Abstract
The nephron progenitor cells (NPCs) give rise to all segments of functional nephrons and are of great interest due to their potential as a source for novel treatment strategies for kidney disease. Fibroblast growth factor (FGF) signal plays pivotal roles in generating and maintaining NPCs during kidney development. However, molecule(s) regulating FGF signal during nephron development is not known. Sprouty (SPRY) is an antagonist of receptor tyrosine kinases. During kidney development, SPRY1 is expressed in the ureteric buds (UBs) and regulates UB branching by antagonizing Ret-GDNF signal. Here, we provide evidence that SPRY1 expressed in NPCs modulates activity of FGF signal in NPCs and regulates NPC stemness. Haploinsufficiency of Spry1 rescues bilateral renal agenesis and premature NPC differentiation caused by loss of Fgf9 and Fgf20. In addition, haploinsufficiency of Spry1 rescues NPC proliferation and cell death defects induced by loss of Fgf9 and Fgf20. In the absence of SPRY1, FGF9 and FGF20, another FGF ligand, FGF8 promotes nephrogenesis. Deleting both Fgf8 and Fgf20 results in kidney agenesis and defects in NPC proliferation and cell death. Rescue of loss of Fgf9 and Fgf20 induced renal agenesis by Spry1 haploinsufficiency was reversed when one copy of Fgf8 was deleted. These findings indicate the importance of the balance between positive and negative signal during NPC maintenance. Failure of the balance may underlie some human congenital kidney malformation.
Significance Statement Nephrons are functional units of kidney to filter blood to excrete wastes and regulate osmolarity and ion concentrations. Nephrons are derived from nephron progenitors. Nephron progenitors are depleted during kidney development which makes it unable to regenerate nephrons. Therefore, understanding signaling molecules regulating nephron progenitor generation and maintenance is of great interest for the future kidney regenerative medicine. Here, we show that Sprouty1 regulates nephron progenitor maintenance by inhibiting FGF signal. Deletion of Sprouty1 rescues renal agenesis and nephron progenitor depletion in the Fgf9/20 loss-of-function kidneys. Further decrease of FGF signal by deleting one copy of Fgf8 makes kidneys irresponsive to Sprouty1 resulting in failure of nephron progenitor maintenance. This study thus identifies the reciprocal function of FGF-Sprouty1 signal during nephron progenitor development.
Introduction
Receptor tyrosine kinases (RTK) are activated upon binding of their cognate ligands and regulate many aspects of organogenesis. The intracellular signals initiated by RTK activation determine cellular behavior such as proliferation, survival, cell fate determination and morphogenesis. Mis-regulation of RTK activity leads to the onset and progression of a wide-range of disease such as diabetes, inflammation, bone disorders, atherosclerosis, angiogenesis and various cancers (1, 2). Therefore, tight regulation of the activity of RTK should be guaranteed during development and homeostasis (3).
RTK plays crucial role during kidney development. glial cell line-derived neurotrophic factor (GDNF) produced in the nephron progenitor cells (NPCs) activates RET/GFRα1 RTK/co-receptor in the ureteric bud (UB) tips (4). Deletion of Gdnf, Ret, or Gfra1 during development results in renal agenesis due to failure of UB induction (5–8). UB branching is sensitive to the level of GDNF since haploinsufficiency of GDNF or decreasing expression of GDNF due to loss of Fras1 produces unilateral renal agenesis or hypoplasia (9, 10). On the other hand, FGF9 and FGF20 activate FGFR1 and FGFR2 in NPCs and maintain the stemness of NPCs. Loss of FGF20 in human, loss of Fgf9 and Fgf20 in mice, or metanephric mesenchyme specific deletion of Fgfr1 and Fgfr2 causes bilateral renal agenesis (11–13). In addition, reduction in Fgf9 and Fgf20 levels results in loss of NPCs and premature differentiation indicating that stemness of NPCs are sensitive to the copy number of Fgf genes (12).
