A comparative molecular analysis of developing mouse forelimbs and hindlimbs using serial analysis of gene expression (SAGE)

Abstract

The analysis of differentially expressed genes is a powerful approach to elucidate the genetic mechanisms underlying the morphological and evolutionary diversity among serially homologous structures, both within the same organism (e.g., hand vs. foot) and between different species (e.g., hand vs. wing). In the developing embryo, limb-specific expression of Pitx1, Tbx4, and Tbx5 regulates the determination of limb identity. However, numerous lines of evidence, including the fact that these three genes encode transcription factors, indicate that additional genes are involved in the Pitx1-Tbx hierarchy. To examine the molecular distinctions coded for by these factors, and to identify novel genes involved in the determination of limb identity, we have used Serial Analysis of Gene Expression (SAGE) to generate comprehensive gene expression profiles from intact, developing mouse forelimbs and hindlimbs. To minimize the extraction of erroneous SAGE tags from low-quality sequence data, we used a new algorithm to extract tags from -analyzed sequence data and obtained 68,406 and 68,450 SAGE tags from forelimb and hindlimb SAGE libraries, respectively. We also developed an improved method for determining the identity of SAGE tags that increases the specificity of and provides additional information about the confidence of the tag-UniGene cluster match. The most differentially expressed gene between our SAGE libraries was Pitx1. The differential expression of Tbx4, Tbx5, and several limb-specific Hox genes was also detected; however, their abundances in the SAGE libraries were low. Because numerous other tags were differentially expressed at this low level, we performed a 'virtual' subtraction with 362,344 tags from six additional nonlimb SAGE libraries to further refine this set of candidate genes. This subtraction reduced the number of candidate genes by 74%, yet preserved the previously identified regulators of limb identity. This study presents the gene expression complexity of the developing limb and identifies candidate genes involved in the regulation of limb identity. We propose that our computational tools and the overall strategy used here are broadly applicable to other SAGE-based studies in a variety of organisms. [SAGE data are all available at GEO (http://www.ncbi.nlm.nih.gov/geo/) under accession nos. GSM55 and GSM56, which correspond to the forelimb and hindlimb raw SAGE data.]

Figure 1

Scatter plot of tag abundance distributions in the forelimb and hindlimb. Note the log scale. Each point represents a tag with a given frequency in the forelimb (X-axis) and hindlimb (Y-axis). The number at each point indicates the number of unique tags with a given frequency. (Dashed and solid lines) 0.05 and 0.01 P values for probability of differential expression, respectively. Tags falling outside these lines are differentially expressed according to the test statistic developed by Audic and Claverie (1997). Seventy SAGE tags fall outside the 1% confidence interval and 317 SAGE tags fall outside the 5% confidence interval. (Dotted line) Equal expression in the forelimb and hindlimb. Zero is plotted as 0.5 to be observed on a log scale.

Figure 2

Fraction of unique SAGE tags added to the database. (X-axis) Cumulative total number of SAGE tags in our combined forelimb/hindlimb database; (Y-axis) average fraction of unique SAGE tags added to the database. eSAGE calculates the fraction of unique SAGE tags extracted from each sequence file. The data points were calculated by averaging sequence file information over the cumulative SAGE tag interval (just after the previous data point and including the current data point). For example, of the last 17,000 tags added to the database (from ∼120,000 to ∼137,000 tags), an average of 16% were unique (or novel entries).

Figure 3

Distribution of Fit values for all tag-UniGene cluster matches. Fit values were calculated as described in Equation 1 and represent the proportion of tag-UniGene cluster matches that come from the given tag sequence plus the count of all other tag matches in the given UniGene cluster. In our experience, Fit values >0.9 generally result from UniGene clusters with numerous EST sequencing errors and usually are indicative of only one SAGE tag matching a UniGene cluster.

Figure 4

Venn diagram representation of the forelimb and hindlimb SAGE libraries. This figure is not drawn to scale. (A) (Light gray circle) Number of unique tags observed in the forelimb; (black circle) number in the hindlimb; (overlapping fraction) number of unique tags observed in both SAGE libraries; (bold numbers) tags observed more than once; (numbers in parentheses) unique tags, including those observed once. (B) Fraction of unique tags (Y-axis) from the subset of 27,131 unique tags observed in only one SAGE library (light gray and black, not including the overlapping fraction), with a cumulative frequency indicated (X-axis). For example, 97% of the unique tags in only one library were observed two times or less.

Figure 4

Venn diagram representation of the forelimb and hindlimb SAGE libraries. This figure is not drawn to scale. (A) (Light gray circle) Number of unique tags observed in the forelimb; (black circle) number in the hindlimb; (overlapping fraction) number of unique tags observed in both SAGE libraries; (bold numbers) tags observed more than once; (numbers in parentheses) unique tags, including those observed once. (B) Fraction of unique tags (Y-axis) from the subset of 27,131 unique tags observed in only one SAGE library (light gray and black, not including the overlapping fraction), with a cumulative frequency indicated (X-axis). For example, 97% of the unique tags in only one library were observed two times or less.

Figure 5

Venn diagram comparing the forelimb and hindlimb SAGE libraries with other libraries listed in Table 5. This figure is not drawn to scale. (Light gray circles) Forelimb SAGE library; (black circle) hindlimb SAGE library (as in Fig. 4); (white circle) pooled, nonlimb SAGE tags from six additional SAGE libraries; (bold numbers) tags observed more than once (tags represented in more than one library are inherently observed more than once); (numbers in parentheses) unique tags, including those observed once. For example, 407 tags were observed two times or more in the forelimb and not observed in either the hindlimb or pooled, nonlimb SAGE libraries.

Figure 6

Representative output from the ehm-tag-Mapping log file for the first two analyzed UniGene clusters. The first entry of the log file has been annotated for explanation purposes after the ###.