Multiple fibroblast growth factors support growth of the ureteric bud but have different effects on branching morphogenesis

Abstract

Together with glial-derived neurotrophic factor (GDNF), soluble factors present in a metanephric mesenchyme (MM) cell conditioned medium (BSN-CM) are necessary to induce branching morphogenesis of the isolated ureteric bud (UB) in vitro (Proc. Natl. Acad. Sci. USA 96 (1999) 7330). Several lines of evidence are presented here in support of a modulating role for fibroblast growth factors (FGFs) in this process. RT-PCR revealed the expression of two FGF receptors, FGFR1(IIIc) and FGFR2(IIIb), in isolated embryonic day 13 rat UBs, which by indirect immunofluorescence displayed a uniform distribution. Rat kidney organ culture experiments in the presence of a soluble FGFR2(IIIb) chimera or a neutralizing antibody to FGF7 suggested an important contribution of FGFs other than FGF7 to the branching program. Several FGFs, including FGF1, FGF2, FGF7 and FGF10, in combination with GDNF and BSN-CM were found to affect growth and branching of the isolated UB, albeit with very different effects. FGF1 and FGF7 were at extreme ends of the spectrum, with FGF10 (more FGF1-like) and FGF2 (more FGF7-like) falling in between. FGF1 induced the formation of elongated UB branching stalks with distinct proliferative ampullary tips, whereas FGF7 induced amorphous buds displaying nonselective proliferation with little distinction between stalks and ampullae. Electron microscopic examination demonstrated that FGF1 treatment induced cytoskeletal organization, intercellular junctions and lumens along the stalk portion of the developing tubules, while the ampullary regions contained 'less differentiated' cells with an abundant secretory apparatus. In contrast, FGF7-induced UBs displayed this 'less differentiated' morphology regardless of position on the structure and were virtually indistinguishable from FGF1-induced ampullae. Consistent with this, GeneChip array analysis (employing a novel nanogram-scale assay consisting of two rounds of amplification and in vitro transcription for analyzing small quantities of RNA) revealed that FGF7-induced UBs expressed more markers of cell proliferation than FGF1, which caused the UB to express cytoskeletal proteins, extracellular matrix proteins, and at least one integrin, some of which may be important in UB branch elongation. Thus, while the various FGFs examined all support UB growth, FGF1 and FGF10 appear to be more important for branching and branch elongation, and may thus play a role in determination of nephron number and patterning in the developing kidney. These in vitro data may help to explain results from knockout and transgenic studies and suggest how different FGFs may, together with GDNF and other factor(s) secreted by MM cells, regulate branching morphogenesis of the UB by their relative effects on its growth, branching and branch elongation and differentiation, thereby affecting patterning in the developing kidney.

Identification of FGF10 Targets in the Embryonic Lung Epithelium during Bud Morphogenesis

“…We had no evidence of mesenchymal cell contamination, because expression of other cardiac genes or vimentin was undetectable. Interestingly, a similar study in rat mesenchyme-free ureteric bud cultures shows that the rat Myh7 ortholog can be also induced in the epithelium by recombinant FGF1 or FGF7 (31). The FGF10 induction of Myh7 is intriguing and raises the possibility of new unsuspected roles for this myosin in epithelial development.…”

Staged in vitro reconstitution and implantation of engineered rat kidney tissue

“…In vitro, these growth factor conditions provided the most robust branch-stimulating conditions (i.e., growth plus most in vivo-like patterning) among the many soluble-factor combinations that we have tested (33)(34)(35)(36)(37). The fact that a combination of BSN-CM, FGF1, and GDNF gave more consistent branching growth than PTN, FGF1, and GDNF suggests that one or more factors within BSN-CM will need to be isolated to achieve ideal minimal conditions.…”

Definition and spatial annotation of the dynamic secretome during early kidney development

“…This implied involvement of FGF1 and FGF7 in developmental changes between E10.5 and E13.5, despite no detected changes in levels of transcripts for these factors. Both FGF1 and FGF2 have previously been impli- cated in metanephric development (Perantoni et al, 1995;Barasch et al, 1997;Qiao et al, 2001;Zhao et al, 2004). While the FGFs are secreted via a mechanism distinct from the use of an ER signal peptide (Prudovsky et al, 2003) and hence would not have been included in the soluble/secreted class using our prediction tools, these transcripts were actually not outliers themselves, highlighting the need for parallel analyses of signalling pathways.…”