Sprouty (SPRY) is a negative feedback regulator of RTK signaling. During kidney development, SPRY negatively regulates activity of GDNF-RET signal. Either knock-out or antisense oligonucleotides treatment of Spry1 generates supernumerary and dilated UBs (14, 15). In addition, ectopic expression of SPRY2 in the developing kidney leads to renal hypoplasia and agenesis (16). Furthermore, deleting Spry1 restores kidneys in the Gdnf (or its expression level) or Ret knock-out mice (17–19). When both GDNF/RET and SPRY signal is depleted, FGF10 promotes UB branching (18). Although these studies point to the importance of SPRY1 in the UB branching, the function of SPRY1 in the NPCs in vivo remains to be determined.
Here, we show that SPRY1 regulates NPC maintenance by modulating FGF signal. Both germ line and NPC specific deletion of Spry1 rescue renal agenesis in Fgf9-/-;Fgf20-/- embryos. We also show that FGF8 is another FGF ligand promoting NPC maintenance. Combinatory gene deletion analyses show that balance of FGFs and Spry1 level is critical to maintain NPCs.
Results
Loss of Spry1 partially restores kidney phenotypes caused by loss of Fgf9 and Fgf20
To test whether Spry1 regulates Fgf9 and Fgf20 activity during kidney development, we generated Fgf9, Fgf20, and Spry1 triple compound mutants. In this study, we used Fgf20-/- embryos as controls. Previously, we identified that Fgf20-/- embryos produce 15% smaller kidneys than Fgf20-/+ with decreased number of nephrons (12). Kidney size of Fgf20-/- and Fgf20-/-;Spry1-/+ embryos were comparable at E18.5 (100.3±8.0% vs. 101.3±9.7%, n=12, ns, Figs. 1A, B, Q, R). Histological morphology appears normal in both genotypes (Figs. 1G and H). In Fgf9-/+;Fgf20-/- embryos, 10 out of 18 kidneys were hypoplastic and 1 kidney was aplastic and kidney size was decreased to 61.8±30.1% compared to Fgf20-/- (p<0.001) (Figs. 1C, Q, R). Fgf9-/+;Fgf20-/- kidneys were also cystic (Fig. 1I). In Fgf9-/+;Fgf20-/-;Spry1-/+ embryos, 5 out of 18 kidneys were hypoplastic and kidney size was significantly restored compared to Fgf9-/+;Fgf20-/- embryos (87.4±15.0%, p<0.005 compared to Fgf9-/+;Fgf20-/- Figs. 1D, Q, R). Histologically, Fgf9-/+:Fgf20-/-;Spry1-/+ kidneys were comparable to controls (Fig. 1J). However, kidney size was not restored back to control size (p<0.05 compared to Fgf20-/-)(Fig. 1R). All kidneys in Fgf9-/-;Fgf20-/- embryos were missing (n=8) (Figs. 1E and Q). Interestingly, all Fgf9-/-;Fgf20-/-;Spry1-/+ embryos generated kidneys. Among 14 kidneys analyzed, 4 kidneys were normal and 8 kidneys were hypoplastic (Figs. 1F and Q). Size of the kidneys in Fgf9-/-;Fgf20-/-;Spry1-/+ embryos was 64±18.9% compared to controls, which is significantly increased compared to in Fgf9-/-;Fgf20-/- embryos (p<0.005) and comparable to in Fgf9-/+;Fgf20-/- embryos (p>0.75) (Fig. 1R). Histological analysis showed that kidney structures were comparable to controls (Fig. 1K). Nephron progenitors were repopulated in Fgf9-/+;Fgf20-/-;Spry1-/+ and Fgf9-/-;Fgf20-/-;Spry1-/+ kidneys compared to Fgf9-/+;Fgf20-/-;Spry1-/+ and Fgf9-/-;Fgf20-/-;Spry1-/+, respectively (Figs. 1L-P’). These data indicate that deleting one copy of Spry1 partially restores nephrogenesis caused by loss of Fgf9 and Fgf20.
Spry1 is reported to be expressed in both UBs and NPCs during kidney development (14, 15). We observed that Spry1 was expressed in both UBs and NPCs at E11.5 and its expression was decreased in NPCs at E13.5 and E14.5 kidneys (Fig. S1). Spry1 expressed in the UBs negatively regulates GDNF-RET induced UB branch morphogenesis (17, 18). We hypothesized that Spry1 expressed in the NPCs antagonizes FGF9 and FGF20 induced NPC maintenance. To investigate this, we conditionally deleted Spry1 in NPCs using Fgf20Cre knock-in mouse line, which serves both loss of Fgf20 and Cre driver of NPC (12). Cre recombinase in Fgf20Cre was active only in NPCs and their derivatives (Fig. S2).
