Abstract
BackgroundBone morphogenetic proteins (BMPs) regulate essential processes during organogenesis, and a functional understanding of these secreted proteins depends on identification of their target cells. In this study, we generate a transgenic reporter for organogenesis studies that we use to define BMP pathway activation in the developing kidney.ResultsMouse strains reporting on BMP pathway activation were generated by transgenically expressing β-galactosidase under the control of BMP responsive elements from Id1. Reporter expression corresponds well with immunoassays for pathway activation in all organs studied, validating the model. Using these reporters we have generated a detailed map of cellular targets of BMP signaling in the developing kidney. We find that SMAD dependent BMP signaling is active in collecting duct trunks, but not tips. Furthermore, glomerular endothelial cells, and proximal nephron tubules from the renal vesicle stage onward show pathway activation. Surprisingly, little activation is detected in the nephrogenic zone of the kidney, and in organ culture BMP treatment fails to activate SMAD dependent BMP signaling in nephron progenitor cells. In contrast, signaling is efficiently induced in collecting duct tips.ConclusionTransgenic reporters driven by control elements from BMP responsive genes such as Id1 offer significant advantages in sensitivity and consistency over immunostaining for studies of BMP pathway activation. They also provide opportunities for analysis of BMP signaling in organ and primary cell cultures subjected to experimental manipulation. Using such a reporter, we made the surprising finding that SMAD dependent BMP signaling is inactive in nephron progenitors, and that these cells are refractory to activation by applied growth factors. Furthermore, we find that the BMP pathway is not normally active in collecting duct tips, but that it can be ectopically activated by BMP treatment, offering a possible explanation for the inhibitory effects of BMP treatment on collecting duct growth and branching.
Highlights
Bone morphogenetic proteins (BMPs) regulate essential processes during organogenesis, and a functional understanding of these secreted proteins depends on identification of their target cells
We find that the BMP signaling pathway is highly activated in collecting ducts, developing nephron tubules and glomeruli, but surprisingly it is inactive in the nephron progenitor cell population, which is thought to respond to BMP signaling
The BMP Responsive Element (BRE)-Hspa1a-lacZ (BRE-lacZ) transgenic reporter strain To generate a mouse in which cells responding to BMP signaling are labeled by β-galactosidase expression, we derived a transgenic strain using a construct containing a concatamerized SMAD1/5/8 binding site from the mouse Id1 gene [15], upstream of the Hspa1a promoter (-873 to +5 relative to the translational start) fused to the β-galactosidase cDNA followed by an SV40 polyadenylation signal [27,28] (Fig. 1A)
Summary
Bone morphogenetic proteins (BMPs) regulate essential processes during organogenesis, and a functional understanding of these secreted proteins depends on identification of their target cells. The SMAD Binding Element (SBE), GTCT and the GC-rich consensus motif GNCGCC [13,14,15], confer relatively low affinity and specificity to the SMAD:DNA interaction, necessitating association with other transcription factors such as ZFP423 (OAZ) [16] for efficient DNA binding. This combinatorial requirement for binding can be bypassed by concatamerizing the basic SMAD binding motifs. This natural concatamer, known as the BMP Responsive Element (BRE) has been used to generate both an in vitro BMP reporter [15] and in vivo reporters [18,19]
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