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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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Related in: MedlinePlus

A model for Shh-mediated integration of growth and patterning.Shh activity in wild-type (WT) and ShhcreERT2/C (mutant)            genital tubercles is shown in top panel, and is based on analysis of Ptch1            expression (see Fig. 1c). During outgrowth of the genital            tubercle, cell populations (red, tan, black coloured circles) defined by regionalized            gene expression are exposed to secreted Shh (blue shaded areas). In wild-type genitalia,            these cells divide approximately every 8.5 h and, as these progenitor pools double in            number, this expands gene expression patterns. Following loss of Shh activity in mutant            genitalia, cells continue to divide but cell cycle length increases to 14.4 h. This            leads to a reduction in both the doubling rate of progenitor pools and the overall size            of the genital tubercle. The general molecular pattern of the mutant tubercle is            retained. Large red circles above and below tubercles represent the doubling time of all            cells in the genital tubercle. Tc, total cell cycle time.
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f6: A model for Shh-mediated integration of growth and patterning.Shh activity in wild-type (WT) and ShhcreERT2/C (mutant) genital tubercles is shown in top panel, and is based on analysis of Ptch1 expression (see Fig. 1c). During outgrowth of the genital tubercle, cell populations (red, tan, black coloured circles) defined by regionalized gene expression are exposed to secreted Shh (blue shaded areas). In wild-type genitalia, these cells divide approximately every 8.5 h and, as these progenitor pools double in number, this expands gene expression patterns. Following loss of Shh activity in mutant genitalia, cells continue to divide but cell cycle length increases to 14.4 h. This leads to a reduction in both the doubling rate of progenitor pools and the overall size of the genital tubercle. The general molecular pattern of the mutant tubercle is retained. Large red circles above and below tubercles represent the doubling time of all cells in the genital tubercle. Tc, total cell cycle time.

Mentions: How could these relatively small proportional shifts in cell cycle phase lead to the large growth differences that result from deletion of Shh? If Shh is involved in regulating G1/S and G2/M transitions, then one possibility is that loss of Shh signalling induces cells to arrest at specific cycle checkpoints, thereby arresting proliferation or inducing apoptosis; however, such changes did not occur after deletion of Shh (Fig. 1d and Supplementary Fig. S2). Alternatively, inactivation of Shh could decrease the rate of progression through G1, which would be reflected by increased cycle length. To calculate cell cycle kinetics of progenitor cells in the developing genital tubercle mesenchyme, we applied the principles developed by Nowakowski et al.31 for quantification of cell cycle length. The relative lengths of S-phase (TS) and the entire cell cycle (Tc) were determined for both ShhcreERT2/C (n=3) and wild-type littermates (n=3) by measuring the proportions of cells in S-phase (BrdU/4,6-diamidino-2-phenylindole (DAPI) positive), S-phase cells that have cycled through G2/M (BrdU/PHH3/DAPI positive) and unlabelled cells (DAPI positive; see formulas in Fig. 5d and labelled cells in Fig. 5e–i). Inactivation of Shh signalling at E11.5 resulted in lengthening of the entire cell cycle (Tc) from 8.5 to 14.4 h (t(4)=2.83; P=0.024) (Fig. 5b). BrdU analysis revealed a greater proportion of unlabelled cells in ShhcreERT2/C mutants (t(4)=1.62; P=0.09), suggesting that this lengthening is not a result of altered S-phase duration but more likely reflects a delay in G1 or the G1/S checkpoint (Fig. 5b). Such a marked increase in cell cycle duration would be expected to reduce the total cell number, which may account for the ∼75% reduction of tubercle volume in ShhcreERT2/C mutants. Indeed, when the proportion of cells in each phase of the cell cycle is weighed against total cell number, the data show that the loss of Shh signalling decreases the cycling cell population by ∼73% (Fig. 5c). Thus, loss of Shh activity lengthens the time that cells spend in G1/G0, thereby reducing the number of cells in S-phase, which, in turn, feeds fewer cells into G2/M-phase and, ultimately, back into the cell cycle (Fig. 6). Taken together, these data indicate that Shh controls the rate of progenitor cell proliferation, and thus progenitor pool size, by regulating the speed of the cell cycle. This highlights a novel mechanism for Shh-mediated control of organ growth.