Kidney size of Fgf20Cre/- and Fgf20Cre/-;Spry1fl/+ embryos were comparable at E18.5 (99.7±14.0% vs. 99.0±10.5%, n=12, ns, Figs. 2A, B, G, H). In Fgf9-/+;Fgf20Cre/- embryos, 3 out of 16 kidneys were hypoplastic and 2 kidney was aplastic and kidney size was decreased to 75.7±32.1% compared to Fgf20Cre/- (p<0.05) (Figs. 2C, G, H). In Fgf9-/+;Fgf20Cre/-;Spry1fl/+ embryos, 2 out of 18 kidneys were hypoplastic and kidney size was 87.6±22.9% compared to Fgf20Cre/- (p>0.22) (Figs. 2D, G, H). All kidneys in Fgf9-/-;Fgf20Cre/- embryos were missing (n=12) (Figs. 2E and G). 6 out of 8 kidneys in Fgf9-/-;Fgf20Cre/-;Spry1fl/+ embryos generated kidneys. Among 8 kidneys analyzed, 2 kidneys were normal, 4 kidneys were hypoplastic, and 2 kidneys were missing (Figs. 2F and G). Size of the kidneys in Fgf9-/-;Fgf20cre/-;Spry1fl/+ embryos was 56.2±41.6% compared to Fgf20Cre/-, which is significantly increased compared to in Fgf9-/-;Fgf20-/- embryos (p<0.005) (Fig 2H). Together, these data indicate that phenotypic rescue of NPC specific Spry1 deletion recapitulates whole body deletion of Spry1 suggesting SPRY1 in NPCs antagonizes activity of FGF9 and FGF20 to regulate kidney development.
FGF9 and FGF20 are required for nephron progenitor survival and proliferation
Previously, we identified that loss of Fgf9 and Fgf20 resulted in NPC cell death (12). To further analyze their roles during kidney development, we performed proliferation and cell death analyses of NPCs at the time of NPC induction. At E10.5, number (67.4±15.3, 54.2±3.9, 61.5±11.2, 58.8±9.8 in Fgf9-/+;Fgf20-/+, Fgf9-/-;Fgf20-/+, Fgf9-/-;Fgf20-/-, Fgf9-/-;Fgf20-/-, respectively, n=4, ns), proliferation rate (22.5±7.4, 18.5±4.4, 20.5±4.5, 17.1±1.1, n=4, ns), and cell death rate (0.0±0.0, 0.0±0.0, 0.2±0.2, 0.5±0.6, n=4, ns) of Six2+ NPCs were comparable to all genotypes (Fig. S3). At E11.5, number of NPCs were comparable in Fgf9-/+;Fgf20-/+, Fgf9-/-;Fgf20-/+, Fgf9+/-;Fgf20-/- embryos (70.9±8.9, 67.3±7.2, 74.4±2.5, respectively, n=3, ns)(Figs. S4A-C and M). However, number of NPCs in Fgf9-/-;Fgf20-/- embryos (38.4±7.6, n=3, p<0.01) was significantly decreased compared to Fgf9-/+;Fgf20-/+ embryos (Figs. S4D and M). Proliferation rate of NPCs in Fgf9-/-;Fgf20-/- embryos (19.5±1.6, n=3, p<0.01) was also significantly decreased compared to Fgf9-/+;Fgf20-/+ embryos (27.9±2.2, n=3) (Figs. S4H and N). Proliferation rates of NPCs in Fgf9-/-;Fgf20-/+ (28.6±1.9, n=3) and Fgf9-/+;Fgf20-/- (27.9±1.4, n=3) embryos were comparable to Fgf9-/+;Fgf20-/+ embryos (Figs. 4E-G and N). Cell death rates of NPCs in Fgf9-/+;Fgf20-/+ and Fgf9-/-;Fgf20-/+ embryos were comparable (0.3±0.3, 0.7±0.6, n=3, respectively, ns) (Figs. S4I, J, O). Cell death rate of NPCs in Fgf9-/+;Fgf20-/- (2.4±0.8, n=3 p<0.05) embryos was increased compared to Fgf9-/+;Fgf20-/+ embryos (Figs. S4K and O). Cell death rate of NPCs was further increased in Fgf9-/-;Fgf20-/- (8.5±3.0, n=3), p<0.01) embryos compared to Fgf9-/+;Fgf20-/+ (p<0.01) and Fgf9-/+;Fgf20-/- (p<0.05) embryos (Figs. S4L and O).
