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, 6 (2), e17286

Identification of Anchor Genes During Kidney Development Defines Ontological Relationships, Molecular Subcompartments and Regulatory Pathways

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Identification of Anchor Genes During Kidney Development Defines Ontological Relationships, Molecular Subcompartments and Regulatory Pathways

Rathi D Thiagarajan et al. PLoS One.

Abstract

The development of the mammalian kidney is well conserved from mouse to man. Despite considerable temporal and spatial data on gene expression in mammalian kidney development, primarily in rodent species, there is a paucity of genes whose expression is absolutely specific to a given anatomical compartment and/or developmental stage, defined here as 'anchor' genes. We previously generated an atlas of gene expression in the developing mouse kidney using microarray analysis of anatomical compartments collected via laser capture microdissection. Here, this data is further analysed to identify anchor genes via stringent bioinformatic filtering followed by high resolution section in situ hybridisation performed on 200 transcripts selected as specific to one of 11 anatomical compartments within the midgestation mouse kidney. A total of 37 anchor genes were identified across 6 compartments with the early proximal tubule being the compartment richest in anchor genes. Analysis of minimal and evolutionarily conserved promoter regions of this set of 25 anchor genes identified enrichment of transcription factor binding sites for Hnf4a and Hnf1b, RbpJ (Notch signalling), PPARγ:RxRA and COUP-TF family transcription factors. This was reinforced by GO analyses which also identified these anchor genes as targets in processes including epithelial proliferation and proximal tubular function. As well as defining anchor genes, this large scale validation of gene expression identified a further 92 compartment-enriched genes able to subcompartmentalise key processes during murine renal organogenesis spatially or ontologically. This included a cohort of 13 ureteric epithelial genes revealing previously unappreciated compartmentalisation of the collecting duct system and a series of early tubule genes suggesting that segmentation into proximal tubule, loop of Henle and distal tubule does not occur until the onset of glomerular vascularisation. Overall, this study serves to illuminate previously ill-defined stages of patterning and will enable further refinement of the lineage relationships within mammalian kidney development.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Identification of candidate anchor genes.
Microarray data generated by Brunskill et al (2008) was analysed to identify potential compartment specific gene expression in eleven kidney compartments - Cortical interstitium (CI), medullary interstitium (MI), loop of Henle (LH), cap mesenchyme (CM), renal vesicle (RV), S-shaped body , renal corpuscle (RC), early proximal tubule (PT), ureteric tip (UT), cortical collecting duct and medullary collecting duct (MC). A) The preliminary selection for candidate anchor genes/markers used for all compartments is exemplified using EPT. i) Identification of differentially expressed genes across all profiled compartments (ANOVA p<0.01) with EPT genes highlighted in red; ii) Genes up-regulated within the compartment of interest were selected based on normalized values (fold-change) >2 (log scale) against the median. iii) Final candidate genes for EPT; Genes were further filtered for absolute restricted expression by excluding probesets that were expressed at ≥2fold in other subcompartments, then ranked on median signal intensity values (<200RFU (raw fluorescent units)) and raw signal intensity values (>100RFU) across all compartments. B) Expression profiles of compartment specific genes selected from CM, RV, RC and MC for validation via SISH. C) Global view of distribution of expression for all 11 compartments analysed.
Figure 2
Figure 2. High resolution SISH validating anchor genes from a variety of developmental compartments within the developing kidney.
Representative SISH images of the 15.5dpc kidney are shown for anchor genes identified for the renal vesicle (RV), renal corpuscle (RC), early proximal tubule (EPT), loop of Henle (LoH), medullary collecting duct (MCD) and ureteric tip (UT). Marker genes for the renal interstitium (RI) and cap mesenchyme (CM) are also shown. Col3a1 is expressed in the cortical (CI), medullary (MI) and perihilar interstitial compartments but absent from the nephrogenic interstitium (NI). Eya1 is expressed in the CM and a subset of the adjacent NI. The location of the high magnification inset images is outlined. All scalebars are 200 mm, P = pelvis, SSB = S-shaped body.
Figure 3
Figure 3. Correlation between anatomical compartments used for microarray analysis with molecular compartments revealed via SISH.
