Genetic analysis reveals an unexpected role of BMP7 in initiation of ureteric bud outgrowth in mouse embryos

Abstract

Background: Genetic analysis in the mouse revealed that GREMLIN1 (GREM1)-mediated antagonism of BMP4 is essential for ureteric epithelial branching as the disruption of ureteric bud outgrowth and renal agenesis in Grem1-deficient embryos is restored by additional inactivation of one Bmp4 allele. Another BMP ligand, BMP7, was shown to control the proliferative expansion of nephrogenic progenitors and its requirement for nephrogenesis can be genetically substituted by Bmp4. Therefore, we investigated whether BMP7 in turn also participates in inhibiting ureteric bud outgrowth during the initiation of metanephric kidney development.

Methodology/principal findings: Genetic inactivation of one Bmp7 allele in Grem1-deficient mouse embryos does not alleviate the bilateral renal agenesis, while complete inactivation of Bmp7 restores ureteric bud outgrowth and branching. In mouse embryos lacking both Grem1 and Bmp7, GDNF/WNT11 feedback signaling and the expression of the Etv4 target gene, which regulates formation of the invading ureteric bud tip, are restored. In contrast to the restoration of ureteric bud outgrowth and branching, nephrogenesis remains aberrant as revealed by the premature loss of Six2 expressing nephrogenic progenitor cells. Therefore, very few nephrons develop in kidneys lacking both Grem1 and Bmp7 and the resulting dysplastic phenotype is indistinguishable from the one of Bmp7-deficient mouse embryos.

Conclusions/significance: Our study reveals an unexpected inhibitory role of BMP7 during the onset of ureteric bud outgrowth. As BMP4, BMP7 and GREM1 are expressed in distinct mesenchymal and epithelial domains, the localized antagonistic interactions of GREM1 with BMPs could restrict and guide ureteric bud outgrowth and branching. The robustness and likely significant redundancy of the underlying signaling system is evidenced by the fact that global reduction of Bmp4 or inactivation of Bmp7 are both able to restore ureteric bud outgrowth and epithelial branching in Grem1-deficient mouse embryos.

Figure 1. Bmp4 and Bmp7 during initiation of ureteric bud outgrowth.

(A, B) Kidney rudiments were isolated at embryonic days E11.5 from pregnant females injected with tamoxifen at E8.75–E9.5 of gestation, which inactivates the remaining conditional Bmp4 allele within 24 hours following injection , i.e. prior to initiation of ureteric bud outgrowth (Bmp4 ^Δ/Δc, TMCre ^tg/+). The genotypes are indicated above the panels and Bmp4 ^Δ/f and Bmp4 ^Δ/+, TMCre ^tg/+ embryos are shown as wild-type controls. Note that all embryos (incl. controls) received tamoxifen. Pax2 expression (A) marks metanephric mesenchyme around the ureteric bud epithelia and the epithelia itself, while Ret expression (B) marks the tips of the invading ureteric buds. Asterisks mark the ectopic epithelial buds observed in 50% of all Bmp4 ^Δ/Δc, TMCre ^tg/+ kidney rudiments at E11.5 (n = 5/10). Note the developmental delay of kidney development in Bmp4 ^Δ/Δc, TMCre ^tg/+ embryos and the ectopic expression of Ret and Pax2. Ectopic buds were never observed in metanephric kidney rudiments isolated from Bmp4 ^Δ/f and Bmp4 ^Δ/+, TMCre ^tg/+ embryos. (C) In Grem1-deficient (Grem1 ^Δ/Δ) metanephric kidney rudiments, Bmp7 expression (black arrowheads) appeared initially normal (E10.5), but was rapidly lost from the metanephric mesenchyme (white arrowhead; E11.0) while expression remained in the developmentally arrested epithelium.

Figure 2. Additional genetic inactivation of Bmp7 in Grem1-deficient mouse embryos only partially restores metanephric kidney development.

Morphological analysis of kidneys of newborn mice (postnatal day P0) revealed the presence of two hypodysplastic kidneys in most cases in Grem1 ^Δ/Δ, Bmp7 ^Δ/Δ mice (see also Table 1). (A) Wild-type (+/+) urogenital system at birth (left panel). Histological analysis revealed the morphology of the ureter, an organized collecting duct system in the medulla and many glomeruli in the cortex (only some are indicated by black arrowheads). (B) In contrast, two small, hypodysplastic kidneys (white arrowheads) formed in Grem1 ^Δ/Δ, Bmp7 ^Δ/Δ mice (left panel). Histological analysis revealed the drastic reduction and disorganization of both cortex and medulla, while the ureter, indicative of ureteric epithelial branching was present. Black arrowheads point to the few glomeruli that formed, some of them located within the rudimentary medulla. (C) The hypodysplastic kidney phenotype of Bmp7-deficient mice (white arrowheads, left panel) , . Note the similarity of the kidney phenotypes of Bmp7 ^Δ/Δ and Grem1 ^Δ/Δ, Bmp7 ^Δ/Δ mice at both gross-morphological and histological levels. White arrowheads: hypodysplastic kidneys; Ad: adrenal glands; Pa: papilla; Ur: ureter; stippled boxes: enlargements shown in the right panels; black arrowheads: glomeruli.

