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

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

Refining transcriptional programs in kidney development by integration of deep RNA-sequencing and array-based spatial profiling

<p>Abstract</p> <p>Background</p> <p>The developing mouse kidney is currently the best-characterized model of organogenesis at a transcriptional level. Detailed spatial maps have been generated for gene expression profiling combined with systematic <it>in situ </it>screening. These studies, however, fall short of capturing the transcriptional complexity arising from each locus due to the limited scope of microarray-based technology, which is largely based on "gene-centric" models.</p> <p>Results</p> <p>To address this, the polyadenylated RNA and microRNA transcriptomes of the 15.5 dpc mouse kidney were profiled using strand-specific RNA-sequencing (RNA-Seq) to a depth sufficient to complement spatial maps from pre-existing microarray datasets. The transcriptional complexity of RNAs arising from mouse RefSeq loci was catalogued; including 3568 alternatively spliced transcripts and 532 uncharacterized alternate 3' UTRs. Antisense expressions for 60% of RefSeq genes was also detected including uncharacterized non-coding transcripts overlapping kidney progenitor markers, Six2 and Sall1, and were validated by section <it>in situ </it>hybridization. Analysis of genes known to be involved in kidney development, particularly during mesenchymal-to-epithelial transition, showed an enrichment of non-coding antisense transcripts extended along protein-coding RNAs.</p> <p>Conclusion</p> <p>The resulting resource further refines the transcriptomic cartography of kidney organogenesis by integrating deep RNA sequencing data with locus-based information from previously published expression atlases. The added resolution of RNA-Seq has provided the basis for a transition from classical gene-centric models of kidney development towards more accurate and detailed "transcript-centric" representations, which highlights the extent of transcriptional complexity of genes that direct complex development events.</p

The epigenome in pluripotency and differentiation.

The ability to culture pluripotent stem cells and direct their differentiation into specific cell types in vitro provides a valuable experimental system for modeling pluripotency, development and cellular differentiation. High-throughput profiling of the transcriptomes and epigenomes of pluripotent stem cells and their differentiated derivatives has led to identification of patterns characteristic of each cell type, discovery of new regulatory features in the epigenome and early insights into the complexity of dynamic interactions among regulatory elements. This work has also revealed potential limitations of the use of pluripotent stem cells as in vitro models of developmental events, due to epigenetic variability among different pluripotent stem cell lines and epigenetic instability during derivation and culture, particularly at imprinted and X-inactivated loci. This review focuses on the two most well-studied epigenetic mechanisms, DNA methylation and histone modifications, within the context of pluripotency and differentiation

Identification of novel markers of mouse fetal ovary development

In contrast to the developing testis, molecular pathways driving fetal ovarian development have been difficult to characterise. To date no single master regulator of ovarian development has been identified that would be considered the female equivalent of Sry. Using a genomic approach we identified a number of novel protein-coding as well as non-coding genes that were detectable at higher levels in the ovary compared to testis during early mouse gonad development. We were able to cluster these ovarian genes into different temporal expression categories. Of note, Lrrc34 and AK015184 were detected in XX but not XY germ cells before the onset of sex-specific germ cell differentiation marked by entry into meiosis in an ovary and mitotic arrest in a testis. We also defined distinct spatial expression domains of somatic cell genes in the developing ovary. Our data expands the set of markers of early mouse ovary differentiation and identifies a classification of early ovarian genes, thus providing additional avenues with which to dissect this process

Expression analysis of somatic cell genes.

<p>ISH with sagittal section of XX and XY mouse embryos from 11.5 to 13.5 dpc, 5 weeks (wk) postnatal mouse ovaries and testes showed that <i>Slitrk1</i> (<b>A</b>) and <i>oncRNA3</i> (<b>B</b>) are expressed in ovarian somatic cells at 11.5 and 12.5 dpc. <i>Slitrk1</i> is also expressed in the granulosa cells of the mature ovary. Scale bars, 100 µm.</p

qRT-PCR validation of genes expressed in the developing ovary identified by microarray.

<p>qRT-PCR analysis of mRNA from isolated XX and XY gonads from 11.5, 12.5 and 13.5 dpc mouse embryos using gene-specific primers for <i>Slitrk1</i> (<b>A</b>), <i>Fam196b</i> (<b>B</b>), <i>D630039A03Rik</i> (<b>C</b>), <i>Tmem174</i> (<b>D</b>), <i>Lrrc34</i> (<b>E</b>), <i>Lypd6</i> (<b>F</b>), <i>Egfl6</i> (<b>G</b>), <i>Magi2</i> (<b>H</b>), <i>Dmrtc1c1</i> (<b>I</b>), <i>Smc1b</i> (<b>J</b>), <i>Spdya</i> (<b>K</b>), <i>D6Mm5e</i> (<b>L</b>), <i>Ccdc41</i> (<b>M</b>), <i>Foxl2</i> (<b>N</b>) and <i>Amh</i> (<b>O</b>) relative to <i>Sdha</i> (mean +SEM of at least three independent experiments; two-tailed, unpaired t-test; *p≤0.05, **p≤0.01, ***p≤0.001). Individual experiments were performed in triplicate on RNA obtained from pooled gonads from 3–4 littermates. All candidate genes were confirmed to be higher expressed in the ovary compared to testis at least at one of the developmental stages investigated. <i>Foxl2</i> served as control gene for ovary, <i>Amh</i> as control gene for testis samples.</p

Molecular compartmentalization of the early embryonic ovary.

<p>(<b>A</b>) ISH with sagittal section of 13.5 dpc XX embryos for <i>Fst</i>, <i>Wnt4</i>, <i>Rspo1</i> and <i>Irx3</i> followed by IHC for FOXL2 (first and second panel) and section ISH for <i>Lypd6</i> (third panel) identified three different expression domains of somatic cell genes as represented in the schematic on the left of each panel. Scale bar, 100 µm. (<b>B</b>) ISH with transverse sections of 12.5 dpc XX embryos for <i>Foxl2</i>, <i>Wnt4</i>, <i>oncRNA3</i> and <i>Lypd6</i> showed the extent to which these genes were expressed in the mesonephric and coelomic domains respectively. (<b>C</b>) High magnification of ISH with sagittal sections of 13.5 dpc XX embryos for <i>Fst</i>, <i>Wnt4</i>, <i>Rspo1</i> and <i>Irx3</i> followed by IHC for FOXL2 suggested that <i>Fst</i> and <i>Wnt4</i>, but not <i>Rspo1</i> and <i>Irx3</i>, are co-expressed to a large extent with FOXL2. Scale bar, 100 µm (<b>A</b> and <b>B</b>), 20 µm (<b>C</b>).</p

Temporal and spatial expression analysis of <i>D6Mm5e</i>.

<p>Whole mount ISH of mouse embryonic XX and XY gonads from 11.5 to 15.5 dpc (<b>A</b>) and ISH with sagittal section of mouse embryonic XX and XY gonads from 11.5 to 13.5 dpc, (<b>B</b>), showing wave-like upregulation of <i>D6Mm5e</i> reminiscent of PGC (arrowhead) entry into meiosis. Scale bar, 100 µm. (<b>C</b>) <i>D6Mm5e</i> section ISH hybridization (purple staining) followed by IHC for the germ cell marker E-cadherin (ECAD, brown staining) of 13.5 dpc mouse ovaries confirmed <i>D6Mm5e</i> expression in XX PGCs (arrowheads). Scale bars, 100 µm (low magnification, top panel) and 20 µm (high magnification, bottom panel).</p