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

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

A model for Shh-mediated integration of growth and patterning.Shh activity in wild-type (WT) and ShhcreERT2/C (mutant)            genital tubercles is shown in top panel, and is based on analysis of Ptch1            expression (see Fig. 1c). During outgrowth of the genital            tubercle, cell populations (red, tan, black coloured circles) defined by regionalized            gene expression are exposed to secreted Shh (blue shaded areas). In wild-type genitalia,            these cells divide approximately every 8.5 h and, as these progenitor pools double in            number, this expands gene expression patterns. Following loss of Shh activity in mutant            genitalia, cells continue to divide but cell cycle length increases to 14.4 h. This            leads to a reduction in both the doubling rate of progenitor pools and the overall size            of the genital tubercle. The general molecular pattern of the mutant tubercle is            retained. Large red circles above and below tubercles represent the doubling time of all            cells in the genital tubercle. Tc, total cell cycle time.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2964453&req=5

f6: A model for Shh-mediated integration of growth and patterning.Shh activity in wild-type (WT) and ShhcreERT2/C (mutant) genital tubercles is shown in top panel, and is based on analysis of Ptch1 expression (see Fig. 1c). During outgrowth of the genital tubercle, cell populations (red, tan, black coloured circles) defined by regionalized gene expression are exposed to secreted Shh (blue shaded areas). In wild-type genitalia, these cells divide approximately every 8.5 h and, as these progenitor pools double in number, this expands gene expression patterns. Following loss of Shh activity in mutant genitalia, cells continue to divide but cell cycle length increases to 14.4 h. This leads to a reduction in both the doubling rate of progenitor pools and the overall size of the genital tubercle. The general molecular pattern of the mutant tubercle is retained. Large red circles above and below tubercles represent the doubling time of all cells in the genital tubercle. Tc, total cell cycle time.
Mentions: How could these relatively small proportional shifts in cell cycle phase lead to the large growth differences that result from deletion of Shh? If Shh is involved in regulating G1/S and G2/M transitions, then one possibility is that loss of Shh signalling induces cells to arrest at specific cycle checkpoints, thereby arresting proliferation or inducing apoptosis; however, such changes did not occur after deletion of Shh (Fig. 1d and Supplementary Fig. S2). Alternatively, inactivation of Shh could decrease the rate of progression through G1, which would be reflected by increased cycle length. To calculate cell cycle kinetics of progenitor cells in the developing genital tubercle mesenchyme, we applied the principles developed by Nowakowski et al.31 for quantification of cell cycle length. The relative lengths of S-phase (TS) and the entire cell cycle (Tc) were determined for both ShhcreERT2/C (n=3) and wild-type littermates (n=3) by measuring the proportions of cells in S-phase (BrdU/4,6-diamidino-2-phenylindole (DAPI) positive), S-phase cells that have cycled through G2/M (BrdU/PHH3/DAPI positive) and unlabelled cells (DAPI positive; see formulas in Fig. 5d and labelled cells in Fig. 5e–i). Inactivation of Shh signalling at E11.5 resulted in lengthening of the entire cell cycle (Tc) from 8.5 to 14.4 h (t(4)=2.83; P=0.024) (Fig. 5b). BrdU analysis revealed a greater proportion of unlabelled cells in ShhcreERT2/C mutants (t(4)=1.62; P=0.09), suggesting that this lengthening is not a result of altered S-phase duration but more likely reflects a delay in G1 or the G1/S checkpoint (Fig. 5b). Such a marked increase in cell cycle duration would be expected to reduce the total cell number, which may account for the ∼75% reduction of tubercle volume in ShhcreERT2/C mutants. Indeed, when the proportion of cells in each phase of the cell cycle is weighed against total cell number, the data show that the loss of Shh signalling decreases the cycling cell population by ∼73% (Fig. 5c). Thus, loss of Shh activity lengthens the time that cells spend in G1/G0, thereby reducing the number of cells in S-phase, which, in turn, feeds fewer cells into G2/M-phase and, ultimately, back into the cell cycle (Fig. 6). Taken together, these data indicate that Shh controls the rate of progenitor cell proliferation, and thus progenitor pool size, by regulating the speed of the cell cycle. This highlights a novel mechanism for Shh-mediated control of organ growth.

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