Fgf9 and Fgf20 regulate ureteric bud branching
We also investigated whether FGF9 and FGF20 regulate genes required for UB branching. At E10.5, Ret was expressed in UBs and its expression was comparable to all the genotypes (Figs. S5A-D). At E11.5, Ret+ UBs were bifurcated in Fgf9-/+;Fgf20-/+ and Fgf9-/-;Fgf20-/+ embryos (Figs. S5E and F). However, in Fgf9-/+;Fgf20-/- embryos, UBs were not yet bifurcated (Fig. S5G) and in Fgf9-/-;Fgf20-/- embryos, UBs started to regress (Fig. S5H). Gdnf was highly expressed in NPCs of Fgf9-/+;Fgf20-/+ and Fgf9-/-;Fgf20-/+ embryos (Figs. SI and J). However, expression of Gdnf was decreased in Fgf9-/+;Fgf20-/- embryos (Fig. S5K) and diminished in Fgf9-/-;Fgf20-/- embryos (Fig. S5L). Etv4 and Etv5 are transcription factors regulated by GDNF/RET during UB branching and responsible for UB branching (18, 20–22). Therefore, we tested whether decrease of Gdnf affect expression of Etv4 and Etv5. At E11.5, expression of Etv4 and Etv5 were significantly decreased in Fgf9-/+;Fgf20-/- embryos and diminished in Fgf9-/-;Fgf20-/- embryos (Figs. S5M-S). Together, these data indicate that Fgf9 and Fgf20 are required for UB branching.
Reduction of Spry1 level restores nephron progenitor proliferation and survival in Fgf9 and Fgf20 double mutant kidneys
Next, we investigated whether deleting one copy of Spry1 rescues NPC number, proliferation and cell death defects caused by loss of Fgf9 and Fgf20. Number of NPCs were comparable in both Fgf20-/- and Fgf20-/-;Spry1-/+ embryos (63.3±5.7, 60.8±9.4, n=3, respectively, ns) (Figs. 3A, D, G). Number of NPCs in Fgf9-/+;Fgf20-/-;Spry1-/+ embryos was increased compared to that of Fgf9-/+;Fgf20-/- embryos (44.6±12.0, 60.0±4.8, n=5, respectively, p<0.05) (Figs. 3C, D, G). Also, Number of NPCs in Fgf9-/-;Fgf20-/-;Spry1-/+ embryos was increased compared to that of Fgf9-/-;Fgf20-/- embryos (24.9±8.6, 54.0±2.0, n=5, n=4 respectively, p<0.001) (Figs. 3E, F, G). Of note, number of NPCs in Fgf9-/+;Fgf20-/-;Spry1-/+ and Fgf9-/-;Fgf20-/-;Spry1-/+ embryos was restored close to that of Fgf20-/- embryos (p>0.5). Proliferation indexes were comparable to both Fgf20-/- and Fgf20-/-;Spry1-/+ embryos (21.9±4.7, 23.2±4.0, n=3, respectively, ns) (Figs. 3H, I, N). Proliferation index in Fgf9-/+;Fgf20-/-;Spry1-/+ embryos was increased compared to that of Fgf9-/+;Fgf20-/- embryos (13.7±4.6, 21.4±3.8, n=5, respectively, p<0.05) (Figs. 3J, K, N). Also, proliferation index in Fgf9-/-;Fgf20-/-;Spry1-/+ embryos was increased compared to that of Fgf9-/-;Fgf20-/- embryos (8.7±4.1, 21.0±1.2, n=5, n=4 respectively, p<0.001) (Fig. 3 L, M, N). Of note, Proliferation indexes in Fgf9-/+;Fgf20-/-;Spry1-/+ and Fgf9-/-;Fgf20-/-;Spry1-/+ embryos was restored close to that of Fgf20-/- embryos (p>0.5). Cell death indexes were comparable to both Fgf20-/- and Fgf20-/-;Spry1-/+ embryos (1.3±0.9, 1.0±0.4, n=4, respectively, ns) (Figs. 3O, P, U). Cell death index in Fgf9-/+;Fgf20-/-;Spry1-/+ embryos was increased compared to that of Fgf9-/+;Fgf20-/- embryos (2.7±1.2, 1.1±0.3, n=4, respectively, p<0.05) (Figs. 3Q, R, U). Also, cell death index in Fgf9-/-;Fgf20-/-;Spry1-/+ embryos was increased compared to that of Fgf9-/-;Fgf20-/- embryos (9.5±2.5, 2.6±1.8, n=4 respectively, p<0.001) (Figs. 3S, T, U). Together these data indicate that SPRY1 negatively regulates FGF9 and FGF20-dependent NPC survival and cell death.
