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Neuron hemilineages provide the functional ground plan for the Drosophila ventral nervous system.

Harris RM, Pfeiffer BD, Rubin GM, Truman JW - Elife (2015)

Bottom Line: The next level was hemilineages of similar projection cells that drove intersegmentally coordinated behaviors such as walking.The highest level involved hemilineages whose activation elicited complex behaviors such as takeoff.These activation phenotypes indicate that the hemilineages vary in their behavioral roles with some contributing to local networks for sensorimotor processing and others having higher order functions of coordinating these local networks into complex behavior.

View Article: PubMed Central - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.

ABSTRACT
Drosophila central neurons arise from neuroblasts that generate neurons in a pair-wise fashion, with the two daughters providing the basis for distinct A and B hemilineage groups. 33 postembryonically-born hemilineages contribute over 90% of the neurons in each thoracic hemisegment. We devised genetic approaches to define the anatomy of most of these hemilineages and to assessed their functional roles using the heat-sensitive channel dTRPA1. The simplest hemilineages contained local interneurons and their activation caused tonic or phasic leg movements lacking interlimb coordination. The next level was hemilineages of similar projection cells that drove intersegmentally coordinated behaviors such as walking. The highest level involved hemilineages whose activation elicited complex behaviors such as takeoff. These activation phenotypes indicate that the hemilineages vary in their behavioral roles with some contributing to local networks for sensorimotor processing and others having higher order functions of coordinating these local networks into complex behavior.

No MeSH data available.


Related in: MedlinePlus

The different strategies that were used to establish lines that showed selective expression in defined hemilineages.The strategies are based on a screen through the large collection of enhancer lines built from cis regulatory modules (CRMs) of CNS expressed genes. (A) For CRMs whose thoracic expression is confined to a hemilineage, gene-switch constructs are combined with feeding larvae the progesterone mimic (RU486) in the last larval stage. The larval expression of flippase then promotes the excision of a STOP cassette from another trans-gene allowing a constitutive promotor (Actin5C) to drive continual expression following excision. (B) When the larval expression pattern includes functional larval neurons as well as a hemilineage, expression in the larval neurons is blocked by including a nSynaptobrevin-GAL80 gene. Gene switch cannot be used in this context because it is not suppressed by GAL80. (C) Spatial and temporal specificity is accomplished using a conditional flippase that is the human progesterone receptor ligand-binding domain (hPR) fused to Flippase. Exposure of third instar larvae to RU486 then confines the flip event to the last larval stage. See text for more details.DOI:http://dx.doi.org/10.7554/eLife.04493.003
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fig1: The different strategies that were used to establish lines that showed selective expression in defined hemilineages.The strategies are based on a screen through the large collection of enhancer lines built from cis regulatory modules (CRMs) of CNS expressed genes. (A) For CRMs whose thoracic expression is confined to a hemilineage, gene-switch constructs are combined with feeding larvae the progesterone mimic (RU486) in the last larval stage. The larval expression of flippase then promotes the excision of a STOP cassette from another trans-gene allowing a constitutive promotor (Actin5C) to drive continual expression following excision. (B) When the larval expression pattern includes functional larval neurons as well as a hemilineage, expression in the larval neurons is blocked by including a nSynaptobrevin-GAL80 gene. Gene switch cannot be used in this context because it is not suppressed by GAL80. (C) Spatial and temporal specificity is accomplished using a conditional flippase that is the human progesterone receptor ligand-binding domain (hPR) fused to Flippase. Exposure of third instar larvae to RU486 then confines the flip event to the last larval stage. See text for more details.DOI:http://dx.doi.org/10.7554/eLife.04493.003

