Oscillatory expression of the notch pathway gene Hes1 in synchronized mouse C2C12 myoblasts (A,B) and HES1 in synchronized human mesenchymal stem cells (C,D). Cells were synchronized by treatment in low-serum conditions, and restoration of normal serum levels induced oscillation of notch pathway genes. Robust multichip averaging methods were used to normalize microarray data, and levels of housekeeping genes such as beta-actin were relatively constant (gray lines in A, MOE430v2; C, HG-U133A). Affymetrix MOE430Av2 (A) and quantitative PCR (B) results for Hes1 expression in mouse C2C12 myoblasts are shown, with an oscillation period of 2 hours, consistent with previously published reports for mouse cells in cell culture (Hirata et al., 2002) and correlating with the average periodicity in mouse embryos (Gossler & Tam, 2002). Affymetrix HG-U133A (C) and quantitative PCR (D) results for HES1 expression in human UCB1 cells are shown with an oscillation period of approximately 5 hours. Quantitative PCR data points are shown with error bars indicating one SD. Gray bars indicate oscillatory peak regions (A-D).

Identification of genes with oscillatory expression in synchronized mesenchymal stem cells by Affymetrix HG-U133 microarray analysis. Genes with oscillatory expression were identified by Fourier analysis and cluster analysis, and the mouse homologues of 20 top candidate genes were selected for embryonic analysis. Shown are the gene expression levels of 9 genes that displayed localized expression in 9.5 dpc embryos: the notch pathway gene MAML3 (A), the wnt pathway genes NKD2 (B) and TCF7L2 (C), the glucocorticoid induced gene GLCCI1 (D), the notch target genes ID1 (E) and ID2 (F), and the genes CTGF (G), NUAK1 (H), and SLC2A3 (I). In addition, the expression time series for the snail homologue SNAI2 (J) is shown. Expression data for 8 hours is shown with microarray results (black line) and quantitative PCR results (gray line). Expression values are shown on a logarithmic scale. Levels of NKD2 were below the threshold of detection by Q-PCR. Gray bars indicate oscillatory peak regions (A-J).

Localized expression of the wnt pathway gene Nkd2 in 9.5 dpc (A, B) and 10.5 dpc (C-F) mouse embryos determined by whole mount in situ hybridization. Nkd2 expression in the caudal presomitic mesoderm is detectable beginning at 9.5 dpc stage (A, B; white arrowheads), with no expression visible at 8.5 dpc (data not shown). Low levels of expression are also present in maturing somites (A; black arrowheads) and in the rostral region of the first pharyngeal arch. Localization of expression of Nkd2 is similar in 10.5 dpc (C; black arrowhead), with higher levels of expression observed in the somites (C). Levels of Nkd2 expression within the PSM were variable (arrowheads), as shown in three PSM regions from 10.5 dpc embryos (D-F). Transverse sections from the caudal PSM of a 9.5 dpc embryo clearly show Nkd2 expression localized to the paraxial mesoderm (G) and in the tailbud mesoderm of a 10.5 dpc embryo (H, dorsal is at top). Localized expression of Nkd2, compared to the rostral PSM marker Mesp2 (I and magnified in J) and known cycling genes in the notch pathway (Lfng; K), wnt pathway (Axin2; L), snail (Snai1; M) in 9.5 dpc microdissected PSM halves. Expression in 9.5 dpc embryos was analyzed by whole mount in situ hybridization of microdissected PSM halves. For Nkd2, the broad region of PSM expression is caudal to the rostral PSM band of Lfng (K; n=8) and Mesp2 (I, J; n=4) and is expressed over a broader region than the caudal zone of expression of Axin2 (L; n=8) and Snai1 (M; n=4).

Expression of Maml3 (A, B), Nkd2 (C, D), and Lfng (E, F) oscillate in levels as assayed in split embryos analyzed by whole mount in situ hybridization (A, C, E) and quantitative PCR (B, D, F). 9.5 dpc embryos were split in half, with the left halves collected immediately and the right halves cultured for 1 hour and then collected for in situ analysis or RNA extraction. Three representative embryos are shown for Maml3, Nkd2, and Lfng (Embryo 1 analyzed by RNA in situ hybridization and Embryos 2 and 3 by Q-PCR). For Q-PCR analysis from Embryos 2 and 3 (B, D, F), expression values were normalized to Gapdh control. For comparison, levels of the housekeeping gene beta-actin (Actb) are shown. Levels of Maml3 were observed to vary in intensity (n=12) by in situ hybridization (A), as confirmed by Q-PCR (B). Levels of Nkd2 were also observed to vary by in situ hybridization (C; n=11), as confirmed by quantitative PCR (D). Nkd2 levels were measured to display 1.4 and 2.1 fold increase in expression by Q-PCR, whereas Maml3 displayed less variability (1.5 and 1.8). The cycling gene Lfng displayed significant changes in expression by in situ hybridization (E), consistent with previous reports (Forsberg et al., 1998; Aulehla et al., 1999), and displayed correspondingly large changes in expression level (2.5 and 3.9, F). By quantitative PCR, we only detected low levels of expression of the other genes present in the PSM, Glcci1 and Tcf7l2, and we did not observe clear evidence of dynamic expression by Q-PCR (data not shown).