Figure 3. Restoration of ureteric bud outgrowth, branching and GNDF/Wnt11 feedback signaling in Grem1 ^Δ/Δ, Bmp7 ^Δ/Δ embryos.

Comparative in situ hybridization analysis of Ret (A), Wnt11 (B), Gdnf (C), Etv4 (D) and Pax2 (E) expression in wild-type (Wt; +/+), Grem1-deficient, Grem1 ^Δ/Δ, Bmp7 ^Δ/Δ and Bmp7 ^Δ/Δ kidneys at embryonic day E11.5–11.75 (49–54 somites). (A, B) Ret and Wnt11 expression were absent from the arrested ureteric bud in Grem1 ^Δ/Δ embryos (Michos et al. 2004, 2007), but their expression was restored in kidney rudiments of Grem1 ^Δ/Δ, Bmp7 ^Δ/Δ embryos. (C, D) In addition, the expression of mesenchymal Gdnf and the transcriptional target Etv4 was propagated in Grem1 ^Δ/Δ, Bmp7 ^Δ/Δ embryos in contrast to their rapid loss from Grem1 ^Δ/Δ kidney rudiments. Note that the labeling of the Wolffian duct and ureteric epithelium by the Gdnf in situ hybridization probe is non-specific. (E) Pax2 expression was also restored in the metanephric mesenchyme of Grem1 ^Δ/Δ, Bmp7 ^Δ/Δ embryos, while its expression is lost from Grem1 ^Δ/Δ mutants concurrent with massive apoptosis .

Figure 4. The massive apoptosis in Grem1-deficient metanephric kidney rudiments is suppressed by the Bmp7 inactivation.

Apoptotic cells were detected by TUNEL (green fluorescence) on serial histological sections of kidney rudiments at E11.5. Concurrently, mitotic cells were revealed by detection of nuclear phospho-histone H3 proteins (pH 3, red fluorescence) and the overall cell density was assessed by staining nuclear DNA with Hoechst 33258 (blue fluorescence). Representative sections are shown for all genotypes. (A) Wild-type metanephros. Note the condensation of mesenchymal cells (blue nuclei) around the invading ureteric tips. (B) Grem1-deficient metanephros. Massive apoptosis was observed in both the mesenchyme and ureteric bud epithelium (green) and mesenchymal cells remained loose (blue). Only few mitotic cells (red) were detected. (C) Grem1 ^Δ/Δ, Bmp7 ^Δ/Δ metanephros. Cellular apoptosis in both compartments was almost completely suppressed and proliferation, mesenchymal condensation and branching of the ureteric epithelium were restored. Note that the apoptosis in the distal part of the branching ureteric epithelium remained increased. (D) The development of the Bmp7-deficient metanephros was comparable to wild-type controls at this developmental stage. Ub: ureteric bud epithelium; Mm: metanephric mesenchyme.

Figure 5. Differential loss of Six2 expressing progenitors from mutant metanephric kidneys.

Six2 expression was analyzed by whole-mount in situ hybridization during initiation (E11.5) and progression of nephrogenesis (E13.5 and E14.5). Representative pairs of developing metanephric kidneys are shown at the same magnifications for the different stages and genotypes to reveal the differences in size. (A) Wild-types. Only one of the two developing kidneys is shown at E14.5 due to size. (B) Grem1 deficiency. Six2 expression was lost by E13.5. At E13.5, the developing gonads became visible due to the renal agenesis. (C) Grem1 ^Δ/Δ, Bmp7 ^Δ/Δ (G1 ^Δ/Δ, B7 ^Δ/Δ) metanephric kidneys. Six2 expression was normal at E11.5, but lost during progression of nephrogenesis (E13.5–E14.5) as in Bmp7-deficient kidneys. (D) Bmp7-deficient metanephric kidneys.

Figure 6. Interactions of GREM1 with BMP7 and BMP4 during ureteric epithelial branching morphogenesis.

(A) GREM1-mediated reduction of overall BMP activity enables initiation of ureteric bud outgrowth. (B) GREM1-mediated BMP antagonism during the onset of epithelial branching morphogenesis. Bmp4 (red) is expressed by the tail bud derived mesenchyme (Tm) enveloping the Wolffian duct (Wd) and ureteric bud epithelia (Ub). Bmp7 (yellow) is expressed by the nascent mesenchyme (panel A), metanephric mesenchyme (Mm, panel B) and the Wolffian duct and ureteric bud epithelia (Figure 1C). Grem1 (green) is initially expressed by the mesenchyme surrounding the nascent ureteric bud, where it likely antagonizes both BMP4 and BMP7 activities (panel A). Subsequently, Grem1 is expressed by the metanephric mesenchyme surrounding the invading ureteric epithelial tips (panel B). The overlap of the Grem1 with the Bmp4 (panel A) and Bmp7 expression domains (panel A, B) is indicated by green stripes.