SUMMARYIn vertebrates, the common expression border of two homeobox genes, Otx2 and Gbx2, demarcates the prospective midbrainhindbrain border (MHB) in the neural plate at the end of gastrulation. The presence of a compartment boundary at the MHB has been demonstrated, but the mechanism and timing of its formation remain unclear. We show by genetic inducible fate mapping using a Gbx2CreER knock-in mouse line that descendants of Gbx2 + cells as early as embryonic day (E) 7.5 do not cross the MHB. Without Gbx2, hindbrain-born cells abnormally populate the entire midbrain, demonstrating that Gbx2 is essential for specifying hindbrain fate. Gbx2 + and Otx2 + cells segregate from each other, suggesting that mutually exclusive expression of Otx2 and Gbx2 in midbrain and hindbrain progenitors is responsible for cell sorting in establishing the MHB. The MHB organizer gene Fgf8, which is expressed as a sharp transverse band immediately posterior to the lineage boundary at the MHB, is crucial in maintaining the lineage-restricted boundary after E7.5. Partial deletion of Fgf8 disrupts MHB lineage separation. Activation of FGF pathways has a cell-autonomous effect on cell sorting in midbrain progenitors. Therefore, Fgf8 from the MHB may signal the nearby mesencephalic cells to impart distinct cell surface characteristics or induce local cell-cell signaling, which consequently prevents cell movements across the MHB. Our findings reveal the distinct function of Gbx2 and Fgf8 in a stepwise process in the development of the compartment boundary at the MHB and that Fgf8, in addition to its organizer function, plays a crucial role in maintaining the lineage boundary at the MHB by restricting cell movement.

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.

SummaryThe mouse homeobox gene, Gbx2, is expressed in discreet domains in the neural tube and plays a key role in forebrain and hindbrain development. Previous studies have demonstrated that mutual inhibition between Gbx2 and Otx2, which are respectively expressed in the anterior and posterior parts of the neural plate, positions the prospective midbrain-hindbrain junction. We describe here a conditional Gbx2 gain-of-function transgenic mouse line, Gbx2-GOF, which expresses Gbx2 and red fluorescence protein, mCherry, upon Cre-mediated recombination. In the absence of Cre, β-galactosidase is broadly expressed in mouse embryos and adult brains carrying the transgene. By combining Gbx2-GOF and En1 Cre knock-in allele, we activated expression of Gbx2 and mCherry throughout the mesencephalon (mes) and rhombomere 1 (r1). The ectopic expression of Gbx2 causes an anterior shift of the mes/r1 junction at embryonic day 10.5. Interestingly, we found that persistent expression of Gbx2 throughout the mes/r1 region largely abolishes expression of the isthmic organizer gene Fgf8, leading to deletion of the midbrain and cerebellum at later stages. Our data suggest that the juxtaposition of the expression domains of Gbx2 and Otx2 within the mes/r1 area is essential for the maintenance of Fgf8 expression. Furthermore, the Gbx2-GOF transgenic line is suitable for functional study of Gbx2 during development. KeywordsGbx2; Fgf8; transcription factor; Cre; conditional gain of function The mouse homeobox gene, Gbx2 (gastrulation and brain homeobox gene) displays a complex and dynamic expression profile in the posterior mesoderm, limb buds and the neural tube during development (Bouillet et al., 1995;Bulfone et al., 1993;Chen et al., 2009). Gbx2 is initially expressed in the posterior part of the mouse embryo during gastrulation (Bouillet et al., 1995;Wassarman et al., 1997). Genetic studies have demonstrated that mutual inhibition between Gbx2 and Otx2, which is expressed in the anterior part of the embryo, positions a signaling center, known as the isthmic organizer, at the prospective mid-hindbrain junction . Mutation of Gbx2 leads to a posterior expansion of the expression domain of Otx2 and deletion of the entire cerebellum (Li and Joyner, 2001;Martinez-Barbera et al., 2001;Millet et al., 1999;Wassarman et al., 1997). Conversely, ectopic expression of Gbx2 in the mesencephalon (mes) in chick embryos by electroporation, or in mouse embryos by a transgene driven by regulatory elements of Wnt1, which is expressed in the mes, results in an anterior shift of the isthmus (Katahira et al., 2000;Millet et al., 1999). At later stages, however, the formation of the (Katahira et al., 2000;Millet et al., 1999). By contrast, ectopic expression of Otx2 in r1 leads to complete transformation of r1 into a midbrain fate (Broccoli et al., 1999; Katahira et al., 2000). The ectopic expression of Gbx2 by electroporation or using a Wnt1-Gbx2 transgene is transient (Katahira et al., 2000;Millet et al., 1999), which precludes an assessment of whether G...

Soluble growth factors play an important role in the coordination and integration of cell proliferation, differentiation, fate determination, and morphogenesis during development of multicellular organisms. Fibroblast growth factors (FGFs) are a large family of polypeptide growth factors that are present in organisms ranging from nematodes to humans. RNA alternative splicing of FGFs and their receptors further enhances the complexity of this ligand-receptor system. The mouse Fgf8 gene produces eight splice variants, which encode isoform proteins with different N-termini and distinct receptor-binding affinity and biological activity. In this article, we review the roles of Fgf8 in vertebrate development and summarize the recent findings on the in vivo function of different Fgf8 splice variants. We propose that multiple Fgf8 isoform proteins act in concert to regulate the overall function of Fgf8 and account for the diverse and essential role of Fgf8 during vertebrate development.J. Cell. Physiol. 226: 1722-1726, 2011. ß 2010 Wiley-Liss, Inc. The FGF Family in VertebratesThe first two fibroblast growth factors (FGFs), Fgf1 and Fgf2, were isolated as mitogens for fibroblast from bovine pituitary and brain (Gospodarowicz et al., 1974(Gospodarowicz et al., , 1978Gospodarowicz, 1975;Maciag et al., 1979). Subsequent studies have identified numerous FGFs in various organisms, except for unicellular organisms (Itoh and Ornitz, 2008). In mice and humans, the FGF family is comprised of 22 members, which are grouped into six or seven subfamilies based on similarity in sequence, gene location, and function. Genome sequence analyses suggest that the FGF family may have expanded in two phases by gene duplication during evolution Ornitz, 2004, 2008). The first phase was associated with early metazoan evolution to generate ancestors of 6-7 FGF subfamilies. Subsequently, large-scale genome duplication during the early evolution of vertebrates further expanded the FGF subfamilies to contain 3-4 members. Most of FGF members are secreted proteins with a clear N-terminal signal peptide. FGFs 1, 2, 9, 16, and 20 are secreted proteins though they lack an obvious cleavable N-terminal signal sequence. FGFs 11-14 are intracellular proteins and thus called intracellular FGF (iFGF) (Itoh and Ornitz, 2008). All FGFs contain a conserved core of approximately120 amino acids arranged into a globular domain composed of 12 antiparallel b strands (Eriksson et al., 1991;Zhu et al., 1991). This core domain is important for receptor binding, while the divergent N-and C-termini of FGF can modify the receptor-binding affinity and account for the different biological function of FGF (Mohammadi et al., 2005). In humans and mice, there are four FGF receptors, FGFR1-4, which are receptor tyrosine kinases and contain an extracellular ligand-binding domain with three immunoglobulin domains (I, II, and III), a single-pass transmembrane domain, and intracellular tyrosine kinase domain (Ornitz and Itoh, 2001;Mohammadi et al., 2005). Alternative splicing of FGF...

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