Fgf8b-containing spliceforms, but not Fgf8a, are essential for Fgf8 function during development of the midbrain and cerebellum

Abstract

Summary The single Fgf8 gene in mice produces eight protein isoforms (Fgf8a–h) with different N-termini by alternative splicing. Gain-of-function studies have demonstrated that Fgf8a and Fgf8b have distinct activities in the developing midbrain and hindbrain (MHB) due to their different binding affinities with FGF receptors. Here we have performed loss-of-function analyses to determine the in vivo requirement for these two Fgf8 spliceforms during MHB development. We showed that deletion of Fgf8b-containing spliceforms (b, d, f and h) leads to loss of multiple key regulatory genes, including Fgf8 itself, in the MHB region. Therefore, specific inactivation of Fgf8b-containing spliceforms, similar to the loss of Fgf8, in MHB progenitors results in deletion of the midbrain, isthmus, and cerebellum. We also created a splice-site mutation abolishing Fgf8a-containing spliceforms (a, c, e, and g). Mice lacking Fgf8a-containing spliceforms exhibit growth retardation and postnatal lethality, and the phenotype is variable in different genetic backgrounds, suggesting that the Fgf8a-containing spliceforms may play a role in modulating the activity of Fgf8. Surprisingly, no discernable defect was detected in the midbrain and cerebellum of Fgf8a-deficient mice. To determine if Fgf17, which is expressed in the MHB region and possesses similar activities to Fgf8a based on gain-of-function studies, may compensate for the loss of Fgf8a, we generated Fgf17 and Fgf8a double mutant mice. Mice lacking both Fgf8a-containing spliceforms and Fgf17 display the same defect in the posterior midbrain and anterior cerebellum as Fgf17 mutant mice. Therefore, Fgf8b-containing spliceforms, but not Fgf8a, are essential for the function of Fgf8 during the development of the midbrain and cerebellum.

Fibroblast Growth Factor 8 Organizes the Neocortical Area Map and Regulates Sensory Map Topography

“…In chick embryos, FGF8-coated beads can induce an ectopic ISO and cerebellum, or, in caudal forebrain, a striking morphological duplication of the midbrain (Crossley et al, 1996; Martinez et al, 1999). Conditional deletion of Fgf8 , or specific deletion of the FGF8b isoform in mice causes loss of the midbrain, isthmus, and cerebellum (Chi et al, 2003; Guo et al, 2009). Similarly, ectopic FGF8 in the early NP induces neocortical area duplications, and augmenting or reducing the endogenous source of FGF8 causes appropriate R/C area shifts in the map (Fukuchi-Shimogori and Grove, 2001; Garel et al, 2003), including major patterning changes in the frontal cortex immediately adjacent to the endogenous FGF8 source (Garel et al, 2003; Cholfin and Rubenstein, 2008).…”

Cubilin, a High Affinity Receptor for Fibroblast Growth Factor 8, Is Required for Cell Survival in the Developing Vertebrate Head

“…Further SPR analysis showed that recombinant mouse Fgf8a and Fgf8b, the two biologically active Fgf8 isomorphs (44,45), bound in a Ca 2ϩ -dependent manner and with equally high affinity to a purified Cubn chip (Fig. 5B).…”

Regulation of neurogenesis by Fgf8a requires Cdc42 signaling and a novel Cdc42 effector protein

“…Fgf8 isoforms (of which Fgf8b is the major form) are co-expressed many tissues in the embryo, including the gastrula, the mesoderm, the midbrain-hindbrain boundary and the limb bud apical ectodermal ridge (Crossley et al ., 1995). Interestingly, elimination of the Fgf8a -containing spliceforms ( a, c, e, g ) in mouse, through gene targeted alteration of the relevant splice acceptor site, failed to cause embryonic defects in Fgf8 -expressing tissues (Guo et al ., 2010), in contrast to deletion of the b -containing forms or complete Fgf8 nulls. Additionally, compound mutants of Fgf8a and Fgf17 , which likely has Fgf8a-like activity, did not result in additional defects (Guo et al ., 2010).…”

“…Interestingly, elimination of the Fgf8a -containing spliceforms ( a, c, e, g ) in mouse, through gene targeted alteration of the relevant splice acceptor site, failed to cause embryonic defects in Fgf8 -expressing tissues (Guo et al ., 2010), in contrast to deletion of the b -containing forms or complete Fgf8 nulls. Additionally, compound mutants of Fgf8a and Fgf17 , which likely has Fgf8a-like activity, did not result in additional defects (Guo et al ., 2010). Residual Fgf8a-like activity could be supplied by Fgf18 in these animals, but triple mutants have not been described.…”