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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.

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Changes in Tv4 network configuration for maintenance.(A–E) eya regulates FMRFa independently of BMP signaling. (A,B) Nuclear pMad accumulation (red) in Tv4 (arrowhead) was downregulated in eyadsRNAi flies (B) compared to w1118 control (A). (C) Expression of eyadsRNAi in the presence of constitutively-activated Thickveins and Saxophone BMP type I receptors (TSA) activated pMad accumulation (red) in all Tv neurons (including Tv4; arrow) but failed to rescue FMRFa (blue) compared to w1118 control. (D) Quantification of pMad immunoreactivity in Tv4 nucleus in w1118 and eyadsRNAi flies. * p<0.0001 eyadsRNAi (n = 30) compared to w1118 control (n = 30). (E) Quantification of FMRFa immunoreactivity in Tv4 in w1118, eyadsRNAi and eyadsRNAi; TSA. * p<0.0001 eyadsRNAi (n = 38) and eyadsRNAi; TSA (n = 42) compared to w1118 control (n = 34). NSD, no significant difference between eyadsRNAi and eyadsRNAi; TSA. (F–H) eya does not regulate dimm in adult Tv4. dimm (red) in Tv neurons (blue) is maintained in eyadsRNAi (G) compared to w1118 control (F). (H) Quantification of Dimm immunoreactivity in Tv4. There is no significant difference between eyadsRNAi (n = 24) and w1118 (n = 25) controls (p = 0.67). (I–K) ap regulates dimm in adult Tv4. dimm (red) in Tv neurons (blue) is downregulated in apdsRNAi (J) compared to w1118 control (I). (K) Quantification of Dimm immunoreactivity in Tv4. * p<0.0001 apdsRNAi (n = 24) compared to w1118 (n = 35). (L) Model depicting regulatory configuration of ap, eya, dimm and BMP signaling from embryonic to adult Tv4. The dependence of dimm on eya expression is not maintained (X). Genotypes: w1118 (A,D, F,H, I,J) (UAS-dicer2/+; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); eyadsRNAi flies (B,D,E, G,H) (UAS-dicer2/UAS-eyadsRNAi; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); eyadsRNAi; TSA flies (C,E) (UAS-dicer2/UAS-eyadsRNAi; apGal4/UAS-tkvA, UAS-saxA; tub-Gal80TS, UAS-nEGFP/+). apdsRNAi flies (J),K (UAS-dicer2/+; apGal4; tub-Gal80TS, UAS-nEGFP/UAS-apdsRNAi).
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pgen-1002501-g004: Changes in Tv4 network configuration for maintenance.(A–E) eya regulates FMRFa independently of BMP signaling. (A,B) Nuclear pMad accumulation (red) in Tv4 (arrowhead) was downregulated in eyadsRNAi flies (B) compared to w1118 control (A). (C) Expression of eyadsRNAi in the presence of constitutively-activated Thickveins and Saxophone BMP type I receptors (TSA) activated pMad accumulation (red) in all Tv neurons (including Tv4; arrow) but failed to rescue FMRFa (blue) compared to w1118 control. (D) Quantification of pMad immunoreactivity in Tv4 nucleus in w1118 and eyadsRNAi flies. * p<0.0001 eyadsRNAi (n = 30) compared to w1118 control (n = 30). (E) Quantification of FMRFa immunoreactivity in Tv4 in w1118, eyadsRNAi and eyadsRNAi; TSA. * p<0.0001 eyadsRNAi (n = 38) and eyadsRNAi; TSA (n = 42) compared to w1118 control (n = 34). NSD, no significant difference between eyadsRNAi and eyadsRNAi; TSA. (F–H) eya does not regulate dimm in adult Tv4. dimm (red) in Tv neurons (blue) is maintained in eyadsRNAi (G) compared to w1118 control (F). (H) Quantification of Dimm immunoreactivity in Tv4. There is no significant difference between eyadsRNAi (n = 24) and w1118 (n = 25) controls (p = 0.67). (I–K) ap regulates dimm in adult Tv4. dimm (red) in Tv neurons (blue) is downregulated in apdsRNAi (J) compared to w1118 control (I). (K) Quantification of Dimm immunoreactivity in Tv4. * p<0.0001 apdsRNAi (n = 24) compared to w1118 (n = 35). (L) Model depicting regulatory configuration of ap, eya, dimm and BMP signaling from embryonic to adult Tv4. The dependence of dimm on eya expression is not maintained (X). Genotypes: w1118 (A,D, F,H, I,J) (UAS-dicer2/+; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); eyadsRNAi flies (B,D,E, G,H) (UAS-dicer2/UAS-eyadsRNAi; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); eyadsRNAi; TSA flies (C,E) (UAS-dicer2/UAS-eyadsRNAi; apGal4/UAS-tkvA, UAS-saxA; tub-Gal80TS, UAS-nEGFP/+). apdsRNAi flies (J),K (UAS-dicer2/+; apGal4; tub-Gal80TS, UAS-nEGFP/UAS-apdsRNAi).

