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Sonic hedgehog controls growth of external genitalia by regulating cell cycle kinetics.

Seifert AW, Zheng Z, Ormerod BK, Cohn MJ - Nat Commun (2010)

Bottom Line: In this study, we show that inactivation of Shh in external genitalia extends the cell cycle from 8.5 to 14.4 h, and genital growth is reduced by ∼75%.Transient Shh signalling establishes pattern in the genital tubercle; however, transcriptional levels of G1 cell cycle regulators are reduced.Cell cycle genes responded similarly to Shh inactivation in genitalia and limbs, suggesting that Shh may regulate growth by similar mechanisms in different organ systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Florida, Gainesville, FL 32611, USA.

ABSTRACT
During embryonic development, cells are instructed which position to occupy, they interpret these cues as differentiation programmes, and expand these patterns by growth. Sonic hedgehog (Shh) specifies positional identity in many organs; however, its role in growth is not well understood. In this study, we show that inactivation of Shh in external genitalia extends the cell cycle from 8.5 to 14.4 h, and genital growth is reduced by ∼75%. Transient Shh signalling establishes pattern in the genital tubercle; however, transcriptional levels of G1 cell cycle regulators are reduced. Consequently, G1 length is extended, leading to fewer progenitor cells entering S-phase. Cell cycle genes responded similarly to Shh inactivation in genitalia and limbs, suggesting that Shh may regulate growth by similar mechanisms in different organ systems. The finding that Shh regulates cell number by controlling the length of specific cell cycle phases identifies a novel mechanism by which Shh elaborates pattern during appendage development.

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Shh inactivation alters cell cycle gene expression in genital tubercles and limb          buds.(a, b) Quantitative comparison of transcript levels in                ShhcreERT2/C and Shh+/c            embryos assayed at three time points (indicated in key) using the RT2-Profiler PCR Array (SABiosciences). Tamoxifen injections were administered at E10.5            and E11.5. Changes in transcript level are expressed as percent change relative to            control embryos for G/S-phase cell cycle genes (a) and S- and G2/M-phase genes              (b). Error bars show ± s.e.m., n = 3 and asterisks denote significant            differences for E11.5 injection/E12.5 collection (Cyclin E1, P=0.030; Dp1,              P=0.055; Cyclin B1, P=0.019; Cdc25a, P=0.034; Mcm3, P=0.031),            E10.5 injection/E12.5 collection (Cyclin A1, P=0.011; E2f1, P=0.013),            E11.5 injection/E13.5 collection (Cyclin A1, P=0.0004; Cyclin E1, P=0.027;              Dp1, P=0.033; E2f1, P=0.010; Mcm2, P=0.052). (c) Graph            shows comparison of transcript levels in limb buds (grey bars) and genital tubercles            (black bars) of ShhcreERT2/C relative to                Shh+/C embryos. Tamoxifen injections were administered            at E10.5 and embryos were collected for qRT–PCR at E12.5. Changes in transcript level            are expressed as percent change relative to control embryos. Error bars show ± s.e.m.,              n = 3 and asterisks denote significant differences in E2f1 (GT,            P=0.010; limb, P=0.008), Dp1 (GT, P=0.012; limb,            P=0.026), Myc (GT, P=0.034; limb, P=0.039) and Rb1            (GT, P=0.040; limb, P=0.040).
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f4: Shh inactivation alters cell cycle gene expression in genital tubercles and limb buds.(a, b) Quantitative comparison of transcript levels in ShhcreERT2/C and Shh+/c embryos assayed at three time points (indicated in key) using the RT2-Profiler PCR Array (SABiosciences). Tamoxifen injections were administered at E10.5 and E11.5. Changes in transcript level are expressed as percent change relative to control embryos for G/S-phase cell cycle genes (a) and S- and G2/M-phase genes (b). Error bars show ± s.e.m., n = 3 and asterisks denote significant differences for E11.5 injection/E12.5 collection (Cyclin E1, P=0.030; Dp1, P=0.055; Cyclin B1, P=0.019; Cdc25a, P=0.034; Mcm3, P=0.031), E10.5 injection/E12.5 collection (Cyclin A1, P=0.011; E2f1, P=0.013), E11.5 injection/E13.5 collection (Cyclin A1, P=0.0004; Cyclin E1, P=0.027; Dp1, P=0.033; E2f1, P=0.010; Mcm2, P=0.052). (c) Graph shows comparison of transcript levels in limb buds (grey bars) and genital tubercles (black bars) of ShhcreERT2/C relative to Shh+/C embryos. Tamoxifen injections were administered at E10.5 and embryos were collected for qRT–PCR at E12.5. Changes in transcript level are expressed as percent change relative to control embryos. Error bars show ± s.e.m., n = 3 and asterisks denote significant differences in E2f1 (GT, P=0.010; limb, P=0.008), Dp1 (GT, P=0.012; limb, P=0.026), Myc (GT, P=0.034; limb, P=0.039) and Rb1 (GT, P=0.040; limb, P=0.040).

