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Identification of anchor genes during kidney development defines ontological relationships, molecular subcompartments and regulatory pathways.

Thiagarajan RD, Georgas KM, Rumballe BA, Lesieur E, Chiu HS, Taylor D, Tang DT, Grimmond SM, Little MH - PLoS ONE (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Australia.

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.

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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).
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pone-0017286-g005: 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).

Mentions: The strength of the anchor gene analysis was especially demonstrated within the EPT compartment in which 25 anchor genes were identified (see Table 1, Figure 3). The EPT genes demonstrated different regional expression patterns across the tubular segments of the nephron, with regional patterns observed within the Stage IV EPT itself, which could be further subdivided into presumptive S1, S2 and S3 segments (see Figure S1). Figure 5 shows representations of the pattern of expression of each of these EPT anchor genes in the Stage IV nephron. The majority of EPT anchor genes maintained this specific pattern of expression in the proximal tubule of the adult kidney, suggesting very early specification of this tubular segment (see Figure 5 B, Figure S2). As well as identifying many EPT anchor genes, a further 34 genes (identified within the RV, SSB, RC, EPT and LOH compartments) allowed the subdivision of cap mesenchyme derived nephron development into distinct temporospatial gene expression patterns including early (Lhx1) and later markers of pan-nephron development (Gpd1, Susd2, Dpep1), early nephron (Bmp2, Tmem100), presumptive podocyte (Stc2) and LOH/distal tubule (Kcnj1, Tmem72) (Figure 3). One group of 12 genes displayed expression in the tubules of Stage III nephrons. All genes in this group were also expressed in Stage IV nephrons (Figure 3, Figure 5A) but were not restricted to the proximal tubule. Other genes do not commence expression until the differentiation of the nephron into Stage IV (maturing nephron). This included the 25 anchor genes whose expression was restricted to the proximal tubules (Figure 3, Figure 5B) together with a further 13 genes whose expression extended beyond the proximal tubule into other tubular segments (immature loop of Henle and/or early distal tubule) (Figure 3, Figure 5C). As with EPT anchor genes, many of these marker genes maintained the same regional domains of expression in the adult proximal tubule (Figure 5, Figure S2). This suggests that segmentation of the early nephron into proximal, distal and LOH elements does not occur until the formation of a Stage IV nephron.


Identification of anchor genes during kidney development defines ontological relationships, molecular subcompartments and regulatory pathways.

Thiagarajan RD, Georgas KM, Rumballe BA, Lesieur E, Chiu HS, Taylor D, Tang DT, Grimmond SM, Little MH - PLoS ONE (2011)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046260&req=5

pone-0017286-g005: 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).
Mentions: The strength of the anchor gene analysis was especially demonstrated within the EPT compartment in which 25 anchor genes were identified (see Table 1, Figure 3). The EPT genes demonstrated different regional expression patterns across the tubular segments of the nephron, with regional patterns observed within the Stage IV EPT itself, which could be further subdivided into presumptive S1, S2 and S3 segments (see Figure S1). Figure 5 shows representations of the pattern of expression of each of these EPT anchor genes in the Stage IV nephron. The majority of EPT anchor genes maintained this specific pattern of expression in the proximal tubule of the adult kidney, suggesting very early specification of this tubular segment (see Figure 5 B, Figure S2). As well as identifying many EPT anchor genes, a further 34 genes (identified within the RV, SSB, RC, EPT and LOH compartments) allowed the subdivision of cap mesenchyme derived nephron development into distinct temporospatial gene expression patterns including early (Lhx1) and later markers of pan-nephron development (Gpd1, Susd2, Dpep1), early nephron (Bmp2, Tmem100), presumptive podocyte (Stc2) and LOH/distal tubule (Kcnj1, Tmem72) (Figure 3). One group of 12 genes displayed expression in the tubules of Stage III nephrons. All genes in this group were also expressed in Stage IV nephrons (Figure 3, Figure 5A) but were not restricted to the proximal tubule. Other genes do not commence expression until the differentiation of the nephron into Stage IV (maturing nephron). This included the 25 anchor genes whose expression was restricted to the proximal tubules (Figure 3, Figure 5B) together with a further 13 genes whose expression extended beyond the proximal tubule into other tubular segments (immature loop of Henle and/or early distal tubule) (Figure 3, Figure 5C). As with EPT anchor genes, many of these marker genes maintained the same regional domains of expression in the adult proximal tubule (Figure 5, Figure S2). This suggests that segmentation of the early nephron into proximal, distal and LOH elements does not occur until the formation of a Stage IV nephron.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Australia.

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.

Show MeSH
Related in: MedlinePlus