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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 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 [46], renal corpuscle (RC), early proximal tubule (PT), ureteric tip (UT), cortical collecting duct [15] 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.
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pone-0017286-g001: 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 [46], renal corpuscle (RC), early proximal tubule (PT), ureteric tip (UT), cortical collecting duct [15] 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.

Mentions: Brunskill et al (2008) [25] reported Affymetrix expression profiling for 15 compartments, 11 of which were isolated from embryonic day (E) 15.5 developing murine kidney. Microarray data from these 11 anatomical compartments represented interstitial/mesenchymal elements (cap mesenchyme, CM; medullary interstitium, MI; cortical interstitium, CI), anatomical subdivisions of the ureteric epithelium (ureteric tip, UT; cortical collecting duct, CCD; medullary collecting duct, MCD) and CM-derived elements of the nephron (renal vesicle/Stage I nephron, RV; S-shaped body/Stage II nephron, SSB; early proximal tubule, EPT; Loop of Henle, LH and renal corpuscle, RC). A list of putative anchor genes were selected as those displaying; a) the most significant differential expression ratio (>2 x median expression fold change), b) high raw signal intensity (>100 RFU) above background and c) low median signal intensity in all other subcompartments examined (Figure 1). As can be seen in Figure 1, there was considerable variation in the median expression fold change and number of putative anchor genes identified between compartments. Early proximal tubule showed the largest number of highly differentially expressed genes whereas compartments representing transient developmental stages (RV, SSB) or probable cellular heterogeneity (MI, CI) showed very few selected genes. As a lack of anchor genes may also have arisen due to a lack of association between the physical compartment selected and any molecular association or due to contamination of samples during isolation, a parallel comparison of a subset of 7 compartments (UT, RC, MCD, RV, EPT, MI, and SSB) was also performed. A complete list of the 251 genes prioritised for validation from both comparisons is listed in Table S1.


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 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 [46], renal corpuscle (RC), early proximal tubule (PT), ureteric tip (UT), cortical collecting duct [15] 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046260&req=5

pone-0017286-g001: 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 [46], renal corpuscle (RC), early proximal tubule (PT), ureteric tip (UT), cortical collecting duct [15] 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.
Mentions: Brunskill et al (2008) [25] reported Affymetrix expression profiling for 15 compartments, 11 of which were isolated from embryonic day (E) 15.5 developing murine kidney. Microarray data from these 11 anatomical compartments represented interstitial/mesenchymal elements (cap mesenchyme, CM; medullary interstitium, MI; cortical interstitium, CI), anatomical subdivisions of the ureteric epithelium (ureteric tip, UT; cortical collecting duct, CCD; medullary collecting duct, MCD) and CM-derived elements of the nephron (renal vesicle/Stage I nephron, RV; S-shaped body/Stage II nephron, SSB; early proximal tubule, EPT; Loop of Henle, LH and renal corpuscle, RC). A list of putative anchor genes were selected as those displaying; a) the most significant differential expression ratio (>2 x median expression fold change), b) high raw signal intensity (>100 RFU) above background and c) low median signal intensity in all other subcompartments examined (Figure 1). As can be seen in Figure 1, there was considerable variation in the median expression fold change and number of putative anchor genes identified between compartments. Early proximal tubule showed the largest number of highly differentially expressed genes whereas compartments representing transient developmental stages (RV, SSB) or probable cellular heterogeneity (MI, CI) showed very few selected genes. As a lack of anchor genes may also have arisen due to a lack of association between the physical compartment selected and any molecular association or due to contamination of samples during isolation, a parallel comparison of a subset of 7 compartments (UT, RC, MCD, RV, EPT, MI, and SSB) was also performed. A complete list of the 251 genes prioritised for validation from both comparisons is listed in Table S1.

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