Mentions: Once a molecular pre-pattern has been established in a developing organ, elaboration of the pattern requires extensive growth. To dissect the role of Shh in the expansion of genital tubercle progenitor cells, we quantified genital tubercle volume and total cell number in ShhcreERT2/C mutant and control embryos (Fig. 3a–f). Twenty-four hours after inactivation of Shh signalling, genital tubercle volume and total cell number were decreased by ∼75% (t(4)=3.64, P=0.01 and t(4)=2.92, P=0.02 respectively; Fig. 3b–e). Neither cell death nor cell density differed significantly (for density, t(4)=1.33, P=0.12; Fig. 3f and Supplementary Fig. S2), indicating that the reduction in progenitor cell number was not due to dying cells. To test the hypothesis that the growth deficiency reflected a disruption of cell proliferation, we first searched for Shh target genes that could mediate its mitogenic effects. The transcription factors Foxf1 and Foxf2, which have been implicated in Shh-mediated control of the cell cycle2526, were downregulated within 24 h of Shh inactivation (Supplementary Fig. S3). We next used the quantitative RT2–PCR Profiler Array to monitor the response of 84 genes involved in cell cycle regulation, and compared ShhcreERT2/C mutant and control genitalia at different time points after inactivation of Shh (Fig. 4a,b). Examination of cell cycle genes 24 h after tamoxifen injection revealed significant reductions in the levels of cell cycle control genes that control both the G1/S transition (Cyclin E1, Dp1) and G2/M-phase progression (Cyclin B1, Cdc25a; Fig. 4a,b). Cyclin E1, Dp1 and the Dp binding partner E2f1 remained significantly reduced for at least 24 h after Shh inactivation, suggesting a sustained disruption in G1 progression (Fig. 4a,b). Myc levels also decreased (see below and Fig. 4c) but three Cdk inhibitors, Cdkn1a, Cdkn1b and Cdkn2a, did not change significantly (P=0.64, 0.959 and 0.492, respectively). We also observed a significant decrease in the S-phase promoting gene Cyclin A1 and a small but significant decrease in the helicase components Mcm2 and Mcm3, which are involved in DNA replication (Fig. 4a,b). These results show that loss of Shh causes a transient downregulation of G2/M promoting genes and a sustained decrease in the expression of genes that govern the G1/S transition. Our discovery that Shh regulates expression of Myc, Rb1, Dp1 and E2f1 suggests a novel mechanism by which Shh can exercise fine-scale control of cyclin levels through control of cyclin modifiers.


Sonic hedgehog controls growth of external genitalia by regulating cell cycle kinetics.

Seifert AW, Zheng Z, Ormerod BK, Cohn MJ - Nat Commun (2010)

