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First published online 16 June 2004
doi: 10.1242/dev.01251

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Gremlin-mediated BMP antagonism induces the epithelial-mesenchymal feedback signaling controlling metanephric kidney and limb organogenesis

Odyssé Michos^1,2, Lia Panman², Kristina Vintersten^3,*, Konstantin Beier⁴, Rolf Zeller¹ and Aimée Zuniga^1,

¹ Developmental Genetics, Dept. of Clinical-Biological Sciences (DKBW), University of Basel Medical School, c/o Anatomy Institute, Pestalozzistrasse 20, CH-4056 Basel, Switzerland
² Department of Developmental Biology, Utrecht University, Padualaan 8, NL-3584CH Utrecht, The Netherlands
³ Transgenic Service, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
⁴ Department of Histology, Anatomy Institute, Pestalozzistrasse 20, CH-4056 Basel, Switzerland

View larger version (34K): [in a new window]   Fig. 1. Generation of a Grem1 loss-of function mutation by gene targeting. (A) The GreORF loss-of-function allele was generated by homologous recombination in ES cells. The entire ORF encoded by exon 2 (e2) was replaced with an IRES-lacZ gene and the NeoR cassette (flanked by loxP sites indicated by black triangles). Exon 1 (e1) is non-coding and located 8.5 kb upstream of exon 2 (UCSC). The 5' and 3' genomic probes used to screen ES-cell clones by Southern blotting are indicated by black and white boxes, respectively. Thin black lines indicate the sizes of the expected genomic bands detected by these probes. Arrowheads indicate the primers used to detect both wild-type (Wt) and mutant (GreORF) alleles. The relevant restriction enzyme sites are indicated as follows: B, BamHI; E, EcoRV; N, NsiI; Nd, NdeI; X, XbaI. (B,C) Analysis of wild-type (+/+) and correctly targeted heterozygous (+/–) ES-cell clones by Southern blotting using 5' and 3' genomic probes. (D) PCR genotyping of embryos of F2 littermate embryos. (E) Whole-mount in situ hybridization using GreORF/+ embryos at embryonic day 11.0 reveals the identical distribution of Grem1 and lacZ transcripts.

View larger version (104K): [in a new window]   Fig. 2. The Grem1 deficiency causes neonatal lethality because of complete bilateral renal agenesis and lung defects in combination with distal limb defects. (A,B) Urogenital defects in GreORF homozygous newborn mice. Mutant mice display complete bilateral agenesis of the kidney (ki) and ureter (ur). Note that gonads (go) and adrenal glands (ad) form correctly, while the bladder (bl) is not filled in mutant mice. (C-F) Lung defects in newborn Grem1-deficient mice. (C,D) Transversal histological sections through the lung of newborn wild-type mouse (C) and Grem1-deficient mouse (D) at the level of the heart. (E,F) High-magnification views of a longitudinal section through the lungs of a wild-type (E) and a Grem1-deficient (F) newborn mice. av, alveoli; bl, bronchiole. (G-J) Limb skeletal abnormalities in GreORF homozygous newborn mice; (G,H) forelimbs, (I,J) hindlimbs. Arrows indicate the zeugopod; arrowheads indicate metacarpal bones. Digit numbers are reduced and identities lost in GreORF/ORF limbs. Asterisk indicates a fused digit 1; question marks indicate posterior digits with unclear identities.

View larger version (76K): [in a new window]   Fig. 3. Grem1 is required for propagation of Shh in the limb bud mesenchyme and for FGF gene expression in the AER. (A) Shh expression in wild-type and Grem1 (GreORF/ORF)-deficient limb buds during E10.0 and E11.0. The Shh expression domain is activated but not propagated in mutant limb buds. Posterior is towards the bottom and distal towards the right. (B) Expression of Fgf8 is activated normally (E9.5), but the domain remains broader and patchy in mutant limb buds (arrowheads, E9.75). (C) Neither Fgf4 nor Fgf9 expression is activated in mutant limb buds (E10.25). All limb buds shown are fore limb buds. In B,C, posterior is towards the bottom and dorsal towards the left.

View larger version (75K): [in a new window]   Fig. 4. BMP signaling and expression in Grem1-deficient limb buds. (A) Expression of the BMP target Msx1 during early limb bud development (E9.75). (B) Expression of the BMP target Msx2 during limb bud development (E10.25). Note ectopic Msx2 expression in the distal to anterior limb bud mesenchyme of Grem1 (GreORF/ORF)-deficient embryos, which is indicative of enhanced BMP signaling. (C) Expression of Bmp2, Bmp4 and Bmp7 in the limb bud mesenchyme (E10.75). Mesenchymal Bmp2 expression is significantly reduced at this stage in Grem1-deficient limb buds, while Bmp4 and Bmp7 are maintained at normal levels. (D) Bmp2, Bmp4 and Bmp7 expression in the AER of wild-type and Grem1 mutant forelimb buds (E10.75). Expression of all three (Bmp2, Bmp4 and Bmp7) is lacking from the AER of mutant limb buds. (E) Scanning electronic microscopy analysis of wild-type and Grem1-deficient forelimb buds (E11.5). Note that the AER of mutant limb buds is poorly differentiated and the anteroposterior limb bud axis is shortened in comparison with the wild type. In D,E, posterior is towards the bottom and dorsal is towards the left. (F) High-power SEM revealing the morphology of AER-type ectodermal cells (outlined in red).

