Limits...
Developmental transcriptional networks are required to maintain neuronal subtype identity in the mature nervous system.

Eade KT, Fancher HA, Ridyard MS, Allan DW - PLoS Genet. (2012)

Bottom Line: We show that certain critical cross-regulatory relationships that had existed between these transcription factors during development were no longer present in the mature adult neuron.This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons.Together, our results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada.

ABSTRACT
During neurogenesis, transcription factors combinatorially specify neuronal fates and then differentiate subtype identities by inducing subtype-specific gene expression profiles. But how is neuronal subtype identity maintained in mature neurons? Modeling this question in two Drosophila neuronal subtypes (Tv1 and Tv4), we test whether the subtype transcription factor networks that direct differentiation during development are required persistently for long-term maintenance of subtype identity. By conditional transcription factor knockdown in adult Tv neurons after normal development, we find that most transcription factors within the Tv1/Tv4 subtype transcription networks are indeed required to maintain Tv1/Tv4 subtype-specific gene expression in adults. Thus, gene expression profiles are not simply "locked-in," but must be actively maintained by persistent developmental transcription factor networks. We also examined the cross-regulatory relationships between all transcription factors that persisted in adult Tv1/Tv4 neurons. We show that certain critical cross-regulatory relationships that had existed between these transcription factors during development were no longer present in the mature adult neuron. This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons. Together, our results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors. These findings have implications for understanding the maintenance of all long-lived cell types and the functional degeneration of neurons in the aging brain.

Show MeSH

Related in: MedlinePlus

dimm maintains peptidergic phenotype and dac has an enhanced maintenance function.(A,B) Representative images of adult Tv4 neurons expressing FMRFa peptide (red), apGal4,UAS-nlsEGFP (green) and Dimm (blue) at A10 at 29°C. FMRFa is downregulated and Dimm is lost in rev4,dimmdsRNAi (B) compared to w1118 control (A). (C,D) Quantification of FMRFa peptide at A10 (C) and FMRFa transcript at A20 (D) in individual adult Tv4 neurons at 29°C. (C)* p<0.0001 rev4,dimmdsRNAi (n = 19) compared to w1118 control (n = 42). (D)* p<0.0001 rev4,dimmdsRNAi (n = 30) compared to w1118 control (n = 58). (E,F) Representative images of Tv4 neurons expressing mature FMRFa peptide (red), apGal4,UAS-nlsEGFP (green) and PHM (blue) in adult Tv4 neurons at A10 at 29°C. PHM is lost in rev4,dimmdsRNAi (n = 26) (F) compared to w1118 control (n = 30) (E). (G,H) Images of adult Tv4 neurons expressing FMRFa peptide (red), apGal4,UAS-nEGFP (green) and Dac (blue) at A10 at 29°C. FMRFa is downregulated and Dac immunoreactivity is lost in dacdsRNAi (H) compared to w1118 control (G). (I,J) Quantification of FMRFa peptide in individual adult Tv4 neurons at A10 at 29°C (I), and in L1 larval Tv4 neurons in dac  mutants. (J) * p<0.0001 dacdsRNAi (n = 26) compared to w1118 control (n = 47). (J) ** P = 0.02 dac−/− (n = 31) compared to w1118 control (n = 22). Genotypes: w1118 (UAS-dicer2/+; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); rev4,dimmdsRNAi (UAS-dicer2/+; apGal4/rev4, UAS-dimmdsRNAi; tub-Gal80TS, UAS-nEGFP/+); dacdsRNAi (UAS-dicer2/+; apGal4/UAS-dacdsRNAi; tub-Gal80TS, UAS-nEGFP/+); dac−/− (dac3/dacDf(3L)EXEL 7066).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3285578&req=5

