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cis-regulatory analysis of the Drosophila pdm locus reveals a diversity of neural enhancers.

Ross J, Kuzin A, Brody T, Odenwald WF - BMC Genomics (2015)

Bottom Line: To facilitate data accessibility, the results of our analysis are catalogued in cisPatterns, an online database of the structure and function of these and other Drosophila enhancers.A subset of these enhancers share conserved elements including sequences that correspond to known TF DNA binding sites.Although comparative analysis of the nubbin and pdm-2 encoding sequences indicate that these two genes most likely arose from a duplication event, we found only partial evidence of sequence duplication between their enhancers, suggesting that after the putative duplication their cis-regulatory DNA diverged at a higher rate than their coding sequences.

View Article: PubMed Central - PubMed

Affiliation: The Neural Cell-Fate Determinants Section, NINDS, NIH, Bethesda, MD, USA. rossje@ninds.nih.gov.

ABSTRACT

Background: One of the major challenges in developmental biology is to understand the regulatory events that generate neuronal diversity. During Drosophila embryonic neural lineage development, cellular temporal identity is established in part by a transcription factor (TF) regulatory network that mediates a cascade of cellular identity decisions. Two of the regulators essential to this network are the POU-domain TFs Nubbin and Pdm-2, encoded by adjacent genes collectively known as pdm. The focus of this study is the discovery and characterization of cis-regulatory DNA that governs their expression.

Results: Phylogenetic footprinting analysis of a 125 kb genomic region that spans the pdm locus identified 116 conserved sequence clusters. To determine which of these regions function as cis-regulatory enhancers that regulate the dynamics of pdm gene expression, we tested each for in vivo enhancer activity during embryonic development and postembryonic neurogenesis. Our screen revealed 77 unique enhancers positioned throughout the noncoding region of the pdm locus. Many of these activated neural-specific gene expression during different developmental stages and many drove expression in overlapping patterns. Sequence comparisons of functionally related enhancers that activate overlapping expression patterns revealed that they share conserved elements that can be predictive of enhancer behavior. To facilitate data accessibility, the results of our analysis are catalogued in cisPatterns, an online database of the structure and function of these and other Drosophila enhancers.

Conclusions: These studies reveal a diversity of modular enhancers that most likely regulate pdm gene expression during embryonic and adult development, highlighting a high level of temporal and spatial expression specificity. In addition, we discovered clusters of functionally related enhancers throughout the pdm locus. A subset of these enhancers share conserved elements including sequences that correspond to known TF DNA binding sites. Although comparative analysis of the nubbin and pdm-2 encoding sequences indicate that these two genes most likely arose from a duplication event, we found only partial evidence of sequence duplication between their enhancers, suggesting that after the putative duplication their cis-regulatory DNA diverged at a higher rate than their coding sequences.

No MeSH data available.


Related in: MedlinePlus

Sequential activation of the nub-46 enhancer and Castor expression during embryonic NB lineage development. a–d Shown are the dorsal views of whole-mount embryos immunostained for the presence of nub-46 enhancer activity (GFP, green) and Castor (red) expression during NB lineage development. a During mid stage 10 (M10), Castor is present in midline precursors in the ventral nerve cord (VNC), whereas the nub-46 enhancer regulates expression in (a’) a subset of Cas− VNC NBs (arrows). The identification of the VNC NBs are based on size and location. a” There is no co-location of nub-46 enhancer activity and Castor expression. b–b” During early stage 11 (E11), nub-46 enhancer activity and Castor expression begins to co-localize in a subset of VNC NBs (arrowheads). c-c” By mid stage 11 (M11), additional VNC NBs express both Cas and the reporter gene (GFP) driven by nub-46. It is worth noting that co-localization may be in part due to perdurance of GFP. d–d” Co-localization of nub-46 activity and Cas expression is also observed in cephalic lobe cells (arrowheads)
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Fig6: Sequential activation of the nub-46 enhancer and Castor expression during embryonic NB lineage development. a–d Shown are the dorsal views of whole-mount embryos immunostained for the presence of nub-46 enhancer activity (GFP, green) and Castor (red) expression during NB lineage development. a During mid stage 10 (M10), Castor is present in midline precursors in the ventral nerve cord (VNC), whereas the nub-46 enhancer regulates expression in (a’) a subset of Cas− VNC NBs (arrows). The identification of the VNC NBs are based on size and location. a” There is no co-location of nub-46 enhancer activity and Castor expression. b–b” During early stage 11 (E11), nub-46 enhancer activity and Castor expression begins to co-localize in a subset of VNC NBs (arrowheads). c-c” By mid stage 11 (M11), additional VNC NBs express both Cas and the reporter gene (GFP) driven by nub-46. It is worth noting that co-localization may be in part due to perdurance of GFP. d–d” Co-localization of nub-46 activity and Cas expression is also observed in cephalic lobe cells (arrowheads)