FGF8 functions together with FGF20 to regulate nephron progenitor maintenance
FGF8 is expressed in the NPCs and required for NPC survival and tubulogenesis at E14.5 (23–26). Since FGF8 is also required for NPC survival, we hypothesize that FGF8 functions together with FGF9 and FGF20 to maintain NPCs. To investigate this hypothesis, we deleted Fgf8 in the NPC together with Fgf20 using Fgf20Cre mouse line and analyzed kidneys. Kidneys in Fgf8fl/-;Fgf20Cre/+ and Fgf8fl/+;Fgf20Cre/- embryos were smaller than those of Fgf8fl/+;Fgf20Cre/+ embryos (Figs. 4A, B, C). Fgf8fl/-;Fgf20Cre/- embryos showed bilateral kidney agenesis (Fig. 4D). Kidneys of Fgf8fl/-;Fgf20Cre/+ contained no nephrons which is consistent with previous publication (Fig. 4F) (23, 24). Fgf8fl/+;Fgf20Cre/- kidneys had less nephrons compared to Fgf8fl/+;Fgf20Cre/+ embryos (Figs. 4E and G). UBs had less branching in both E14.5 Fgf8fl/-;Fgf20Cre/+ and Fgf8fl/+;Fgf20Cre/- kidneys as notified by Wnt9b staining, the marker of UB (27) (Figs. S6A-C). Wnt4 expression was diminished in the Fgf8fl/-;Fgf20Cre/+ kidneys which is consistent with previous publication (Figs. 6E) (23, 24). However, Wnt4 expression in the Fgf8fl/+;Fgf20Cre/- kidneys was comparable to Fgf8fl/+;Fgf20Cre/+ (Figs. 6D and F) indicating that Fgf20 does not regulate Wnt4 expression.
In NPC maintenance, Six2+ NPCs were decreased in E18.5 kidneys of Fgf8fl/-;Fgf20Cre/+ and Fgf8fl/+;Fgf20Cre/- embryos compared to controls (Figs. 4H-J’). At E11.5, number of NPCs were comparable in Fgf8fl/+;Fgf20Cre/+, Fgf8fl/-;Fgf20Cre/+, Fgf9fl/+;Fgf20Cre/- embryos (61.8±5.5, 65.1±14.7, 55.9±3.0, respectively, n=4, 5, 5, respectively, ns)(Figs. 4K-M and O). However, number of NPCs in Fgf8fl/-;Fgf20Cre/- embryos (34.8±10.2, n=5, p<0.05) was significantly decreased compared to Fgf8fl/+;Fgf20Cre/+ embryos (Figs. 4N and O). Proliferation rates of NPCs in Fgf8fl/-;Fgf20Cre/+, Fgf8fl/+;Fgf20Cre/-, and Fgf8fl/-;Fgf20Cre/- embryos (13.3±1.4, p<0.01, 17.0±5.5, p<0.05, 10.8±4.0, p<0.01, n=5, respectively) were significantly decreased compared to Fgf9-/+;Fgf20-/+ embryos (26.1±3.2, n=4) (Figs. 4P-S and T). Cell death rate of NPCs in Fgf8fl/-;Fgf20Cre/- embryos were comparable (0.1±0.1, ns) compared to controls (0.0±0.0) (Figs. 4U, V, and Y). Cell death rate of NPCs in Fgf8fl/+;Fgf20Cre embryos (1.1±1.0, n=5 p<0.05) embryos was increased (Figs. 4W and Y). Cell death rate of NPCs was further increased in Fgf8fl/-;Fgf20Cre/- (7.4±0.7, n=5, p<0.01) embryos compared to controls (p<0.01) and Fgf8fl/+;Fgf20Cre/- (p<0.01) embryos (Figs. 4X and Y). In addition, similar to Fgf9-/-;Fgf20-/- (12), Pax2 expression in the NPCs were decreased in the Fgf8fl/-;Fgf20Cre/- embryos (Fig. S7A-D).