pgen-1002501-g003: dimm maintains peptidergic phenotype and dac has an enhanced maintenance function.(A,B) Representative images of adult Tv4 neurons expressing FMRFa peptide (red), apGal4,UAS-nlsEGFP (green) and Dimm (blue) at A10 at 29°C. FMRFa is downregulated and Dimm is lost in rev4,dimmdsRNAi (B) compared to w1118 control (A). (C,D) Quantification of FMRFa peptide at A10 (C) and FMRFa transcript at A20 (D) in individual adult Tv4 neurons at 29°C. (C)* p<0.0001 rev4,dimmdsRNAi (n = 19) compared to w1118 control (n = 42). (D)* p<0.0001 rev4,dimmdsRNAi (n = 30) compared to w1118 control (n = 58). (E,F) Representative images of Tv4 neurons expressing mature FMRFa peptide (red), apGal4,UAS-nlsEGFP (green) and PHM (blue) in adult Tv4 neurons at A10 at 29°C. PHM is lost in rev4,dimmdsRNAi (n = 26) (F) compared to w1118 control (n = 30) (E). (G,H) Images of adult Tv4 neurons expressing FMRFa peptide (red), apGal4,UAS-nEGFP (green) and Dac (blue) at A10 at 29°C. FMRFa is downregulated and Dac immunoreactivity is lost in dacdsRNAi (H) compared to w1118 control (G). (I,J) Quantification of FMRFa peptide in individual adult Tv4 neurons at A10 at 29°C (I), and in L1 larval Tv4 neurons in dac mutants. (J) * p<0.0001 dacdsRNAi (n = 26) compared to w1118 control (n = 47). (J) ** P = 0.02 dac−/− (n = 31) compared to w1118 control (n = 22). Genotypes: w1118 (UAS-dicer2/+; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); rev4,dimmdsRNAi (UAS-dicer2/+; apGal4/rev4, UAS-dimmdsRNAi; tub-Gal80TS, UAS-nEGFP/+); dacdsRNAi (UAS-dicer2/+; apGal4/UAS-dacdsRNAi; tub-Gal80TS, UAS-nEGFP/+); dac−/− (dac3/dacDf(3L)EXEL 7066).

Mentions: We induced dimmdsRNAi at A1 and found that immunoreactivity to the mature amidated FMRFa peptide was rapidly and profoundly reduced by dimmdsRNAi to 24.0±3.2% of control by A10 (p<0.0001), and this was enhanced to 9.8±1.6% of control in dimm heterozygotes (p<0.0001 to control and dimm heterozygotes, p<0.001 to dimmdsRNAi alone) (Figure 3A–3C, Figure S3). Immunoreactivity to Dimm demonstrated that it had been eliminated (Table S4). In contrast, FMRFa transcript in adults was downregulated in dimm heterozygotes to 67.1±2.9% of control at A20 (p<0.0001) (Figure 3D). Similar effects were observed using a dimmdsRNAi that targets different dimm sequences (Table S2). It is notable that downregulation of the transcript was only observed after 20 days of dimmdsRNAi induction but the peptide was profoundly reduced after only 10 days of induction. In late Stage 17 embryonic dimm mutants, immunoreactivity to the mature amidated FMRFa peptide was profoundly reduced, but the extent to which FMRFa transcript was affected had not been quantified [10], [18]. Here, we find that FMRFa transcript was only modestly downregulated in late Stage 17 embryonic dimm mutants to 71.6±3.9% of controls (wild type control n = 54, dimm mutant n = 34 (p<0.0001)). Thus, we conclude that dimm retains its role in the initiation and maintenance of both FMRFa transcript and mature peptide. Why is the mature peptide more responsive to dimmdsRNAi than is the transcript? We postulated that this was due to dimm's regulation of proprotein convertases and peptide amidases in secretory neurons, both of which are required to process the FMRFa prepropeptide into amidated neuropeptides [10], [20], [25]. We tested this in adults by examining expression of peptidylglycine α-hydroxylating monooxygenase after dimmdsRNAi induction (PHM). Confirming our hypothesis, dimmdsRNAi entirely eliminated PHM immunoreactivity in Tv4 neurons (Figure 3E, 3F). Thus, the maintenance of neuropeptide-processing enzyme expression and biosynthesis of the amidated FMRFa peptide is highly dependent upon persistent dimm function in adult Tv neurons.


