In this report we present an atlas of gene expression in the developing kidney. This quantitative, sensitive and global definition of the gene expression profiles of the major components of the developing kidney provides a foundation for further analysis of the genetic mechanisms of nephrogenesis. For example, this atlas of normal gene expression provides a baseline that can be used to better understand abnormalities in kidney development present in mutant mice. This could be through improved in situ
hybridization analysis, now equipped with new sets of molecular markers, or through a LCM-microarray based analysis of the mutant phenotype, allowing a more universal characterization of altered gene expression patterns (Potter et al., 2007
; Schwab et al., 2006a
). The atlas also promotes the generation of additional useful genetic tools for the analysis of kidney development, with for example component specific expression of Cre and/or GFP. Global expression analysis of developmental processes can also reveal potential functional redundancies and guide genetic analyses. Mutant screens and targeted mutation studies often fail to realize expected developmental defects. The microarray atlas data presented here can help identify co-expressed genes with overlapping function.
This work considerably extends previous microarrays studies examining kidney development. Microarrays were first used to examine changing gene expression patterns of entire rat (Stuart et al., 2001
) and then mouse (Challen et al., 2005
; Schwab et al., 2003
) kidneys as a function of developmental time. Some spatial definition of microarray expression profiles has been added by physical dissection of E11.5 metanephric mesenchyme and ureteric bud (Schwab et al., 2006b
; Stuart et al., 2003
), and in two cases by FACS sorting, of entire E15.5 UB (Challen et al., 2005
) using Hoxb7
-GFP, and of mesenchyme using Sal1-GFP positive cells (Takasato et al., 2004
). Nevertheless, the study presented here represents the first comprehensive microarray analysis of kidney development, using laser capture or FACS to allow the analysis of all the major elements of nephrogenesis.
It is important to note some of the limitations of the microarray-based dataset. First, over 60% of genes can be alternatively processed, and the microarrays used in this study do not reveal patterns of exon usage. Second, several of the components examined in this study consist of mixtures of different cell types. One extreme example is the S-shaped body, which includes cells that are differentiating into podocytes (visceral epithelium), proximal tubules and other diverse structures. The individual laser-captured sections will include different proportions of these variant cell types, and the final microarray results will represent an average of the gene expression patterns of the different cells collected. It is to be expected therefore, that there will be some heterogeneity in the microarray readouts of the biological triplicates performed for each component. Ideally one would like to extend the microarray analysis to the level of single cell types. This is becoming possible as the combination of microarray data and in situ hybridization results identify genes with increasingly restricted domains of expression. This facilitates the generation of transgenic mice with more localized GFP expression, which can then be used in combination with laser capture microdissection and/or FACS to produce gene expression profiles of more restricted cell types.
The microarray-based atlas that we have developed also provides deeper insight into the mechanisms of kidney development, and organogenesis in general. One emerging theme is that while some genes do display extremely component specific expression, their number is surprisingly small. Different developmental compartments generally exhibit extensive overlap in gene expression patterns, with the differences more quantitative than qualitative in nature. The results suggest an analog model of organogenesis, with differences in gene expression levels often more important than differences in gene expression on/off states. The combinatorial codes of gene expression that drive compartment specific development appear to have an important quantitative element. Microarrays, with their ability to define quantitative gene expression levels, are particularly powerful tools for the characterization of such analog gene expression codes.
It is also possible to use the expression data to begin to provide a global view of the genetic circuitry of kidney development. The microarray results provide a comprehensive analysis of the expressed transcription factors, as well as all other genes expressed. It is possible to begin to connect individual transcription factors with their targets by looking for the presence of evolutionarily conserved transcription factor binding sites within promoters of expressed genes. For example we show a very strong statistical association between the expression of Hnf1 in the developing proximal tubules and the presence of a well-conserved Hnf1 binding sites in the promoters of many genes with proximal tubule enriched expression. However, it is important to note that this microarray expression based dissection of genetic circuitry is not all inclusive, as some target genes will carry transcription factor binding sites outside of the scanned 4 Kb proximal promoter region, some genes will be regulated by non-canonical binding sites, and only a few hundred transcription factor binding sites have been well-defined. In addition, the presence of a highly conserved binding site alone does not guarantee biological function for a transcription factor. Ideally one would like to combine microarray expression analysis with global chromatin immunoprecipitation studies to better define transcription factor positioning in the genome, but with the laser capture approach primarily used in this report the limiting amounts of material available make this impractical. Nevertheless, this one example, examining HNF1β targets in the proximal tubules, demonstrates the power of a bioinformatics based approach. By combining observed coordinate expression with analysis of evolutionarily conserved transcription factor binding sites within promoters we find a number of genes previously confirmed to be regulated by HNF1β, and identify many additional candidate targets.
The microarray atlas presented here establishes a precedent for the global analysis of organogenesis, similar in scope to the Allen Brain Atlas (Lein et al., 2007
), only focused on a developing organ, and made using a different set of tools. The LCM/FACS-microarray strategy offers several advantages over an approach using only in situ
hybridizations. First it is very sensitive, as microarrays can detect transcripts present in only a few copies per cell. Second, it is more quantitative, as single channel microarrays give a numerical measure of gene expression level, while in situ
hybridizations give only a relative staining intensity. In addition, using microarrays is far more efficient and cost effective than performing tens of thousands of in situ
hybridizations. Nevertheless, there is a significant price to be paid, as the microarray analysis of laser captured components does not provide the potentially single cell spatial resolution achievable with in situ
In summary, we describe an atlas of gene expression patterns of the developing kidney generated by using either LCM or FACS to purify components of the developing kidney, followed by hybridization to microarrays to provide comprehensive and quantitative gene expression profiles. This resource provides a global definition of the gene expression program of nephrogenesis, setting the stage for further genetic dissection of this remarkable process.