Date Published: April 12, 2019
Publisher: Public Library of Science
Author(s): Kyle K. Dickinson, Leah C. Hammond, Courtney M. Karner, Nicholas D. Hastie, Thomas J. Carroll, Paul Goodyer, Rajeev Samant.
Primed nephron progenitor cells (NPCs) appear in metanephric mesenchyme by E11.5 and differentiate in response to the inductive WNT9b signal from the ureteric bud. However, the NPC WNT-receptor complex is unknown. We obtained M15 cells from E10.5 mesonephric mesenchyme and systematically analyzed components required for canonical WNT9b-responsiveness. When M15 cells were transfected with a β-catenin luciferase reporter plasmid, exposure to recombinant WNT9b resulted in minimal luciferase activity. We then analyzed mRNA-expression of WNT-pathway components and identified Fzd1-6 and Lrp6 transcripts but not Rspo1. When M15 cells were treated with recombinant RSPO1 the response to transfected WNT9b was augmented 4.8-fold. Co-transfection of M15 cells with Fzd5 (but no other Fzd family member) further increased the WNT9b signal to 16.8-fold and siRNA knockdown of Fzd5 reduced the signal by 52%. Knockdown of Lrp6 resulted in 60% WNT9b signal reduction. We confirmed Fzd5, Lrp6 and Rspo1 mRNA expression in CITED1(+) NPCs from E15.5 embryonic mouse kidney. Thus, while many WNT signaling-pathway components are present by E10.5, optimum responsiveness of E11.5 cap mesenchyme requires that NPCs acquire RSPO1, FZD5 and LRP6.
The mammalian kidneys are derived from progenitor cells in the embryonic intermediate mesoderm, expressing the transcription factor, OSR1. Fate mapping studies of the embryonic kidney reveal that cells labeled by the Osr1 promoter at embryonic day E7.5 give rise to all elements of the maturing kidney  and Osr1 knockout mice are anephric [2, 3]. Around E8.5-E9, a subset of OSR1-positive kidney progenitor cells are transformed into polarized epithelia, forming the paired nephric duct structures that elongate down the embryo . Concurrently, another subset of cells upregulate Wilms’ tumor 1 (WT1) while retaining a mesenchymal phenotype. [5, 6]. The columns of WT1(+) cells flanking each nephric duct are committed to the nephron progenitor cell (NPC) fate; interestingly, Wt1 knockout mice fail to develop functional kidneys . Development of the metanephric kidney begins in earnest when ureteric buds emerge from each nephric duct (E10.5), begins to arborize as it grows into the adjacent column of metanephric mesenchyme and induces local NPCs to begin nephrogenesis.
Around embryonic day E9.0 of mouse development, a lineage of WT1-expressing progenitor cells emerge within the OSR1(+) intermediate mesoderm. To model this early NPC prior to the arrival of the ureteric bud, we studied the M15 cell line isolated from E10.5 mouse kidneys . These cells express Osr1, WT1 and Cited1, placing them in the early NPC lineage. Previous studies from our lab showed the essential role of WT1 for responsiveness to the inductive WNT9b signal through suppression of EZH2, a histone H3K27 methyltransferase. EZH2 suppression in turn opens up chromatin, permitting exit from the stem cell state [15, 28]. Thus, WT1 is essential for maturation of the nephron progenitor cell lineage. Nevertheless, we found that M15 cells were unresponsive to WNT9b in vitro. This suggests that WT1 expression alone is not sufficient to prime the NPC for WNT-responsiveness and that the early NPC must acquire additional molecular properties by the time the ureteric bud arrives at E10.5-E11.