Figure 1.
Fore- and hind-limb buds were cut away from the embryo body as indicated by the red dash and RNA was processed from the full appendages outlined for stages E9.5 to E13.5. Light blue indicate mesenchymal condensations, and cartilage as determined by alcian blue staining is marked by thick black lines. Hindlimbs are morphologically delayed by ∼half a day.
Table 1.
Differentially Expressed Genes.
Figure 2.
Analysis of the Top 100 Up-regulated Genes.
279 genes were examined for their putative protein function, available in situ expression in the limb, and description of null mouse phenotype (A). Most known genes previously uncharacterized in the limb with (B), or novel genes (C) showed unique and restrictive expression pattern in the limb. A survey of in situs of known genes with musculoskeletal defects in the knockout mouse are in Figure S1.
Table 2.
Enrichment of functionally related genes in groups of co-expressed genes.
Figure 3.
C130021I20Rik is a Novel Gene Co-transcribed from the Lmx1b Promoter, a well Known Limb Gene.
C130021I20Rik was mapped to the Lmx1b gene locus (A) and found to be transcribed on the opposite strand. Time course in situ analysis revealed that C130021I20Rik (C) expression pattern is indistinguishable from its neighbor, Lmx1b (B), in the dorsal mesenchyme (b–c) suggesting that they are transcribed from the same divergent promoter and share limb-specific regulatory elements.
Figure 4.
Temporal Expression Pattern Analysis in the Developing Forelimb.
Clusters of co-transcribed genes were grouped into early genes (A), peak genes (B), late genes (C) and oscillating genes (D) based on their profiles. The number of genes corresponding to each curve are indicated below the trace.
Figure 5.
Differentiation Events Corresponding to Stages of Limb Development and Functional Category Enrichments that Describe some of these Events.
Here we graphically depict the change in cell types and tissue formation as a function of developmental stage, highlighting key events during bone, muscle, nerve and skin formation (A). Some key molecules known to participate in these processes are also included along with the cell type morphologies for different stages of skeletal development. GO analysis identified several enrichment categories including osteoblast differentiation (B), epithelial differentiation (C), anterior-posterior patterning (D), neuronal differentiation (E), digit morphogenesis (F), epithelial to mesenchymal transition (G), cartilage development (H), embryonic skeletal system morphogenesis (I) and limb morphogenesis (J). For each plot log2 intensity is on the y axis, and developmental stages are on the x-axis. For each gene the solid trace depicts limb expression and the dashed trace depicts expression found in the whole embryo. Each gene corresponds to a different symbol along the traces.
Figure 6.
Analysis of Stage Specific Gene Expression.
1140 transcripts were found to be significantly up-regulated exclusively at one developmental time point. A large fraction of these stage specific genes were found to be novel (A), in particular at E13.5 only 5% of the transcripts had confirmed musculoskeletal knockout phenotypes and 6% of the genes have been validated by in situ hybridization. A survey of available in situ expression profiles for genes with peak expression levels at E9.5 (B), E10.5 (C), E11.5 (D), E12.5 (E) and E13.5 (F) revealed that they display a wide range of patterns, and include closely related members of gene families such as Irx, Smarc and zinc finger transcription factors.
Figure 7.
Identifying Genes that Contribute to Limb Identity.
To identify genes specifically enriched in the forelimb or hindlimb, we adjusted for the developmental delay of hindlimbs by comparing forelimbs to same stage hindlimbs as well as to hindlimbs from a subsequent time point (A). More forelimb enriched genes were identified (B), and the genes with >2 fold up-regulation in the forelimb (C) or hindlimb (D) are displayed. Genes previously known to function as a limb-identity determinant are marked by red arrows (Tbx5; Hoxc10, Tbx4, Pitx1).
Figure 8.
Expression Patterns of Fore- and Hind-limb Specific Genes.
Qualitatively some genes identified as forelimb or hindlimb specific do not appear to be restricted to one limb such as Sp6, SmarcD2 and Hoxc10, yet quantitatively these transcripts are >2 fold up-regulated in one limb in relationship to the other.