediated genome engineering , 2014, Nature Protocols.

[5]  A. Nagy.  Manipulating the mouse embryo , 2013 .

[6]  M. Mohammadi,et al.  Exploring mechanisms of FGF signalling through the lens of structural biology , 2013, Nature Reviews Molecular Cell Biology.

[7]  M. Capecchi,et al.  Signaling by FGF4 and FGF8 is required for axial elongation of the mouse embryo. , 2012, Developmental biology.

[8]  A. Moon,et al.  Redundant and dosage sensitive requirements for Fgf3 and Fgf10 in cardiovascular development. , 2011, Developmental biology.

[9]  Y. C. Long,et al.  Hormone-like fibroblast growth factors and metabolic regulation. , 2011, Biochimica et biophysica acta.

[10]  M. Lewandoski,et al.  FGF4 and FGF8 comprise the wavefront activity that controls somitogenesis , 2011, Proceedings of the National Academy of Sciences.

[11]  N. Itoh.  Hormone-like (endocrine) Fgfs: their evolutionary history and roles in development, metabolism, and disease , 2010, Cell and Tissue Research.

[12]  A. Moon,et al.  Dosage-dependent hedgehog signals integrated with Wnt/β-catenin signaling regulate external genitalia formation as an appendicular program , 2009, Development.

[13]  Olivier Pourquié,et al.  Control of segment number in vertebrate embryos , 2008, Nature.

[14]  Tracy J. Wright,et al.  Fgf3 is required for dorsal patterning and morphogenesis of the inner ear epithelium , 2007, Development.

[15]  M. Lewandoski,et al.  Inactivation of FGF8 in early mesoderm reveals an essential role in kidney development , 2005, Development.

[16]  M. Goldfarb.  Fibroblast growth factor homologous factors: evolution, structure, and function. , 2005, Cytokine & growth factor reviews.

[17]  Benjamin R. Arenkiel,et al.  The roles of Fgf4 and Fgf8 in limb bud initiation and outgrowth. , 2004, Developmental biology.

[18]  D. Riethmacher,et al.  Requirements for FGF3 and FGF10 during inner ear formation , 2003, Development.

[19]  Tracy J. Wright,et al.  Expression of mouse fibroblast growth factor and fibroblast growth factor receptor genes during early inner ear development , 2003, Developmental dynamics : an official publication of the American Association of Anatomists.

[20]  Shaun K Olsen,et al.  Fibroblast Growth Factor (FGF) Homologous Factors Share Structural but Not Functional Homology with FGFs* , 2003, Journal of Biological Chemistry.

[21]  G. Martin,et al.  Functions of FGF signalling from the apical ectodermal ridge in limb development , 2002, Nature.

[22]  B. Wold,et al.  The bHLH regulator pMesogenin1 is required for maturation and segmentation of paraxial mesoderm. , 2000, Genes & development.

[23]  Reynaldo Sequerra,et al.  High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP , 2000, Nature Genetics.

[24]  P. Gruss,et al.  Uncx4.1 is required for the formation of the pedicles and proximal ribs and acts upstream of Pax9. , 2000, Development.

[25]  G. Martin,et al.  Conditional inactivation of Fgf4 reveals complexity of signalling during limb bud development , 2000, Nature Genetics.

[26]  M. Capecchi,et al.  Normal limb development in conditional mutants of Fgf4. , 2000, Development.

[27]  R. Jaenisch,et al.  Utility of C57BL/6J × 129/SvJae embryonic stem cells for generating chromosomal deletions: tolerance to γ radiation and microsatellite polymorphism , 1998, Mammalian Genome.

[28]  V. Papaioannou,et al.  Requirement of FGF-4 for postimplantation mouse development , 1995, Science.

[29]  G. Martin,et al.  Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. , 1992, Development.

[30]  Mario R. Capecchi,et al.  Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes , 1988, Nature.

[31]  O. Klein,et al.  Fibroblast growth factor signaling in mammalian tooth development , 2013, Odontology.

[32]  Lino Tessarollo,et al.  Isolation, microinjection and transfer of mouse blastocysts. , 2009, Methods in molecular biology.

[33]  V. Coppola,et al.  Gene targeting in mouse embryonic stem cells. , 2009, Methods in molecular biology.

[34]  P. Gruss,et al.  Uncx 4 . 1 is required for the formation of the pedicles and proximal ribs and acts upstream of Pax 9 , 2000 .

[35]  M. Capecchi,et al.  Mice homozygous for a targeted disruption of the proto-oncogene int-2 have developmental defects in the tail and inner ear. , 1993, Development.