Philippe Soriano

The fibroblast growth factor (FGF) pathway is a conserved signaling pathway required for embryonic development. Activated FGF receptor 1 (FGFR1) drives multiple intracellular signaling cascade pathways, including ERK/MAPK and PI3K/AKT, collectively termed canonical signaling. However, unlikeFgfr1-null embryos, embryos containing hypomorphic mutations inFgfr1lacking the ability to activate canonical downstream signals are still able to develop to birth but exhibit severe defects in all mesodermal-derived tissues. The introduction of an additional signaling mutation further reduces the activity ofFgfr1, leading to earlier lethality, reduced somitogenesis, and more severe changes in transcriptional outputs. Genes involved in migration, ECM interaction, and phosphoinositol signaling were significantly downregulated, proteomic analysis identified changes in interactions with endocytic pathway components, and cells expressing mutant receptors show changes in endocytic trafficking. Together, we identified processes regulating early mesoderm development by mechanisms involving both canonical and noncanonicalFgfr1pathways, including direct interaction with cell adhesion components and endocytic regulation.

The Fibroblast growth factor (FGF) pathway is a conserved signaling pathway required for embryonic development. Activated FGF receptor 1 (FGFR1) drives multiple intracellular signaling cascade pathways, including ERK/MAPK and PI3K/AKT, collectively termed canonical signaling. However, unlike Fgfr1 null embryos, embryos containing hypomorphic mutations in Fgfr1 lacking the ability to activate canonical downstream signals are still able to develop to birth, but exhibit severe defects in all mesodermal-derived tissues. The introduction of an additional signaling mutation further reduces the activity of Fgfr1, leading to earlier lethality, reduced somitogenesis, and more severe changes in transcriptional outputs. Genes involved in migration, ECM-interaction, and phosphoinositol signaling were significantly downregulated, proteomic analysis identified changes in interactions with endocytic pathway components, and cells expressing mutant receptors show changes in endocytic trafficking. Together, we identify processes regulating early mesoderm development by mechanisms involving both canonical and non-canonical Fgfr1 pathways, including direct interaction with cell adhesion components and endocytic regulation.

FGF activation is known to engage canonical signals, including ERK/MAPK and PI3K/AKT, through various effectors including FRS2 and GRB2. Fgfr2FCPG/FCPG mutants that abrogate canonical intracellular signaling exhibit a range of mild phenotypes but are viable, in contrast to embryonic lethal Fgfr2−/− mutants. GRB2 has been reported to interact with FGFR2 through a non-traditional mechanism, by binding to the C-terminus of FGFR2 independently of FRS2 recruitment. To investigate whether this interaction provides functionality beyond canonical signaling, we generated mutant mice harboring a C-terminal truncation (T). We found that Fgfr2T/T mice are viable and have no distinguishable phenotype, indicating that GRB2 binding to the C-terminal end of FGFR2 is not required for development or adult homeostasis. We further introduced the T mutation on the sensitized FCPG background but found that Fgfr2FCPGT/FCPGT mutants did not exhibit significantly more severe phenotypes. We therefore conclude that, although GRB2 can bind to FGFR2 independently of FRS2, this binding does not have a critical role in development or homeostasis.

FGF activation is known to engage canonical signals, including ERK/MAPK and PI3K/AKT, through various effectors including FRS2 and GRB2. Fgfr2 ^FCPG/FCPG mutants that abrogate canonical intracellular signaling exhibit a range of mild phenotypes but are viable in contrast to embryonic lethal Fgfr2 ^-/- mutants. GRB2 has been reported to interact with FGFR2 through a non-traditional mechanism, by binding to the C-terminus of FGFR2 independently of FRS2 recruitment. To investigate if this interaction provides functionality beyond canonical signaling, we generated mutant mice harboring a C-terminal truncation (T). We found that Fgfr2 ^T/T mice are viable and have no distinguishable phenotype, indicating that GRB2 binding to the C-terminal end of FGFR2 is not required for development or adult homeostasis. We further introduced the T mutation on the sensitized FCPG background but found that Fgfr2 ^FCPGT/FCPGT mutants did not exhibit significantly more severe phenotypes. We therefore conclude that, while GRB2 can bind to FGFR2 independently of FRS2, this binding does not have a critical role in development or homeostasis.

Loss of FGF signaling leads to defects in salivary gland branching, but the mechanisms underlying this phenotype remain largely unknown. We disrupted expression of Fgfr1 and Fgfr2 in salivary gland epithelial cells and found that both receptors function coordinately in regulating branching. Strikingly, branching morphogenesis in double knockouts is restored by Fgfr1 and Fgfr2 (Fgfr1/2) knock-in alleles incapable of engaging canonical RTK signaling, suggesting that additional FGF-dependent mechanisms play a role in salivary gland branching. Fgfr1/2 conditional null mutants showed defective cell-cell and cell-matrix adhesion, both of which have been shown to play instructive roles in salivary gland branching. Loss of FGF signaling led to disordered cell-basement membrane interactions in vivo as well as in organ culture. This was partially restored upon introducing Fgfr1/2 wild-type or signaling alleles that are incapable of eliciting canonical intracellular signaling. Together, our results identify non-canonical FGF signaling mechanisms that regulate branching morphogenesis through cell-adhesion processes.

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Loss of FGF signaling leads to defects in salivary gland branching, but the mechanisms underlying this phenotype remain largely unknown. We disrupted expression of Fgfr1 and Fgfr2 in salivary gland epithelial cells and find that both receptors function coordinately in regulating branching. Strikingly, branching morphogenesis in double knockouts is restored by Fgfr1/2 knockin alleles incapable of engaging canonical RTK signaling, suggesting that additional FGF dependent mechanisms play a role during salivary gland branching. Fgfr1/2 conditional null mutants showed defective cell-cell and cell-matrix adhesion, both of which have been shown to play instructive roles in salivary gland branching. Loss of FGF signaling led to disordered cellbasement membrane interactions in vivo as well as in organ culture. This was partially restored upon introducing Fgfr1/2 wild type or signaling alleles incapable of eliciting canonical intracellular signaling. Together, our results identify non-canonical FGF signaling mechanisms that regulate branching morphogenesis through cell adhesion processes.

Summary: The Wnt1‐Cre transgenic mouse line is widely used to express the CRE recombinase in neural crest lineages, but it overexpresses WNT1 itself, which can cause undesired phenotypes. To address this, we and others previously developed a Wnt1‐Cre2 line based on the same regulatory elements as Wnt1‐Cre but without ectopic Wnt1 expression. However, while Wnt1‐Cre2 exhibits normal activity when transmitted from female mice, it exhibits unexpected activity in the male germline. The Wnt1‐Cre2 transgene was previously mapped to the E2f1 locus. Several genes in this genomic region exhibit significant expression in spermatogonia or spermatocytes, suggesting that local regulatory elements may be driving ectopic transgene expression. The Wnt1‐Cre2 line can therefore be used both as a neural crest specific and a general deleter, and care should be taken when setting up genetic crosses.