Mentions: Because of the diversity of expression patterns in the driver lines, we explored three different methods for implementing this strategy (summarized in Figure 1). The results of the first method (see Figure 1A) is illustrated in Figure 2. In the last instar larva, line R24B02-GAL4 drives expression in the cells of hemilineage 12A from the subesophageal through the A1 segments (Figure 2A). However, this expression pattern wanes during metamorphosis and is replaced by a different set of cells in the adult CNS (Figure 2B). To maintain lineage expression into the adult, we used R24B02-GAL4 to drive pJFRC180-20XUAS-IVS-Flp2::PEST, hereafter referred to as UAS-Flippase, to remove the transcriptional stop from an Actin5C-FRT>-dSTOP-FRT>-GAL4 construct. As shown in Figure 2C, when combined with the reporter construct, pJFRC2-10XUAS-IVS-mCD8::GFP (Pfeiffer et al., 2010), expression in the hemilineage 12A cells was maintained into the adult. A complication, though, was that expression was observed in other cells, presumably ones in which the stop cassette had been removed in earlier larval or embryonic stages, when this CRM drives a different expression pattern. To circumvent this early expression we used a GeneSwitch strategy (Osterwalder et al., 2001; Roman et al., 2001) in which we fused a Drosophila codon-optimized ligand-binding domain of the human progesterone receptor to the DNA-binding domain of GAL4. R24B02-GeneSwitch should then drive expression only after larvae are treated with a progesterone mimic, such as RU486. As seen in Figure 2D,E feeding larvae RU486 during the third instar resulted in strong expression of immature 12A interneurons. Removal of the drug at the start of metamorphosis was followed by a waning of GFP expression so that it was lost by adult emergence (Harris, 2012). Feeding adults with RU486 reinduced GFP expression but only in the expected adult pattern and not in the 12A interneurons. Consequently, the RU486 given to feeding larvae is effectively cleared from the animal during metamorphosis (this study). We then used the R24B02-GeneSwitch line in conjunction with UAS-flippase, Actin5C-FRT>-dSTOP-FRT>-GAL4, and pJFRC2-10XUAS-IVS-mCD8::GFP to reveal the adult morphology of the hemilineage 12A neurons. Without treatment with RU486 in the larva, we saw no GFP expression at any stage (Figure 2D,F), but feeding them with the drug during the third larval instar resulted in expression in the 12A interneurons that persisted through metamorphosis and continued to be robust in the adult (Figure 2G,H). Importantly, this adult expression pattern did not also contain the adult-specific cells characteristic of R24B02 because the RU486 was cleared from the system before these cells started to express late in metamorphosis.10.7554/eLife.04493.003Figure 1.The different strategies that were used to establish lines that showed selective expression in defined hemilineages.


Neuron hemilineages provide the functional ground plan for the Drosophila ventral nervous system.

Harris RM, Pfeiffer BD, Rubin GM, Truman JW - Elife (2015)

The different strategies that were used to establish lines that showed selective expression in defined hemilineages.The strategies are based on a screen through the large collection of enhancer lines built from cis regulatory modules (CRMs) of CNS expressed genes. (A) For CRMs whose thoracic expression is confined to a hemilineage, gene-switch constructs are combined with feeding larvae the progesterone mimic (RU486) in the last larval stage. The larval expression of flippase then promotes the excision of a STOP cassette from another trans-gene allowing a constitutive promotor (Actin5C) to drive continual expression following excision. (B) When the larval expression pattern includes functional larval neurons as well as a hemilineage, expression in the larval neurons is blocked by including a nSynaptobrevin-GAL80 gene. Gene switch cannot be used in this context because it is not suppressed by GAL80. (C) Spatial and temporal specificity is accomplished using a conditional flippase that is the human progesterone receptor ligand-binding domain (hPR) fused to Flippase. Exposure of third instar larvae to RU486 then confines the flip event to the last larval stage. See text for more details.DOI:http://dx.doi.org/10.7554/eLife.04493.003
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Related In: Results  -  Collection