Developmental transcriptional networks are required to maintain neuronal subtype identity in the mature nervous system.

Eade KT, Fancher HA, Ridyard MS, Allan DW - PLoS Genet. (2012)

dimm maintains peptidergic phenotype and dac has an enhanced maintenance function.(A,B) Representative images of adult Tv4 neurons expressing FMRFa peptide (red), apGal4,UAS-nlsEGFP (green) and Dimm (blue) at A10 at 29°C. FMRFa is downregulated and Dimm is lost in rev4,dimmdsRNAi (B) compared to w1118 control (A). (C,D) Quantification of FMRFa peptide at A10 (C) and FMRFa transcript at A20 (D) in individual adult Tv4 neurons at 29°C. (C)* p<0.0001 rev4,dimmdsRNAi (n = 19) compared to w1118 control (n = 42). (D)* p<0.0001 rev4,dimmdsRNAi (n = 30) compared to w1118 control (n = 58). (E,F) Representative images of Tv4 neurons expressing mature FMRFa peptide (red), apGal4,UAS-nlsEGFP (green) and PHM (blue) in adult Tv4 neurons at A10 at 29°C. PHM is lost in rev4,dimmdsRNAi (n = 26) (F) compared to w1118 control (n = 30) (E). (G,H) Images of adult Tv4 neurons expressing FMRFa peptide (red), apGal4,UAS-nEGFP (green) and Dac (blue) at A10 at 29°C. FMRFa is downregulated and Dac immunoreactivity is lost in dacdsRNAi (H) compared to w1118 control (G). (I,J) Quantification of FMRFa peptide in individual adult Tv4 neurons at A10 at 29°C (I), and in L1 larval Tv4 neurons in dac  mutants. (J) * p<0.0001 dacdsRNAi (n = 26) compared to w1118 control (n = 47). (J) ** P = 0.02 dac−/− (n = 31) compared to w1118 control (n = 22). Genotypes: w1118 (UAS-dicer2/+; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); rev4,dimmdsRNAi (UAS-dicer2/+; apGal4/rev4, UAS-dimmdsRNAi; tub-Gal80TS, UAS-nEGFP/+); dacdsRNAi (UAS-dicer2/+; apGal4/UAS-dacdsRNAi; tub-Gal80TS, UAS-nEGFP/+); dac−/− (dac3/dacDf(3L)EXEL 7066).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285578&req=5