Shh inactivation alters cell cycle gene expression in genital tubercles and limb          buds.(a, b) Quantitative comparison of transcript levels in                ShhcreERT2/C and Shh+/c            embryos assayed at three time points (indicated in key) using the RT2-Profiler PCR Array (SABiosciences). Tamoxifen injections were administered at E10.5            and E11.5. Changes in transcript level are expressed as percent change relative to            control embryos for G/S-phase cell cycle genes (a) and S- and G2/M-phase genes              (b). Error bars show ± s.e.m., n = 3 and asterisks denote significant            differences for E11.5 injection/E12.5 collection (Cyclin E1, P=0.030; Dp1,              P=0.055; Cyclin B1, P=0.019; Cdc25a, P=0.034; Mcm3, P=0.031),            E10.5 injection/E12.5 collection (Cyclin A1, P=0.011; E2f1, P=0.013),            E11.5 injection/E13.5 collection (Cyclin A1, P=0.0004; Cyclin E1, P=0.027;              Dp1, P=0.033; E2f1, P=0.010; Mcm2, P=0.052). (c) Graph            shows comparison of transcript levels in limb buds (grey bars) and genital tubercles            (black bars) of ShhcreERT2/C relative to                Shh+/C embryos. Tamoxifen injections were administered            at E10.5 and embryos were collected for qRT–PCR at E12.5. Changes in transcript level            are expressed as percent change relative to control embryos. Error bars show ± s.e.m.,              n = 3 and asterisks denote significant differences in E2f1 (GT,            P=0.010; limb, P=0.008), Dp1 (GT, P=0.012; limb,            P=0.026), Myc (GT, P=0.034; limb, P=0.039) and Rb1            (GT, P=0.040; limb, P=0.040).
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f4: Shh inactivation alters cell cycle gene expression in genital tubercles and limb buds.(a, b) Quantitative comparison of transcript levels in ShhcreERT2/C and Shh+/c embryos assayed at three time points (indicated in key) using the RT2-Profiler PCR Array (SABiosciences). Tamoxifen injections were administered at E10.5 and E11.5. Changes in transcript level are expressed as percent change relative to control embryos for G/S-phase cell cycle genes (a) and S- and G2/M-phase genes (b). Error bars show ± s.e.m., n = 3 and asterisks denote significant differences for E11.5 injection/E12.5 collection (Cyclin E1, P=0.030; Dp1, P=0.055; Cyclin B1, P=0.019; Cdc25a, P=0.034; Mcm3, P=0.031), E10.5 injection/E12.5 collection (Cyclin A1, P=0.011; E2f1, P=0.013), E11.5 injection/E13.5 collection (Cyclin A1, P=0.0004; Cyclin E1, P=0.027; Dp1, P=0.033; E2f1, P=0.010; Mcm2, P=0.052). (c) Graph shows comparison of transcript levels in limb buds (grey bars) and genital tubercles (black bars) of ShhcreERT2/C relative to Shh+/C embryos. Tamoxifen injections were administered at E10.5 and embryos were collected for qRT–PCR at E12.5. Changes in transcript level are expressed as percent change relative to control embryos. Error bars show ± s.e.m., n = 3 and asterisks denote significant differences in E2f1 (GT, P=0.010; limb, P=0.008), Dp1 (GT, P=0.012; limb, P=0.026), Myc (GT, P=0.034; limb, P=0.039) and Rb1 (GT, P=0.040; limb, P=0.040).
Mentions: Once a molecular pre-pattern has been established in a developing organ, elaboration of the pattern requires extensive growth. To dissect the role of Shh in the expansion of genital tubercle progenitor cells, we quantified genital tubercle volume and total cell number in ShhcreERT2/C mutant and control embryos (Fig. 3a–f). Twenty-four hours after inactivation of Shh signalling, genital tubercle volume and total cell number were decreased by ∼75% (t(4)=3.64, P=0.01 and t(4)=2.92, P=0.02 respectively; Fig. 3b–e). Neither cell death nor cell density differed significantly (for density, t(4)=1.33, P=0.12; Fig. 3f and Supplementary Fig. S2), indicating that the reduction in progenitor cell number was not due to dying cells. To test the hypothesis that the growth deficiency reflected a disruption of cell proliferation, we first searched for Shh target genes that could mediate its mitogenic effects. The transcription factors Foxf1 and Foxf2, which have been implicated in Shh-mediated control of the cell cycle2526, were downregulated within 24 h of Shh inactivation (Supplementary Fig. S3). We next used the quantitative RT2–PCR Profiler Array to monitor the response of 84 genes involved in cell cycle regulation, and compared ShhcreERT2/C mutant and control genitalia at different time points after inactivation of Shh (Fig. 4a,b). Examination of cell cycle genes 24 h after tamoxifen injection revealed significant reductions in the levels of cell cycle control genes that control both the G1/S transition (Cyclin E1, Dp1) and G2/M-phase progression (Cyclin B1, Cdc25a; Fig. 4a,b). Cyclin E1, Dp1 and the Dp binding partner E2f1 remained significantly reduced for at least 24 h after Shh inactivation, suggesting a sustained disruption in G1 progression (Fig. 4a,b). Myc levels also decreased (see below and Fig. 4c) but three Cdk inhibitors, Cdkn1a, Cdkn1b and Cdkn2a, did not change significantly (P=0.64, 0.959 and 0.492, respectively). We also observed a significant decrease in the S-phase promoting gene Cyclin A1 and a small but significant decrease in the helicase components Mcm2 and Mcm3, which are involved in DNA replication (Fig. 4a,b). These results show that loss of Shh causes a transient downregulation of G2/M promoting genes and a sustained decrease in the expression of genes that govern the G1/S transition. Our discovery that Shh regulates expression of Myc, Rb1, Dp1 and E2f1 suggests a novel mechanism by which Shh can exercise fine-scale control of cyclin levels through control of cyclin modifiers.

Bottom Line: In this study, we show that inactivation of Shh in external genitalia extends the cell cycle from 8.5 to 14.4 h, and genital growth is reduced by ∼75%.Transient Shh signalling establishes pattern in the genital tubercle; however, transcriptional levels of G1 cell cycle regulators are reduced.Cell cycle genes responded similarly to Shh inactivation in genitalia and limbs, suggesting that Shh may regulate growth by similar mechanisms in different organ systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Florida, Gainesville, FL 32611, USA.

ABSTRACT
During embryonic development, cells are instructed which position to occupy, they interpret these cues as differentiation programmes, and expand these patterns by growth. Sonic hedgehog (Shh) specifies positional identity in many organs; however, its role in growth is not well understood. In this study, we show that inactivation of Shh in external genitalia extends the cell cycle from 8.5 to 14.4 h, and genital growth is reduced by ∼75%. Transient Shh signalling establishes pattern in the genital tubercle; however, transcriptional levels of G1 cell cycle regulators are reduced. Consequently, G1 length is extended, leading to fewer progenitor cells entering S-phase. Cell cycle genes responded similarly to Shh inactivation in genitalia and limbs, suggesting that Shh may regulate growth by similar mechanisms in different organ systems. The finding that Shh regulates cell number by controlling the length of specific cell cycle phases identifies a novel mechanism by which Shh elaborates pattern during appendage development.

Show MeSH
Related in: MedlinePlus