Global Gene Expression Analysis of Murine Limb Development

Abstract

Detailed information about stage-specific changes in gene expression is crucial for understanding the gene regulatory networks underlying development and the various signal transduction pathways contributing to morphogenesis. Here we describe the global gene expression dynamics during early murine limb development, when cartilage, tendons, muscle, joints, vasculature and nerves are specified and the musculoskeletal system of limbs is established. We used whole-genome microarrays to identify genes with differential expression at 5 stages of limb development (E9.5 to 13.5), during fore- and hind-limb patterning. We found that the onset of limb formation is characterized by an up-regulation of transcription factors, which is followed by a massive activation of genes during E10.5 and E11.5 which levels off at later time points. Among the 3520 genes identified as significantly up-regulated in the limb, we find ∼30% to be novel, dramatically expanding the repertoire of candidate genes likely to function in the limb. Hierarchical and stage-specific clustering identified expression profiles that are likely to correlate with functional programs during limb development and further characterization of these transcripts will provide new insights into specific tissue patterning processes. Here, we provide for the first time a comprehensive analysis of developmentally regulated genes during murine limb development, and provide some novel insights into the expression dynamics governing limb morphogenesis.

Let-7 represses Nr6a1 and a mid-gestation developmental program in adult fibroblasts

“…4C). We confirmed the anti-correlation between let-7 and its ''high-confidence'' targets in a second, independent published time course of mouse embryonic limb bud development (Taher et al 2011). As before, the majority of ''highconfidence'' targets decreased as the embryonic limb bud developed (Supplemental Fig.…”

Retinoic acid receptor regulation of epimorphic and homeostatic regeneration in the axolotl

“…This might be due to the fact that many RA synthesis genes are upregulated after LE135 treatment. Many cluster 2/3 genes (n=101) are expressed in proximal developing limb buds in other limbed vertebrates or required for proper limb development [cluster 2 expressed in proximal limb: Meis1 (Mercader et al, 2000(Mercader et al, , 2005, Meis2 (Mercader et al, 2000(Mercader et al, , 2005, Pbx1 (Selleri et al, 2001), Arid5b (Ristevski et al, 2001); cluster 2 expressed in limb bud: Mia3 (Bosserhoff et al, 2004), Rac1 (Bell et al, 2004;Suzuki et al, 2013), Asph (Patel et al, 2014), Neo1 (Hong et al, 2012), Cyp26B1 (MacLean et al, 2001), Flrt2 (Haines et al, 2006), Rarγ (Pennimpede et al, 2010), Rbp1 (Gustafson et al, 1993), KIAA1217 (Semba et al, 2006); cluster 3 (Table S1) expressed in proximal limb: Fibin (Taher et al, 2011;Wakahara et al, 2007), Epha7 (Araujo et al, 1998), Nrip1 (Smith et al, 2014), Rnd3 (Bell et al, 2004); cluster 3 expressed in limb bud: Apcdd1 (Jukkola et al, 2004), Zfn638 (Bell et al, 2004), Stat3 (Gray et al, 2004), Tsh2 (Caubit et al, 2000;Erkner et al, 1999)]. The association of these genes with limb patterning in other vertebrates supports the idea that RA reprograms the distal cells to resemble a proximal limb cell fate.…”

“…Alox5 was the only exception because it was exclusively upregulated in RARβ antagonized limbs (LE135 versus DMSO +1.55-fold; RA versus DMSO −1.17). Seven of the 13 genes downregulated in RA-treated limbs are known to be expressed in the distal portion of the developing or regenerating vertebrate limb including Lhx9 (Gu and Kania, 2010;Tzchori et al, 2009), Zic5 (Merzdorf, 2007), Lmo1 (Taher et al, 2011), Lhx2 (Taher et al, 2011;Tzchori et al, 2009), Spry1 (Minowada et al, 1999;Wang and Beck, 2014), Msx2 (Bell et al, 2003;Carlson et al, 1998;Tribioli et al, 2002), HoxA13 (Gardiner et al, 1995;Haack and Gruss, 1993;Scotti et al, 2015), most of which are required for distal identity in developing mouse limbs. This suggests that distal-identity genes are silenced only in limbs undergoing PD duplication, similar to the transcriptional activation of proximalidentity genes during PD duplication.…”

Chromatin state signatures associated with tissue-specific gene expression and enhancer activity in the embryonic limb

“…H3K27ac regions more strongly marked in E10.5 forelimb or hindlimb compared with E11.5 were weakly enriched near genes up-regulated at this timepoint, while H3K27ac regions more strongly marked in E11.5 limb showed no significant enrichment near E11.5 up-regulated genes (Supplemental Tables S14, S15; Supplemental Fig. S9; Taher et al 2011). This is consistent with the small number of genes differentially expressed between E10.5 and E11.5 limbs (328 genes in forelimb and 348 in hindlimb, BHP #0.01; see Methods).…”

“…Statistics for all sequencing runs and resulting alignments are listed in Supplemental Table S17. We identified genes differentially expressed between E10.5 and E11.5 using mouse-limb microarray data from Taher et al (2011). Differential expression analysis was performed using the limma R package (Smyth 2004).…”