pgen-1002501-g003: dimm maintains peptidergic phenotype and dac has an enhanced maintenance function.(A,B) Representative images of adult Tv4 neurons expressing FMRFa peptide (red), apGal4,UAS-nlsEGFP (green) and Dimm (blue) at A10 at 29°C. FMRFa is downregulated and Dimm is lost in rev4,dimmdsRNAi (B) compared to w1118 control (A). (C,D) Quantification of FMRFa peptide at A10 (C) and FMRFa transcript at A20 (D) in individual adult Tv4 neurons at 29°C. (C)* p<0.0001 rev4,dimmdsRNAi (n = 19) compared to w1118 control (n = 42). (D)* p<0.0001 rev4,dimmdsRNAi (n = 30) compared to w1118 control (n = 58). (E,F) Representative images of Tv4 neurons expressing mature FMRFa peptide (red), apGal4,UAS-nlsEGFP (green) and PHM (blue) in adult Tv4 neurons at A10 at 29°C. PHM is lost in rev4,dimmdsRNAi (n = 26) (F) compared to w1118 control (n = 30) (E). (G,H) Images of adult Tv4 neurons expressing FMRFa peptide (red), apGal4,UAS-nEGFP (green) and Dac (blue) at A10 at 29°C. FMRFa is downregulated and Dac immunoreactivity is lost in dacdsRNAi (H) compared to w1118 control (G). (I,J) Quantification of FMRFa peptide in individual adult Tv4 neurons at A10 at 29°C (I), and in L1 larval Tv4 neurons in dac mutants. (J) * p<0.0001 dacdsRNAi (n = 26) compared to w1118 control (n = 47). (J) ** P = 0.02 dac−/− (n = 31) compared to w1118 control (n = 22). Genotypes: w1118 (UAS-dicer2/+; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); rev4,dimmdsRNAi (UAS-dicer2/+; apGal4/rev4, UAS-dimmdsRNAi; tub-Gal80TS, UAS-nEGFP/+); dacdsRNAi (UAS-dicer2/+; apGal4/UAS-dacdsRNAi; tub-Gal80TS, UAS-nEGFP/+); dac−/− (dac3/dacDf(3L)EXEL 7066).
Mentions: We induced dimmdsRNAi at A1 and found that immunoreactivity to the mature amidated FMRFa peptide was rapidly and profoundly reduced by dimmdsRNAi to 24.0±3.2% of control by A10 (p<0.0001), and this was enhanced to 9.8±1.6% of control in dimm heterozygotes (p<0.0001 to control and dimm heterozygotes, p<0.001 to dimmdsRNAi alone) (Figure 3A–3C, Figure S3). Immunoreactivity to Dimm demonstrated that it had been eliminated (Table S4). In contrast, FMRFa transcript in adults was downregulated in dimm heterozygotes to 67.1±2.9% of control at A20 (p<0.0001) (Figure 3D). Similar effects were observed using a dimmdsRNAi that targets different dimm sequences (Table S2). It is notable that downregulation of the transcript was only observed after 20 days of dimmdsRNAi induction but the peptide was profoundly reduced after only 10 days of induction. In late Stage 17 embryonic dimm mutants, immunoreactivity to the mature amidated FMRFa peptide was profoundly reduced, but the extent to which FMRFa transcript was affected had not been quantified [10], [18]. Here, we find that FMRFa transcript was only modestly downregulated in late Stage 17 embryonic dimm mutants to 71.6±3.9% of controls (wild type control n = 54, dimm mutant n = 34 (p<0.0001)). Thus, we conclude that dimm retains its role in the initiation and maintenance of both FMRFa transcript and mature peptide. Why is the mature peptide more responsive to dimmdsRNAi than is the transcript? We postulated that this was due to dimm's regulation of proprotein convertases and peptide amidases in secretory neurons, both of which are required to process the FMRFa prepropeptide into amidated neuropeptides [10], [20], [25]. We tested this in adults by examining expression of peptidylglycine α-hydroxylating monooxygenase after dimmdsRNAi induction (PHM). Confirming our hypothesis, dimmdsRNAi entirely eliminated PHM immunoreactivity in Tv4 neurons (Figure 3E, 3F). Thus, the maintenance of neuropeptide-processing enzyme expression and biosynthesis of the amidated FMRFa peptide is highly dependent upon persistent dimm function in adult Tv neurons.

Bottom Line: We show that certain critical cross-regulatory relationships that had existed between these transcription factors during development were no longer present in the mature adult neuron.This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons.Together, our results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada.

ABSTRACT
During neurogenesis, transcription factors combinatorially specify neuronal fates and then differentiate subtype identities by inducing subtype-specific gene expression profiles. But how is neuronal subtype identity maintained in mature neurons? Modeling this question in two Drosophila neuronal subtypes (Tv1 and Tv4), we test whether the subtype transcription factor networks that direct differentiation during development are required persistently for long-term maintenance of subtype identity. By conditional transcription factor knockdown in adult Tv neurons after normal development, we find that most transcription factors within the Tv1/Tv4 subtype transcription networks are indeed required to maintain Tv1/Tv4 subtype-specific gene expression in adults. Thus, gene expression profiles are not simply "locked-in," but must be actively maintained by persistent developmental transcription factor networks. We also examined the cross-regulatory relationships between all transcription factors that persisted in adult Tv1/Tv4 neurons. We show that certain critical cross-regulatory relationships that had existed between these transcription factors during development were no longer present in the mature adult neuron. This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons. Together, our results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors. These findings have implications for understanding the maintenance of all long-lived cell types and the functional degeneration of neurons in the aging brain.

Show MeSH
Related in: MedlinePlus