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fig1: The different strategies that were used to establish lines that showed selective expression in defined hemilineages.The strategies are based on a screen through the large collection of enhancer lines built from cis regulatory modules (CRMs) of CNS expressed genes. (A) For CRMs whose thoracic expression is confined to a hemilineage, gene-switch constructs are combined with feeding larvae the progesterone mimic (RU486) in the last larval stage. The larval expression of flippase then promotes the excision of a STOP cassette from another trans-gene allowing a constitutive promotor (Actin5C) to drive continual expression following excision. (B) When the larval expression pattern includes functional larval neurons as well as a hemilineage, expression in the larval neurons is blocked by including a nSynaptobrevin-GAL80 gene. Gene switch cannot be used in this context because it is not suppressed by GAL80. (C) Spatial and temporal specificity is accomplished using a conditional flippase that is the human progesterone receptor ligand-binding domain (hPR) fused to Flippase. Exposure of third instar larvae to RU486 then confines the flip event to the last larval stage. See text for more details.DOI:http://dx.doi.org/10.7554/eLife.04493.003
Mentions: Because of the diversity of expression patterns in the driver lines, we explored three different methods for implementing this strategy (summarized in Figure 1). The results of the first method (see Figure 1A) is illustrated in Figure 2. In the last instar larva, line R24B02-GAL4 drives expression in the cells of hemilineage 12A from the subesophageal through the A1 segments (Figure 2A). However, this expression pattern wanes during metamorphosis and is replaced by a different set of cells in the adult CNS (Figure 2B). To maintain lineage expression into the adult, we used R24B02-GAL4 to drive pJFRC180-20XUAS-IVS-Flp2::PEST, hereafter referred to as UAS-Flippase, to remove the transcriptional stop from an Actin5C-FRT>-dSTOP-FRT>-GAL4 construct. As shown in Figure 2C, when combined with the reporter construct, pJFRC2-10XUAS-IVS-mCD8::GFP (Pfeiffer et al., 2010), expression in the hemilineage 12A cells was maintained into the adult. A complication, though, was that expression was observed in other cells, presumably ones in which the stop cassette had been removed in earlier larval or embryonic stages, when this CRM drives a different expression pattern. To circumvent this early expression we used a GeneSwitch strategy (Osterwalder et al., 2001; Roman et al., 2001) in which we fused a Drosophila codon-optimized ligand-binding domain of the human progesterone receptor to the DNA-binding domain of GAL4. R24B02-GeneSwitch should then drive expression only after larvae are treated with a progesterone mimic, such as RU486. As seen in Figure 2D,E feeding larvae RU486 during the third instar resulted in strong expression of immature 12A interneurons. Removal of the drug at the start of metamorphosis was followed by a waning of GFP expression so that it was lost by adult emergence (Harris, 2012). Feeding adults with RU486 reinduced GFP expression but only in the expected adult pattern and not in the 12A interneurons. Consequently, the RU486 given to feeding larvae is effectively cleared from the animal during metamorphosis (this study). We then used the R24B02-GeneSwitch line in conjunction with UAS-flippase, Actin5C-FRT>-dSTOP-FRT>-GAL4, and pJFRC2-10XUAS-IVS-mCD8::GFP to reveal the adult morphology of the hemilineage 12A neurons. Without treatment with RU486 in the larva, we saw no GFP expression at any stage (Figure 2D,F), but feeding them with the drug during the third larval instar resulted in expression in the 12A interneurons that persisted through metamorphosis and continued to be robust in the adult (Figure 2G,H). Importantly, this adult expression pattern did not also contain the adult-specific cells characteristic of R24B02 because the RU486 was cleared from the system before these cells started to express late in metamorphosis.10.7554/eLife.04493.003Figure 1.The different strategies that were used to establish lines that showed selective expression in defined hemilineages.

Bottom Line: The next level was hemilineages of similar projection cells that drove intersegmentally coordinated behaviors such as walking.The highest level involved hemilineages whose activation elicited complex behaviors such as takeoff.These activation phenotypes indicate that the hemilineages vary in their behavioral roles with some contributing to local networks for sensorimotor processing and others having higher order functions of coordinating these local networks into complex behavior.

View Article: PubMed Central - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.

ABSTRACT
Drosophila central neurons arise from neuroblasts that generate neurons in a pair-wise fashion, with the two daughters providing the basis for distinct A and B hemilineage groups. 33 postembryonically-born hemilineages contribute over 90% of the neurons in each thoracic hemisegment. We devised genetic approaches to define the anatomy of most of these hemilineages and to assessed their functional roles using the heat-sensitive channel dTRPA1. The simplest hemilineages contained local interneurons and their activation caused tonic or phasic leg movements lacking interlimb coordination. The next level was hemilineages of similar projection cells that drove intersegmentally coordinated behaviors such as walking. The highest level involved hemilineages whose activation elicited complex behaviors such as takeoff. These activation phenotypes indicate that the hemilineages vary in their behavioral roles with some contributing to local networks for sensorimotor processing and others having higher order functions of coordinating these local networks into complex behavior.

No MeSH data available.


Related in: MedlinePlus