Mentions: BMP signaling in embryonic Tv4 neurons is dramatically reduced in eya mutants [11]. We expressed eyadsRNAi in adults until A15 and found that nuclear pMad, an indicator of BMP activity [12], was significantly downregulated to 47.9%±2.9 of control (Figure 4A, 4B, 4D). As BMP signaling is required for FMRFa expression in embryos and adults, we asked whether eya-dependence of FMRFa in adults is due to reduced BMP signaling. To do this, we simultaneously expressed eyadsRNAi and restored BMP signaling, using constitutively-activated type I BMP-receptors, thickveins and saxophone. Even though nuclear pMad was robustly activated in all Tv neurons, eyadsRNAi-induced FMRFa downregulation was not rescued (Figure 4C, 4E). Thus, in adults, eya independently maintains both BMP signaling and FMRFa expression.


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)

Changes in Tv4 network configuration for maintenance.(A–E) eya regulates FMRFa independently of BMP signaling. (A,B) Nuclear pMad accumulation (red) in Tv4 (arrowhead) was downregulated in eyadsRNAi flies (B) compared to w1118 control (A). (C) Expression of eyadsRNAi in the presence of constitutively-activated Thickveins and Saxophone BMP type I receptors (TSA) activated pMad accumulation (red) in all Tv neurons (including Tv4; arrow) but failed to rescue FMRFa (blue) compared to w1118 control. (D) Quantification of pMad immunoreactivity in Tv4 nucleus in w1118 and eyadsRNAi flies. * p<0.0001 eyadsRNAi (n = 30) compared to w1118 control (n = 30). (E) Quantification of FMRFa immunoreactivity in Tv4 in w1118, eyadsRNAi and eyadsRNAi; TSA. * p<0.0001 eyadsRNAi (n = 38) and eyadsRNAi; TSA (n = 42) compared to w1118 control (n = 34). NSD, no significant difference between eyadsRNAi and eyadsRNAi; TSA. (F–H) eya does not regulate dimm in adult Tv4. dimm (red) in Tv neurons (blue) is maintained in eyadsRNAi (G) compared to w1118 control (F). (H) Quantification of Dimm immunoreactivity in Tv4. There is no significant difference between eyadsRNAi (n = 24) and w1118 (n = 25) controls (p = 0.67). (I–K) ap regulates dimm in adult Tv4. dimm (red) in Tv neurons (blue) is downregulated in apdsRNAi (J) compared to w1118 control (I). (K) Quantification of Dimm immunoreactivity in Tv4. * p<0.0001 apdsRNAi (n = 24) compared to w1118 (n = 35). (L) Model depicting regulatory configuration of ap, eya, dimm and BMP signaling from embryonic to adult Tv4. The dependence of dimm on eya expression is not maintained (X). Genotypes: w1118 (A,D, F,H, I,J) (UAS-dicer2/+; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); eyadsRNAi flies (B,D,E, G,H) (UAS-dicer2/UAS-eyadsRNAi; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); eyadsRNAi; TSA flies (C,E) (UAS-dicer2/UAS-eyadsRNAi; apGal4/UAS-tkvA, UAS-saxA; tub-Gal80TS, UAS-nEGFP/+). apdsRNAi flies (J),K (UAS-dicer2/+; apGal4; tub-Gal80TS, UAS-nEGFP/UAS-apdsRNAi).