View larger version (73K): [in a new window]   Fig. 5. Grem1, but not SHH, rescues Fgf8 and Bmp2 expression in the AER of GreORF/ORF limb buds. All grafted limb buds are forelimb buds (E10.5) of Grem1 mutant embryos. Limb buds either received Shh (red arrow) or Grem1 (green arrow)-expressing cell aggregates and were cultured for 15 hours prior to analysis. White arrowheads indicate the endogenous expression domains; blue arrowheads and asterisks indicate the induced expression. (A) Bmp2 expression in a non-grafted control limb bud of a mutant embryo. (B) Posterior grafts of Shh-expressing cell aggregates fail to rescue Bmp2 expression. (C,D) Posterior grafts of Grem1-expressing cells induce Bmp2 expression in the AER (D), while no Bmp2 transcripts are detected in the AER of non-grafted mutant limb buds (C). Note also the enhancement of mesenchymal Bmp2 expression (D). (E,F) Posterior grafts of Shh-expressing cells do not rescue Fgf8 expression in the AER (F) in comparison with a non-grafted mutant limb buds (E). (G,H) Posterior grafts of Grem1-expressing cells induce upregulation of Fgf8 expression in the AER (H) in comparison with endogenous Fgf8 expression in non-grafted mutant limb buds (G). (A-D) Dorsal views with posterior towards the bottom and distal towards the right; (E-H) posterior is towards the bottom and dorsal towards the left.

View larger version (118K): [in a new window]   Fig. 6. Disruption of metanephric kidney morphogenesis. (A-C) Distribution of Grem1 transcripts during kidney morphogenesis. (A) Grem1 expression in the Wolffian duct and mesonephric tubules during E10.0. (B) Distribution of Grem1 transcripts during initiation of metanephric development (E11.0). The distribution of lacZ transcripts in a GreORF heterozygous embryonic kidney is shown. Note expression by both posterior Wolffian duct and metanephric mesenchyme. The broken line indicates the approximate position corresponding to the section shown in C. (C) In situ analysis on section reveals Grem1 expression locally in the metanephric mesenchyme surrounding the ureter tips. (D) Growth of the ureter and invasion of the metanephric mesenchyme in wild-type embryos occurs by E11.25. This growth and invasion results in upregulation of Pax2 expression in the induced mesenchyme in wild type, while it remains low in mutant mesenchyme. (E) In contrast to the wild type, Pax2 expression is lost from mutant mesenchyme by E12.5, while it remains similar to the wild type in the Wolffian duct. (F) In contrast to the wild type, Bmp2 fails to be expressed by the nephrogenic regions of mutant embryos. (G) In wild-type embryos, Bmp7 expression is induced to high levels within the condensing mesenchyme. This induction is completely disrupted in Grem1-deficient embryos. In D-G, only the metanephric region is shown. cm, condensing metanephric mesenchyme; gr, genital ridge; mm, metanephric mesenchyme; ub, ureteric bud; ur, ureter; wd, Wolffian duct.

View larger version (107K): [in a new window]   Fig. 7. Disruption of induction of ureter growth and RET/GDNF feedback signaling during metanephric organogenesis. (A) During onset of ureter growth (arrow) in wild-type embryos, Gdnf transcription is upregulated in the induced metanephric mesenchyme. (B) Ureter growth and Gdnf upregulation are not induced in mutant embryos (E11.25). (C) By E11.5, the ureter has branched once and expresses high levels of Ret (arrowheads) and Gdnf is maintained in the induced metanephric mesenchyme. (D) By contrast, ureter development is arrested and Gdnf expression lost in Grem1-deficient embryos. (E,F) Analysis of Ret and Gdnf expression on sections of E11.5 embryos confirms the disruption of RET/GDNF epithelial-mesenchymal feedback signaling in mutant embryos. cm, condensing mesenchyme; ub, ureteric bud; wd, Wolffian duct. (E,F) Transverse sections at the level of hind limb buds.

View larger version (100K): [in a new window]   Fig. 8. Grem1 is required for cell survival during both limb and kidney organogenesis. (A) TUNEL assay to reveal apoptotic cell death on histological sections. In the absence of Grem1, cells in core limb bud mesenchyme undergo massive cell death by E11.0. (B) lacZ transcripts are detected in E11.0 forelimb buds (whole mount) to follow the fate of cells normally expressing Grem1 in both heterozygous and homozygous mutant limb buds. Note that lacZ-expressing cells survive in GreORF/ORF limb buds. White arrowheads indicate the anterior and posterior domain boundaries. Forelimb buds in A,B are shown with ventral towards the bottom and distal towards the right. (C) Massive abnormal cell death is detected by TUNEL assay in the metanephric mesenchyme (mm) of Grem1 mutant kidneys by E11.5. Note that both wild-type and Grem1 mutant mesonephric mesenchyme (ms) undergoes normal apoptosis at this stage. (D) Pax2 expression in the nephrogenic tissue of a wild-type and GreORF/ORF embryo. The sections shown are adjacent to the ones shown in C. Pax2 expression fails to be upregulated in the metanephric mesenchyme of mutant kidneys, while expression in mesonephric mesenchyme (ms) is similar to Wt. (C,D) Ventral views, posterior towards the bottom.

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