A, Diagram illustrating oscillatory expression of Maml3 and Nkd2 compared to cycling and stage specific genes. Cycling genes such as Lfng display caudal to rostral changes in localized expression within the PSM. Stage-specific genes such as Mesp2 display the stripe of expression with variable length that changes during the somite cycle. Genes such as Maml3 and Nkd2 do not fit neatly into either category are in a category of genes with stationary oscillatory expression. Embryos are shown at cycling stages A, C, E (Forsberg et al., 1998). B, Diagram modeling expression of Nkd2 at the high and low ends of the oscillatory period. While the peak of expression can remain stationary, the expression levels can vary and expression boundaries can display some minor shifts.

Diagrams illustrating the levels of candidate oscillatory expression genes, assayed by Affymetrix microarray. Red indicates increased expression and green decreased expression relative to the mean level in the displayed HEAT map. Candidates were identified by Fourier analysis of expression time series (A, 824 gene probes on the HG-U133A array and B, 585 gene probes on the HG-U133B array). Additional analysis was used to identify the top 20 gene candidates, illustrated on C (HG-U133A array) and D (HG-U133B array).

Localized expression of the notch pathway gene Maml3 in 8.5 dpc (A-C), 9.0 dpc (D), 9.5 dpc (E-G) and 10.5 dpc (H) mouse embryos, as determined by whole mount in situ hybridization. Maml3 expression in the rostral presomitic mesoderm is detectable beginning at 8.5 dpc stage (A-C), with levels of expression within the PSM varying (arrowheads) relative to expression in the neural tube (asterisks). Expression in 9.5 dpc embryos was also present in the first pharyngeal arch and caudal midbrain (asterisks in E). Localization of expression of Maml3 within the PSM is similar in 10.5 dpc embryos (H). Transverse section from the rostral PSM demonstrates that Maml3 is expressed in the paraxial mesoderm and ventral neural of a 9.5 dpc embryo (I). Localized expression of Maml3, compared to the rostral PSM marker Mesp2 (J and magnified in K) and known cycling genes in the notch pathway (Lfng; L), wnt pathway (Axin2; M), snail (Snai1; N), in microdissected PSM halves. Expression of 9.5 dpc embryos was analyzed by whole mount in situ hybridization of microdissected PSM halves, with gene expression patterns compared. The broad region of Maml3 expression extends further rostrally than Mesp2 (J,K; n=4) and uniquely marks a region within the presomitic mesoderm extending from the rostral bands of expression of Lfng (L; n=5), Axin2 (M; n=4), and Snai1 (N; n=3) but diminishing prior to the tailbud region.

Expression of additional oscillatory gene candidates in 9.5 dpc mouse embryos. Mouse genes homologous to oscillatory genes identified in the human MSC microarray screen were determined as described. Whole mount in situ hybridization was used to determine expression patterns of 20 candidate genes. Selected genes with localized expression in 9.5 dpc embryos, including the wnt pathway gene Tcf7l2 (A,B), the glucocorticoid induced transcript 1 (Glcci1; C), the notch pathway genes Id1 and Id2 (D,E), connective tissue growth factor (Ctgf; F), the protein kinase Nuak1 (G), and Slc2a3 (H), are shown. We observed genes with expression in the paraxial mesoderm. Specifically, low levels of expression in the presomitic mesoderm are observed in Tcf7l2 (A, 9.5 dpc; B, 8.5 dpc; indicated with arrowheads) and Glcci1 (C). Somitic expression is observed in Glcci1 (C), Id1 (D), and Id2 (E), marked with arrowheads. Expression in the developing neural tube and/or pharyngeal arches (indicated by asterisks) is also observed for Id2 (E), Ctgf (F), Nuak1 (G), and Slc2a3 (H). Slc2a3 is also expressed in the surface ectoderm (H).

Expression of Maml3 and Nkd2 in Dll3^pu and Wnt3a^tm1Amc mutant embryos. The Dll3^pu mutation has been previously shown to disrupt oscillatory expression of Lfng but not Hes7 (Kusumi et al., 2004), but does not cause the complete loss of gene expression as observed for Dll1 mutants (Barrantes et al., 1999). All embryos are at 9.5 dpc stage, unless indicated otherwise. Expression of Maml3 (A-C) is observed in 10.5 dpc Dll3^pu/pu embryos (A, B; n=5) at levels comparable to that in wild-type (WT; C) embryos. Variation in Maml3 levels was observed in Dll3^pu mutant embryos, consistent with dynamic expression. In contrast, the more severe PSM disruptions in Wnt3a^tm1Amc targeted mutant embryos (D, E; n=5) resulted in a loss of Maml3 expression compared to wild type embryos (F). For Nkd2 (G-I), variation in expression levels is observed in Dll3^pu/pu embryos (G,H; n=8), consistent with potential oscillatory expression. In contrast, Nkd2 levels are severely decreased in Wnt3a mutant embryos (J, K; n=5) compared to wild-type embryos (L).