We also analyzed early UB branching. At E11, Ret was expressed in UBs and its expression was comparable to all the genotypes suggesting UB induction is not affected (Figs. S7E-H). At E11.5, Ret+ UBs were bifurcated in Fgf8fl/+;Fgf20Cre/+ embryo (Fig. S7J).
However, in Fgf8fl/-;Fgf20Cre/+, Fgf8fl/+;Fgf20Cre/-, and Fgf8fl/-;Fgf20Cre/- embryos, UBs were not yet bifurcated (Figs. S7J-L). Gdnf expression was comparable to Fgf8fl/+;Fgf20Cre/+ and Fgf8fl/-;Fgf20Cre/+ embryos (Figs. S7Q and R). However, expression of Gdnf was decreased in Fgf8fl/+;Fgf20Cre/- embryos (Fig. S7O) and diminished in Fgf8fl/-;Fgf20Cre/- embryos (Fig. S7P). In addition, Etv4 and Etv5 were diminished in Fgf8fl/-;Fgf20Cre/- embryos (Figs. S7Q-X). Together, these data indicate that Fgf8, similar to Fgf9, functions together with Fgf20 to regulate kidney development.
Spry1 haploinsufficiency does not rescue kidney defects of Fgf8 and Fgf20 double knock-out
Next, we questioned whether loss of Spry1 also rescue renal phenotypes caused by loss of Fgf8 and Fgf20. Therefore, we generated Spry1, Fgf8, Fgf20 compound mutants and analyzed kidneys. In Fgf8fl/-;Fgf20Cre/- embryos, among 16 kidneys, 2 very small kidneys were observed and 14 kidneys were missing (Figs. S8A and C). In Fgf8fl/-;Fgf20Cre/-;Spry1-/+ embryos, 2 out of 10 kidneys were severely hypoplastic and 8 kidneys were missing (Figs. S8B and C). In addition, NPC number (34.8±10.2 in Fgf8fl/-;Fgf20Cre/-, n=5, and 35.4±1.7 in Fgf8fl/-;Fgf20Cre/-;Spry1-/+, n=3, ns), proliferation rate (10.8±4.0 in Fgf8fl/-;Fgf20Cre/-, n=5, and 10.3±2.3 in Fgf8fl/-;Fgf20Cre/-;Spry1-/+, n=3, ns), and cell death index (7.3±0.7 in Fgf8fl/-;Fgf20Cre/-, n=5, and 5.9±2.2 in Fgf8fl/-;Fgf20Cre/-;Spry1-/+, n=3, ns) were not rescued in the Fgf8fl/-;Fgf20Cre/-;Spry1-/+ embryos compared to Fgf8fl/-;Fgf20Cre/- embryos (Figs. S8D-F).
SPRY1 antagonizes FGFs in dose dependent manner
We identified that FGF8, FGF9, and FGF20 function together to regulate NPC maintenance. We also identified that deletion of Spry1 did not rescue renal phenotypes caused by loss of both Fgf8 and Fgf20. These results suggest that antagonism of SPRY1 to the FGF signal is limited by Fgf genes copy number. To investigate this, we generated Fgf8, Fgf9, Fgf20, and Spry1 compound mutants and analyzed kidneys. In Fgf8fl/+;Fgf9-/+;Fgf20Cre/- embryos, 1 out of 10 kidney was normal, 6 were hypoplastic and 3 were missing (Figs. 5A and E). Kidney size was 34.8±34.3 (p<0.001) compared to Fgf8fl/+;Fgf9-/+;Fgf20Cre/-Spry1-/+ embryos (Fig. 5F). In Fgf8fl/+;Fgf9-/+;Fgf20Cre/-Spry1-/+ embryos, all kidneys (10 out of 10) were increased in size compared to Fgf8fl/+;Fgf9-/+;Fgf20Cre/- (Figs. 5B, E, F). All the kidneys (4 out of 4) in Fgf8fl/+;Fgf9-/-;Fgf20Cre/- embryos were missing (Figs. 5C, E, F). Fgf8fl/+;Fgf9-/-;Fgf20Cre/-;Spry1-/+ embryos also lost all kidneys (Figs. 5C, D, E, F). These data indicate that rescue of kidney phenotypes in the haploinsufficiency of Spry1 is depends on the dosage of gene copy numbers of Fgfs.
