Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice - PubMed (original) (raw)

Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice

D L Miller et al. Mol Cell Biol. 2000 Mar.

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Abstract

Fibroblast growth factor 1 (FGF1) and FGF2, the prototypic members of the FGF family of growth factors, have been implicated in a variety of physiological and pathological processes. Unlike most other FGFs, FGF1 and FGF2 are ubiquitously expressed and are not efficiently secreted. Gene knockouts in mice have previously demonstrated a role for FGF2 in brain development, blood pressure regulation, and wound healing. The relatively mild phenotypic defects associated with FGF2 deletion led to the hypothesis that the continued expression of other FGFs partially compensated for the absence of FGF2 in these mice. We now report our generation of mice lacking FGF1 and their use, in combination with our previously described FGF2 null mice, to produce mice lacking both FGF1 and FGF2. FGF1-FGF2 double-knockout mice are viable and fertile and do not display any gross phenotypic defects. In the double-knockout mice we observed defects that were similar in extent to those previously described for the FGF2 null mice. Differences in the organization of neurons of the frontal motor cortex and in the rates of wound healing were observed. We also observed in FGF2(-/-) mice and in FGF1-FGF2 double-knockout mice novel impairments in hematopoiesis that were similar in severity. Essentially no abnormalities were found in mice lacking only FGF1. Our results suggest that the relatively mild defects in FGF2 knockout animals are not a consequence of compensation by FGF1 and suggest highly restricted roles for both factors under normal developmental and physiological conditions.

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Figures

FIG. 1

FIG. 1

(A) The pPNT replacement vector was used to construct a targeting plasmid to delete approximately 5 kb of genomic DNA, including the entire first coding exon of FGF1 (black box). Probes used in Southern analysis are indicated. Abbreviations for restriction enzyme sites are as follows: B, _Bam_HI; N, _Nco_I; Not, _Not_I; RI, _Eco_RI; RV, _Eco_RV; and X, _Xba_I. The _Not_I site at the 3′ end of the genomic sequence is derived from the phage DNA. The sites in parentheses were destroyed during cloning. (B) (Top) The presence of the recombined targeting plasmid in a clone (+/−) is detected by using an external probe. A normal clone (+/+) is shown for comparison. (Center and bottom) Litters from crosses of heterozygous mice contain wild-type (+/+), heterozygous (+/−), and FGF1 null (−/−) mice. Southern analysis was performed with the indicated restriction enzymes and probes. Positions of molecular size standards (in kilobases) are shown at the left. (C) Extracts from brain (lanes 2 to 4) and heart (lanes 5 to 7) tissues were analyzed for FGF1 and FGF2 expression. Tissues from wild-type (lanes 2 and 5), heterozygous (lanes 3 and 6), and FGF1 null (lanes 4 and 7) mice were assayed. (Top) Forty micrograms of whole-cell extract; immunoblotting with an anti-FGF1 polyclonal antibody. Recombinant FGF1, which contains a small N-terminal truncation, is in lane 1. The arrow indicates the migration position of FGF1. (Center) Five milligrams of total protein, concentrated by using heparin-Sepharose beads; immunoblotting with an anti-FGF1 polyclonal antibody. Recombinant FGF1 is in lane 1. The arrow indicates the migration position of FGF1. (Bottom) Five milligrams of total protein, concentrated by using heparin-Sepharose beads; immunoblotting with an anti-FGF2 polyclonal antibody. Recombinant FGF2 is in lane 1. The three unmarked arrows indicate the migration positions of the three FGF2 isoforms present in mice. The starred arrow indicates a band in lanes 5 and 6 arising from cross-reactivity of the antibody with the FGF1 present. Migration positions of molecular mass standards (in kilodaltons) are shown at the left. (D) Extracts were prepared from the brains of wild-type, FGF1−/− (FGF1 KO), FGF2−/− (FGF2 KO), and FGF1−/− FGF2−/− (Double KO) mice. (Left) Forty micrograms of whole-cell extract; immunoblotting with an anti-FGF1 polyclonal antibody. Recombinant FGF1 (rFGF1) was used as a control. (Right) Five milligrams of total protein, concentrated by using heparin-Sepharose beads; immunoblotting with an anti-FGF2 polyclonal antibody. Recombinant FGF2 was used as a control. Neither protein is present in FGF1-FGF2 double-knockout mice. The migration positions of molecular mass standards (in kilodaltons) are shown at the left.

FIG. 2

FIG. 2

Matched coronal sections of brain tissue from wild-type (A, E, and I), FGF1 null (FGF1 KO) (B, F, and J), FGF2 null (FGF2 KO) (C, G, and K), and double-knockout (Double KO) (D, H, and L) mice were microscopically analyzed. Sections were stained for Nissl substance (A to D) or analyzed by immunohistochemical techniques using antibodies against parvalbumin (E to H) or calbindin (I to L). No differences between the brains of wild-type and FGF1−/− mice were noted. Brains of FGF2−/− mice show a thickening of the motor cortex (C) and a decrease in the number of parvalbumin (G)- and calbindin (K)-positive cells. No significant further thickening of the cortex (D) or decrease in neuronal subpopulations (H and L) was apparent in the brains of FGF1−/− FGF2−/− mice.

FIG. 3

FIG. 3

Ten mice of each genotype were wounded on day zero, and the degree of healing was assessed visually according to a semiquantitative scale (described in Materials and Methods). Wild-type (WT) and FGF1 null mice healed at a similar rate; FGF2 null and double-knockout mice healed more slowly than wild-type mice. Data were plotted as degree of healing over time (wound score) (A) and as percentage of completely healed animals over time (B). In both cases, there was no significant further impairment in the healing of double-knockout mice compared to that of FGF2 null mice.

FIG. 4

FIG. 4

(A) LTBMCs were prepared from wild-type, FGF1−/−, and FGF2−/− mice, and CFU-c were assayed at weekly intervals. No statistically significant differences between cultures derived from wild-type and FGF1−/− animals were noted, while cultures from FGF2−/− mice produced fewer myeloid progenitors. (B) LTBMCs from FGF1-FGF2 double-knockout (KO) mice exhibit diminished production of CFU-c compared to cultures from wild-type (WT) mice, but the degree of impairment is similar to that seen in cultures prepared from FGF2−/− mice. (C) LTBMCs were prepared from wild-type (WT) and FGF1-FGF2 double-knockout (DKO) mice. After the stromal layers became confluent, endogenous hematopoiesis was suppressed by irradiation. Cultures were subsequently recharged with either WT or DKO BMCs. WT stromal cells support hematopoiesis in both WT (WT on WT) and DKO (DKO on WT) BMC, while DKO stromal cells poorly support hematopoiesis in WT (WT on DKO) and DKO (DKO on DKO) BMCs.

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