In vivo clonal analysis reveals lineage-restricted progenitor characteristics in mammalian kidney development, maintenance, and regeneration

Abstract

The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maintenance, and regeneration. We show that the adult mammalian kidney undergoes continuous tubulogenesis via expansions of fate-restricted clones. Kidneys recovering from damage undergo tubulogenesis through expansions of clones with segment-specific borders, and renal spheres developing in vitro from individual cells maintain distinct, segment-specific fates. Analysis of mice derived by transfer of color-marked embryonic stem cells (ESCs) into uncolored blastocysts demonstrates that nephrons are polyclonal, developing from expansions of singly fated clones. Finally, we show that adult renal clones are derived from Wnt-responsive precursors, and their tracing in vivo generates tubules that are segment specific. Collectively, these analyses demonstrate that fate-restricted precursors functioning as unipotent progenitors continuously maintain and self-preserve the mouse kidney throughout life.

Figure 1

Clonal analysis during adult kidney growth. (A) Line chart depicting the mean change in kidney mass over time. X-axis represents age in days. Y-axis represents kidney mass in grams. (B–D) Composite (Rainbow & DAPI) images from Actin^CreER; R26^VT2/GK3 mice that was chased for 7 months. Singly colored clones contribute to distinct segments in the renal cortex (B, single red clone is outlined), medulla (C, single yellow clone is outlined), papilla (D, blue clone is outlined). (E–G) Composite images from Actin^CreER; R26^VT2/GK3 mice following a low dose regimen of tamoxifen, showing single and sparse clones of 8–10 cells (E–G, dotted white line). (H–J) Isolated nephron segments from Actin^CreER; R26^VT2/GK3 mice that were chased for 7 months reveal some tubule segments lack any clonal expansions (H, arrows), while others show extensive tubulogenesis by single colored clones (I, J, dotted white line). Scale bar: B–D, H–J (50μm); E–G (25μm).

Figure 2

Fate-restriction of clones during adult kidney maintenance. (A) Illustration of a single nephron tubule with distal tubule, loop of henle, proximal tubule and collecting duct segments. Segment specific markers (provided in the image) were used to characterize the composition of individually colored clones. (B–D) Long-lived clones that emerge following 7 months of chase are entirely retained within the segment-specific domains of label. (B) PNA⁺ illustrates a clone with a DT fate. (C, D) AQP2 and AQP3 illustrate clones with a CD fate (E–G) Double immunostaining of segment-specific markers shows that clones are fate-restricted to a single tubule type. (E) LTA⁻ MUC1⁻ illustrates a clone with a DT fate. (F) LTA⁺ PNA⁻ illustrates a clone with a PT fate. (G) Calbindin⁺ LTA⁻ illustrates a clone with DT fate. PT, proximal tubule; DT, distal tubule, CD, collecting duct. (B–G) Numbers on top right hand side of images represent the clone size. Scale bar: B–G (50μm).

Figure 3

Clonal analysis of the developing kidney. (A–D) Composite images (Rainbow & DAPI) from Actin^CreER; R26^VT2/GK3 mice that were traced from gestational stage of E13.5 up to postnatal day 1 (A–D). Lineage traced cells in the Cap mesenchyme are intermixed at the cortex prior to differentiation (A, white line). At post-MET stages, tubules are expanding from mixed progenitors (B, C, dotted white line) creating the future polyclonal nephrons. (D) A later stage in a developing tubule showing separate red and green clones contributing to a single tubule segment. (E–H) Composite (Rainbow & DAPI) images of renal tubules from a tetrachimeric mouse. (E) An image of the medulla showing polyclonal nephron and CD tubules. White arrows indicate boarders between clones within individual nephrons. (F) High power image showing non-dividing cells (white arrows) and cell processes interspersed within a green clone. Dotted white line indicates the boarder of the green clone within adjacent and separately colored tubule. (G, H) Separately colored clones are retained within tubules of the CD. (I) Confocal fluorescent microscopy image of the medulla and a glomerular mesangium showing separate red and green clones of mesangial cells (J–J″). Singly colored clones from tetrachimeric mice are fate-restricted to PTs (K–M, LTA⁺ PNA⁻), DT (N, O, PNA⁺), or CD-fates (P, AQP3⁺). PT, proximal tubule; DT, distal tubule, CD, collecting duct. PT, proximal tubule; DT, distal tubule, CD, collecting duct. Scale bar F, I–P (50μm); A–D, E–H (25μm).