Discussion
SPRY was originally identified to regulate development of Drosophila trachea through antagonizing branchless and breathless, which are orthologs of mammalian FGF and FGFR, respectively (28). The mammalian SPRYs antagonize FGF signaling in many developing organs including lung, mandible, external genitalia, long bone, auditory, and tooth (29–34). However, in kidney development, SPRY1 seems to function mainly as an antagonist of Ret-GDNF dependent UB branching. (15, 17). Of note, Brown et al, showed that ectopic expression of SPRY1 in NPCs resulted in loss of NPC due to NPC cell death (35), suggesting a possible role of SPRY1 in NPCs. Indeed, we present data indicating that haploinsufficiency of Spry1 partially rescued renal phenotypes due to loss of Fgf9 and Fgf20 during NPC maintenance. NPC cell death, proliferation defect, and premature depletion was rescued. NPC specific Spry1 deletion also rescued the Fgf9 and Fgf20 loss of function deletion indicating antagonistic function of Spry1 is in a cell autonomous manner within NPCs.
FGF8 also functions together with FGF20 to maintain NPCs. At E11.5 Fgf8 is expressed in the NPCs and its expression is restricted to pre-tubular aggregate (PTA) and renal vesicle as the embryo develops (23, 24). Previous studies indicate that Fgf8 is required for NPC and PTA survival at later stage of development (E14.5 and later) but dispensable for NPC proliferation (23, 24). Interestingly, in this study, we identified that at early developmental stage (E11.5), proliferation of NPCs was decreased in all Fgf8, Fgf20 compound mutants (Fgf8-/fl;Fgf20Cre/+, Fgf8-/+;Fgf20Cre/-, and Fgf8-/fl;Fgf20Cre/-). And NPC cell death was mostly pronounced in the Fgf8 and Fgf20 double mutant kidneys. Fgf20 does not seem to function together with Fgf8 to maintain PTA, as PTA marker (Wnt4) was not changed in the Fgf8fl/+;Fgf20-/- kidneys. Different from Fgf9 and Fgf20, deleting one copy of Spry1 did not rescue renal phenotypes caused by loss of Fgf8 and Fgf20. No kidneys developed in Fgf8, Fgf20, and Spry1 compound mutant (Fgf8fl/-;Fgf20Cre/-;Spry1-/+). Also, no rescue of NPC proliferation and cell death occurs. More intriguingly, antagonism of SPRY1 to FGFs is dose dependent as haploinsufficiency of Spry1 rescues renal agenesis in Fgf8fl/+;Fgf9-/+;Fgf20-/- but not in the Fgf8fl/+;Fgf9-/-;Fgf20Cre/-. Based on this, we propose a model (Fig. 5G) of FGFs and SPRY1 functions for NPC maintenance during kidney development. FGF8, FGF9, and FGF20 bind to FGFR1 and FGFR2 in the NPCs to activate downstream signaling pathways, including RAS-MAPK and PI3K-AKT, that supports NPC proliferation, survival and stemness (35, 36). Upon activation of FGF signaling, Spry1 is upregulated and fine-tune FGF signal by antagonizing RAS-MAPK and PI3K-AKT signaling cascade (37).
Another interesting observation is that Gdnf, Etv4 and Etv5 expression were lost in both Fgf9/20 and Fgf8/20 double mutants. As GDNF is critical activator of RET for UB branching morphogenesis and ETV4 and ETV5 are downstream effector of RET for UB branching (20), it would be interesting to investigate whether lowering FGF signal in both NPC and UB causes renal defects. FGF10 functions to promote UB branching in the absence of Ret-GDNF/SPRY1 (18). However, Fgf10 null embryos has mild effect of UB branching generating small kidneys (38). Deleting both Fgf10 and Fgf20 would give an insight for the combination FGF function in both NPC and UB.
