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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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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.
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pone-0017286-g004: 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.

Mentions: While it is known that specific factors secreted from the tips of the ureteric tree are critical for the induction of nephron formation, the specification, segmentation and differentiation of non-tip regions of the ureteric epithelium is not well understood. Many well known ureteric tree markers are ubiquitously expressed throughout the ureteric epithelia during development, including Calb1 and Hoxb7 [37], [38]. While previous studies have sought differential markers of ureteric trunk versus tip [21], [29], [30], few have been well defined other than Wnt11 in the tip [39]. Even the widely reported tip marker, Ret, while enriched in the ureteric tip, is also expressed weakly in the ureteric trunk [39]. Three spatially defined compartments were isolated from the ureteric epithelium in the initial microarray analysis; UT, CCD and MCD. Validation of predicted anchor genes within these subcompartments identified Slco4c1 as a novel UT anchor gene, five MCD anchor genes (Gsdmc4, Clmn, AI83600, Fam129a, and Upk3a) but no CCD anchor genes. However, a cohort of seven additional genes revealed a complex set of overlapping gene expression patterns indicating a much greater level of segmentation of this epithelial compartment (Figure 4). Two genes, Tesc and Scnn1b were expressed in the ureteric epithelia as well as the adjacent distal nephron tubules. Genes specific to or enriched in MCD included genes involved in epithelial differentiation and specialisation. Gsdmc4, part of the Gsdmc gene cluster, also shows epithelial-specific expression in the skin and gut [40]. Mutations in Upk3a, which encodes a protein critical to urothelial plaque formation, can result in renal adysplasia including vesicoureteral reflux and echogenic cystic kidneys [41]. This set of markers can be used to further understand functional differentiation along the ureteric tree.


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)

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.
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Related In: Results  -  Collection

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pone-0017286-g004: 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.
Mentions: While it is known that specific factors secreted from the tips of the ureteric tree are critical for the induction of nephron formation, the specification, segmentation and differentiation of non-tip regions of the ureteric epithelium is not well understood. Many well known ureteric tree markers are ubiquitously expressed throughout the ureteric epithelia during development, including Calb1 and Hoxb7 [37], [38]. While previous studies have sought differential markers of ureteric trunk versus tip [21], [29], [30], few have been well defined other than Wnt11 in the tip [39]. Even the widely reported tip marker, Ret, while enriched in the ureteric tip, is also expressed weakly in the ureteric trunk [39]. Three spatially defined compartments were isolated from the ureteric epithelium in the initial microarray analysis; UT, CCD and MCD. Validation of predicted anchor genes within these subcompartments identified Slco4c1 as a novel UT anchor gene, five MCD anchor genes (Gsdmc4, Clmn, AI83600, Fam129a, and Upk3a) but no CCD anchor genes. However, a cohort of seven additional genes revealed a complex set of overlapping gene expression patterns indicating a much greater level of segmentation of this epithelial compartment (Figure 4). Two genes, Tesc and Scnn1b were expressed in the ureteric epithelia as well as the adjacent distal nephron tubules. Genes specific to or enriched in MCD included genes involved in epithelial differentiation and specialisation. Gsdmc4, part of the Gsdmc gene cluster, also shows epithelial-specific expression in the skin and gut [40]. Mutations in Upk3a, which encodes a protein critical to urothelial plaque formation, can result in renal adysplasia including vesicoureteral reflux and echogenic cystic kidneys [41]. This set of markers can be used to further understand functional differentiation along the ureteric tree.

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