Figure 4

Clonal analysis of the mammalian kidney following Ischemia/Reperfusion (I.R.). (A–C) Composite Rainbow images from Actin^CreER; R26^VT2/GK3 mice following two months of tracing. (A-A″) Singly colored red and green clones contributing to the damaged renal cortex. (B-B″) Singly colored yellow and red clones contributing to the damaged renal medulla. (C-C″) High power image showing red and green clones contributing to the damaged collecting ducts. (D, D′). Confocal Rainbow images of the renal medulla showing clones are retained within segments following renal damage. (E–I) Singly colored clones that emerge following renal damage are fate-restricted. Clones are either completely absent (PNA⁺ for DT fate) or entirely express (DBA⁺ for CD fate) the segment-specific markers. Double immunostaining of segment-specific markers showing a green clone that is LTA⁺ DBA⁻ (H) and LTA⁺ AQP3⁻ (I) both representing a DT fate. PT, proximal tubule; DT, distal tubule, CD, collecting duct. Scale bar: A, C–I (50μm); B (25μm).

Figure 5

Renal spheres that develop from individual cells are lineage-restricted in-vitro. (A-A⁗) Composite fluorescent images of spheres from single cell suspensions of Actin^CreER; R26^VT2/GK3 kidneys. (B-B″) Representative sections of renal spheres stained with Hematoxylin and eosin. (C) Histogram that graphically represents the frequency of spheres formed from cells cultured in limited dilution. (D) Graphical representation of the frequency of monoclonal/polyclonal spheres emerging after serial passaging. Following three passages, most emerging spheres are monoclonal (red line), and not polyclonal (blue line). (E, F) Images of sections from renal spheres immunostained with antibodies to distinct renal segments reveal that each segment-specific marker stains some but not all spheres. (G–I) Immunostaining of 3 representative spheres with antibodies for segment-specific markers. Each renal sphere is fate-restricted to DTs (PNA⁺), PTs (LTA⁺) or CDs (AQP3⁺). PT, proximal tubule; DT, distal tubule, CD, collecting duct. Scale bar: A-A⁗, B-B″, E–I (50μm).

Figure 6

Wnt-Responsive Cells (WRCs) are clone forming cells and lineage-restricted in-vivo. Sections from Axin^lacZ/lacZ kidneys stained with beta-galactosidase and counter-stained with eosin showing Axin2 expression within the collecting system and proximal tubules (A, A′). Axin2^CreER;R26^mTmG kidneys that were pulsed for 4 days show GFP expression within the collecting system and proximal tubules (A″, white arrows). (B-B‴) Axin2^CreER;R26^mTmG kidneys that were pulsed from E17.5 up to the 3^rd postnatal month show large GFP⁺ clones within proximal tubules and collecting ducts. (C–D) Intact PT segments from Axin2^CreER;R26^mTmG kidneys show large GFP⁺ clones confined to individual segments. Axin2^CreER; R26^VT2/GK3 kidneys that were pulsed from E17.5 up to the 5^th postnatal month show singly colored large clones within proximal tubules (F, F′) and collecting ducts (F″, F‴). (G) Histogram which graphically represent the difference in clonal outcomes of all cells (blue) vs. WRCs (red). (E,F) Clones derived from WRCs are lineage restricted to either PT or CD fates. Within the cortex, clones are LTA⁺, PNA⁻ indicating PT fate. (G–I) Within the renal papilla, clones are DBA⁺, AQP2⁺, AQP3⁺ indicating CD fate. PT, proximal tubule; DT, distal tubule, CD, collecting duct. Scale bar: A′, A″ (50μm); A, B-B‴, F-F‴, C–E (25μm).