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pgen-1002501-g004: Changes in Tv4 network configuration for maintenance.(A–E) eya regulates FMRFa independently of BMP signaling. (A,B) Nuclear pMad accumulation (red) in Tv4 (arrowhead) was downregulated in eyadsRNAi flies (B) compared to w1118 control (A). (C) Expression of eyadsRNAi in the presence of constitutively-activated Thickveins and Saxophone BMP type I receptors (TSA) activated pMad accumulation (red) in all Tv neurons (including Tv4; arrow) but failed to rescue FMRFa (blue) compared to w1118 control. (D) Quantification of pMad immunoreactivity in Tv4 nucleus in w1118 and eyadsRNAi flies. * p<0.0001 eyadsRNAi (n = 30) compared to w1118 control (n = 30). (E) Quantification of FMRFa immunoreactivity in Tv4 in w1118, eyadsRNAi and eyadsRNAi; TSA. * p<0.0001 eyadsRNAi (n = 38) and eyadsRNAi; TSA (n = 42) compared to w1118 control (n = 34). NSD, no significant difference between eyadsRNAi and eyadsRNAi; TSA. (F–H) eya does not regulate dimm in adult Tv4. dimm (red) in Tv neurons (blue) is maintained in eyadsRNAi (G) compared to w1118 control (F). (H) Quantification of Dimm immunoreactivity in Tv4. There is no significant difference between eyadsRNAi (n = 24) and w1118 (n = 25) controls (p = 0.67). (I–K) ap regulates dimm in adult Tv4. dimm (red) in Tv neurons (blue) is downregulated in apdsRNAi (J) compared to w1118 control (I). (K) Quantification of Dimm immunoreactivity in Tv4. * p<0.0001 apdsRNAi (n = 24) compared to w1118 (n = 35). (L) Model depicting regulatory configuration of ap, eya, dimm and BMP signaling from embryonic to adult Tv4. The dependence of dimm on eya expression is not maintained (X). Genotypes: w1118 (A,D, F,H, I,J) (UAS-dicer2/+; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); eyadsRNAi flies (B,D,E, G,H) (UAS-dicer2/UAS-eyadsRNAi; apGal4/+; tub-Gal80TS, UAS-nEGFP/+); eyadsRNAi; TSA flies (C,E) (UAS-dicer2/UAS-eyadsRNAi; apGal4/UAS-tkvA, UAS-saxA; tub-Gal80TS, UAS-nEGFP/+). apdsRNAi flies (J),K (UAS-dicer2/+; apGal4; tub-Gal80TS, UAS-nEGFP/UAS-apdsRNAi).
Mentions: BMP signaling in embryonic Tv4 neurons is dramatically reduced in eya mutants [11]. We expressed eyadsRNAi in adults until A15 and found that nuclear pMad, an indicator of BMP activity [12], was significantly downregulated to 47.9%±2.9 of control (Figure 4A, 4B, 4D). As BMP signaling is required for FMRFa expression in embryos and adults, we asked whether eya-dependence of FMRFa in adults is due to reduced BMP signaling. To do this, we simultaneously expressed eyadsRNAi and restored BMP signaling, using constitutively-activated type I BMP-receptors, thickveins and saxophone. Even though nuclear pMad was robustly activated in all Tv neurons, eyadsRNAi-induced FMRFa downregulation was not rescued (Figure 4C, 4E). Thus, in adults, eya independently maintains both BMP signaling and FMRFa expression.

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