In conclusion, fine-tuning of FGF signaling is required for proper NPC maintenance and number of nephrons. In recent advent of kidney organoids, many protocols use FGF9 to promote NPC from intermediate mesoderm using both mouse and human embryonic stem cells (39–42). This information would provide importance of fine-tuning FGF signal during kidney development and may be used to make better organoid protocol.
Materials and Methods
Mice
Spry1-/+ (15), Spry1fl/+ (MMRRC:029870) (15), Fgf8fl/+ (43), Fgf8-/+ (23), Fgf9-/+ (44), Fgf20-/+ (45), Fgf20Cre/+ (46), RosaTdTomato/+ (Jax#007905) were used. All the mice were maintained in the University of Nebraska Medical Center animal facility according to animal care regulations, and the Animal Care and Use Committee (protocol number 16-005-02-EP).
Supporting Materials and Methods
Histology
For histological analysis, E18.5 kidneys were dissected from embryos, fixed overnight with 4% paraformaldehyde (PFA) overnight at 4°C, and dehydrated with ethanol gradients. Dehydrated kidneys were embedded in paraffin and sectioned. The paraffin embedded sections were de-paraffinized, hydrated and stained with hematoxylin and eosin. Stained slides were dehydrated, mounted, and photographed with Zeiss microscope.
Immunohistochemistry
E10.5 and 11.5 embryos were incubated with 30% sucrose solution. For E18.5 and P0 samples, kidneys were isolated from embryos and incubated with series of sucrose solutions (10%, 20%, and 30%). Samples were frozen sectioned and stored at −80°C for storage. For antibody staining, sections were washed with PBST (PBS + 0.5% Triton-X 100) for 30 min at RT and incubated with blocking solution (5% donkey serum, in PBST) for 1hr at RT. Sections were incubated with primary antibodies in PBST with 1% donkey serum overnight at 4°C. Sections were washed 3X with PBS and incubated with secondary antibodies for 30min at RT. After washing 3X with PBS, slides were mounted with vectorshield (Vector labs). Images were acquired with Zeiss Axioimage Z2 equipped with ApoTome. Antibodies used were Six2 (Proteintech 1:500), Cytokeratin-8 (DSHB, 1:40), Biotylated-DBA (Vector labs 1:500) FoxD1 (Santa Cruz, 1:200), and Pax2 (Covance, 1:200). Secondary antibodies conjugated with Alexa488 and Alexa555 (Molecular Probes 1:500) were used.
EdU and TUNEL staining
For EdU pulse-labeling, moms containing E10.5 and E11.5 were injected (xxx mg/mg). 2hrs after injection, moms were sacrificed and embryos were collected and processed for frozen section. Staining of EdU was followed by Click-iT® EdU imaging kit protocol (Invitrogen, C10338). TUNEL staining was performed according to the manufacturer’s recommendation (Roche, In situ Cell Death Dection Kit).
In situ hybridization
For sectioning in situ hybridization, paraffinized slides were hydrated with Diethylpyrocarbonate (DEPC) treated ethanol series and water, washed with hybridization solution, and incubated overnight with digoxigenin-labeled Spry1 probe. After washing, samples were incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche) and the color reaction was performed using alkaline phosphate substrate (Roche). For whole mount in situ hybridization, embryos were dissected in DEPC treated PBS and fixed with 4% PFA. After washing, samples were dehydrated with methanol series and stored until usage. Probes used for these studies were Wnt9b, Wnt4, Pax2, Ret, Gdnf, Etv4, and Etv5.
Statistical analysis
GraphPad Prism was used to perform a Welch’s t-test or a non-parametric Kruskal-Wallis test with Dunn’s test compensating for multiple comparisons where appropriate. A value of P < 0.05 was considered statistically significant. For comparative analysis of kidney sizes, kidney perimeter was measured and individual measurements were used for statistical analysis. Three or more animals from at least two independent experiments were examined.
Supporting Figure Legends
Acknowledgements
We thank Dr. Gail Marin for the Fgf8fl, Fgf8-, and Spry1- mice. This work was supported by the NIH P30 DK074038 (S.H). We are grateful to Dr. Kameswaran Surendran (Sanford Research) for helpful discussion.