The 3 major tissue compartments of the kidney are represented by the top row of painted bars (yellow, purple, blue). Below this, the second row indicates the anatomical subdivision of these into 14 compartments. The 11 compartments profiled by microarray are painted and the method used to isolate each is indicated below the bar . PI and EDT were not isolated for microarray, and CI and NI were isolated as one compartment. The bars in the rows below these indicate the regional gene expression patterns observed by high resolution SISH. The expression patterns are shown from broadest (top) to the most restricted (bottom) and the genes observed with each of these patterns are listed below the bar. The painted gene expression bars (yellow, purple, blue) indicate restricted expression in only one compartment and the genes listed below each of these are the anchor genes (bold type). PI, MI, CI and NI = perihilar, renal medullary, renal cortical and nephrogenic interstitium; CM = cap mesenchyme; RV = renal vesicle; SSB = S-shaped body; RC = renal corpuscle (StageIII-IV); EPT = early proximal tubule; LoH = immature and anlage of loop of Henle; EDT = early distal tubule; MCD and CCD = medullary and cortical collecting duct; UT = ureteric tip. Below each of the interstitial/mesenchyme expression bars, MI: and CI: indicate the microarrary compartment used to identify the genes. Some genes identified in the renal interstitium were also expressed in the vasculature of the kidney and/or the mesangial tissue of renal corpuscles and these genes are listed (bottom, left).
Figure 4
Figure 4. Expression of marker genes in the ureteric tree at 15.5dpc.
Three compartments were analysed for anchor genes in the ureteric tree; the ureteric tip including the uretric tree terminal branch (UT), the cortical collecting duct (CCD) and medullary collecting duct (MCD). A) Examples of expression patterns seen by SISH are shown for UT and MCD genes. The top panel shows kidney sections with the region enlarged shown below. Arrows in b,d  =  UT and f,h  =  MCD expression. The genes identified as potentially CCD-specific did not validate by SISH. SISH images and text-annotated expression patterns for all genes are available on the GUDMAP website (http://www.gudmap.org). B) Schematic of 15.5dpc kidney divided into nephrogenic zone (top), renal cortex, and medulla showing segmentation of the ureteric tree. *indicates the regions collected by LCM representing the UT compartment. The expression of each gene giving either a specific or enriched expression pattern was painted onto the schematic and is shown in C) UT genes D) MCD specific anchor genes and E) MCD enriched genes. Slco4c1 was identified as the only UT-specific anchor gene.
Figure 5
Figure 5. Identification of distinct ontological markers of renal tubule segmentation and patterning.
For the early proximal tubule (EPT) and loop of Henle (LOH) compartments of the nephron, expression patterns determined by SISH at 15.5dpc were painted onto a schematic of stage III and IV nephrons (see Figure S1). Both tubular and renal corpuscle (RC) structures were painted and expression strength is indicated by colour. Expression in the adult nephron is indicated in text on the right of each schematic (renal proximal tubule S1 S2 S3 segments, DCT distal convoluted tubule, DST distal straight tubule and LOH) and – indicates expression is absent. Unknown  =  expression was not examined?  =  uncertain expression. Red* indicates that these EPT genes also showed expression in a subset of the distal tubules in the adult kidney, Slc6a13 (DST) and Aldh11 (DCT, DST). EPT genes with restricted expression in the nephron tubules were divided into 3 categories; (A) early genes showing expression in Stage III and IV tubules, (B) late genes restricted to EPT of Stage IV tubules, the EPT anchor genes and (C) late genes with expression in EPT and other tubular segments (immature loop of Henle and/or early distal tubule). (D) EPT genes which showed additional expression in the renal corpuscle nephron compartment represent an additional category of enriched EPT genes. (A′–D′) Expression patterns in the kidney at 15.5dpc by SISH are shown for representative genes from each of the EPT spatiotemporal categories. In C′, arrowheads indicate expression in immature LOH. For the LOH compartment, three genes were identified (E) Umod a LOH-specific anchor gene and (F) Kcnj1 and Tmem72 LOH markers also expressed in the early distal tubule (EDT). SISH images and text-annotated expression patterns for all genes are available on the GUDMAP website (http://www.gudmap.org).
Figure 6
Figure 6. Ingenuity/GO analyses of relationships between proximal tubule anchor genes.
Functional network analysis of early proximal tubule anchor genes. Network contains gene/gene products represented as nodes and biological relationship between the nodes as edges as curated in the Ingenuity Knowledge Base. Anchor gene nodes (orange) and transcription factors predicted from TF binding site motif analysis (blue) are highlighted. Kidney related functions and disease are represented in white text boxes. Functions directly related to proximal tubule development are outlined in purple, green and red.

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