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The Bone morphogenetic proteins (BMPs) mediate a wide range of diverse cellular behaviors throughout development. Previous studies implicated an important role for BMP signaling during the differentiation of the definitive mammalian kidney, the metanephros. In order to examine whether BMP signaling also plays an important role during the patterning of earlier renal systems, we examined the development of the earliest nephric system, the pronephros. Using the amphibian model system Xenopus laevis, in combination with reagents designed to inhibit BMP signaling during specific stages of nephric development, we revealed an evolutionarily conserved role for this signaling pathway during renal morphogenesis. Our results demonstrate that conditional BMP inhibition after specification of the pronephric anlagen is completed, but prior to the onset of morphogenesis and differentiation of renal tissues, results in the severe malformation of both the pronephric duct and tubules. Importantly, the effects of BMP signaling on the developing nephron during this developmental window are specific, only affecting the developing duct and tubules, but not the glomus. These data, combined with previous studies examining metanephric development in mice, provide further support that BMP functions to mediate morphogenesis of the specified renal field during vertebrate embryogenesis. Specifically, BMP signaling is required for the differentiation of two types of nephric structures, the pronephric tubules and duct.
Figure 1. Stage-dependent ablation of the pronephric anlage by Delta Smad7-mediated BMP inhibition. mRNAs encoding the Delta Smad7tevGR construct (300-400 pg) and the TEV2GR construct (10 pg) were injected into a single ventralblastomere at the 4-cell stage. The injected side of the embryo was identified via the presence of the lineage marker beta -galactosidase (pink). A,C: Anterior, uninjected side of embryos facing right. B,D: Anterior-injected side of embryos facing left. After injection, dexamethasone was added to the culture medium at stage 14, which is after the completion of renal field specification. The effects of stage-specific BMP inhibition on the development of the renal field were examined via in situ hybridization between stages 22-25 using the pronephric tubule and duct anlage markers (A,B) Xlim-1 and (C,D) Xpax-8. Following conditional inhibition of BMP signaling, expression of both genes was markedly reduced or ablated (B,D; black arrows) when compared to the uninjected-control side of the embryo (A,C; green arrows). Specifically, reduction or ablation of Xlim-1 expression was observed in 72% of injected embryos (n = 160), and reduction or ablation of Xpax-8 expression was observed in 67% of injected embryos.
Figure 2. Effects of Delta Smad7-mediated stage-dependent BMP inhibition on pronephric tubule and duct patterning and differentiation. A-F, H-K: Tadpoles are oriented laterally with dorsal at the top of all panels. Percent indicates the percentage of injected, DEX-treated embryos in which decreased expression of the examined marker was observed. Green arrows denote normal pronephric tubule or ducttissue on uninjected side of embryos. Black arrows mark region on the injected side of the embryo where the renal tissue should be located. Embryos were injected with Delta Smad7tevGR (300-400 pg) and the TEV2GR (10 pg) mRNA and cultured in the presence of DEX from stage 14 until they were harvested for analysis by in situ hybridization or immunohistochemistry. To analyze tubule patterning and differentiation, injected embryos were harvested between stages 32-39 to evaluate early markers, or between stages 38-42 to evaluate markers of terminal differentiation. beta -galactosidase (pink or light blue) was used as a lineage tracer. BMP inhibition resulted in reduction in expression of the general markers of tubule differentiation SMP-30 (B) expression on the injected side of tadpoles when compared to uninjected side (A). This is confirmed by analysis of later markers of tubule differences: XSGLT1K (D) and NKCC-2 (F). Expression of each of these later markers of tubule differentiation is dramatically reduced on the injected side following regulated inhibition of BMP signaling. The reduced expression of NKCC-2 was further examined in section (G, section at line in E, F). Pronephric duct development was examined via immunohistochemistry using renal duct antibodies Na,K-ATPase alpha 5 (H, I), and 4A6 (J, K). Stage-dependent inhibition of BMP signaling resulted in a dramatic reduction of all duct markers examined (black arrows indicate location of missing pronephric duct in I, K), when compared to the uninjected control side of the embryo (green arrows in H, J).
Figure 3. Stage-dependent BMP inhibition in the developing pronephros does not alter expression of glomal-specific genes. Embryos were co-injected with 300-400 pg Delta Smad7tevGR, 10 pg TEV2GR, and beta -galactosidase, and cultured in the presence of DEX starting at stage 14. The effect of BMP inhibition in the pronephric glomus was assessed by in situ hybridization to evaluate expression of the glomal markers WT-1 (A-C), nephrin (D,E), and neph1 (F,G). No observable difference in glomal marker expression between the injected and uninjected control side was observed following regulated inhibition of BMP signaling (A-G). C: Because the glomus is located more medially and is thus more difficult to unequivocally evaluate in whole mount, we examined glomal marker expression in section. Following in situ hybridization, embryos were embedded in JB-4 plastic and sectioned transversely into 30- mu M sections (at the line in B). C shows a representative embryo exhibiting similar levels of WT-1 expression on both the injected and uninjected sides of the tadpole. Nt, neural tube; nc, notochord. Percent indicates the percentage of injected, DEX-treated embryos in which the reported phenotype was observed.
Figure 5. Perturbing BMP signaling at earlier stages of renal development, before the pronephric field is specified, results in the ablation of all three components of a functional nephron. mRNAs encoding Delta Smad7tevGR (300-400 pg), TEV2GR (10 pg), and the lineage marker beta -galactosidase (pink) were injected into a single blastomere at the 4-cell stage. Embryos were cultured until the onset of gastrulation (stages 9-10) when DEX was added to induce injected constructs. At the appropriate stages, renal genes normally expressed in the glomus (WT-1, nephrin; A-D), duct (Xlim-1; E,F), or tubules (3G8; G,H) were examined by either in situ hybridization or immunohistochemistry. In contrast to what was observed when BMP signaling was inhibited starting at stage 14 (post-gastrulation and after initial specification of renal mesoderm), inhibition of BMP signaling during gastrulation affected all three components of the developing nephron. Specifically, following inhibition of BMP signaling during gastrulation, a dramatic reduction in the expression of markers of the tubules, glomus, and duct was observed in 70-100% of injected, DEX-treated embryos. Black arrows indicate the normal position of the renal gene or protein.
Figure 6. BMP inhibition has varying effects on somitic and lateral plate mesoderm pre- and post-gastrulation. mRNAs encoding Delta Smad7tevGR (300-400 pg), TEV2GR (10 pg), and the lineage marker beta -galactosidase (B,C,F: pink; E: light blue) were injected into a single blastomere at the 4-cell stage. DEX was added to induce Delta Smad7-mediated BMP inhibition either at onset of gastrulation (stages 9-10) or at early neurula (stage 14). At the appropriate stages, embryos were assessed for somitic protein 12/101 or heart (lateral plate derivative) transcript cardiac actin by immunohistochemistry or in situ hybridization, respectively. Panels A and D show the uninjected sides of embryos. Inhibition of BMP signaling at either stage 10 or stage 14 in the lateral plate mesoderm resulted in a loss of cardiac actin expression (C,F, black arrows). BMP inhibition in somitic mesoderm at stage 10 resulted in mispatterned somites (B, arrowheads), while similar inhibition at stage 14 had no effect on somitic mesoderm protein 12/101 (E). Percentage of embryos in each panel refers to the percentage of embryos expressing indicated phenotype.