Mentions: We also found that enhancer activity was consistent with the temporal order of Pdm and Cas expression during NB lineage development (Fig. 6). For example, we observed the staggered onset of the initial nub-46 enhancer activity followed by Cas expression in the developing CNS (Fig. 6). nub-46 regulated expression in a subset of VNC NB lineages, whereas Cas was restricted to a separate group of NBs located at the VNC midline (Fig. 6a’, a”). In agreement with the transient overlap of Nub and Cas expression [3], co-localization of nub-46 enhancer activity and Cas in NBs was observed during late developmental time points (Fig. 6b–d). While the costaining of nub-46 enhancer activity and Cas shows correct temporal expression, additional work is needed to characterize the temporal window of the other NB enhancers.Fig. 6


cis-regulatory analysis of the Drosophila pdm locus reveals a diversity of neural enhancers.

Ross J, Kuzin A, Brody T, Odenwald WF - BMC Genomics (2015)

Sequential activation of the nub-46 enhancer and Castor expression during embryonic NB lineage development. a–d Shown are the dorsal views of whole-mount embryos immunostained for the presence of nub-46 enhancer activity (GFP, green) and Castor (red) expression during NB lineage development. a During mid stage 10 (M10), Castor is present in midline precursors in the ventral nerve cord (VNC), whereas the nub-46 enhancer regulates expression in (a’) a subset of Cas− VNC NBs (arrows). The identification of the VNC NBs are based on size and location. a” There is no co-location of nub-46 enhancer activity and Castor expression. b–b” During early stage 11 (E11), nub-46 enhancer activity and Castor expression begins to co-localize in a subset of VNC NBs (arrowheads). c-c” By mid stage 11 (M11), additional VNC NBs express both Cas and the reporter gene (GFP) driven by nub-46. It is worth noting that co-localization may be in part due to perdurance of GFP. d–d” Co-localization of nub-46 activity and Cas expression is also observed in cephalic lobe cells (arrowheads)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4574355&req=5

Fig6: Sequential activation of the nub-46 enhancer and Castor expression during embryonic NB lineage development. a–d Shown are the dorsal views of whole-mount embryos immunostained for the presence of nub-46 enhancer activity (GFP, green) and Castor (red) expression during NB lineage development. a During mid stage 10 (M10), Castor is present in midline precursors in the ventral nerve cord (VNC), whereas the nub-46 enhancer regulates expression in (a’) a subset of Cas− VNC NBs (arrows). The identification of the VNC NBs are based on size and location. a” There is no co-location of nub-46 enhancer activity and Castor expression. b–b” During early stage 11 (E11), nub-46 enhancer activity and Castor expression begins to co-localize in a subset of VNC NBs (arrowheads). c-c” By mid stage 11 (M11), additional VNC NBs express both Cas and the reporter gene (GFP) driven by nub-46. It is worth noting that co-localization may be in part due to perdurance of GFP. d–d” Co-localization of nub-46 activity and Cas expression is also observed in cephalic lobe cells (arrowheads)
Mentions: We also found that enhancer activity was consistent with the temporal order of Pdm and Cas expression during NB lineage development (Fig. 6). For example, we observed the staggered onset of the initial nub-46 enhancer activity followed by Cas expression in the developing CNS (Fig. 6). nub-46 regulated expression in a subset of VNC NB lineages, whereas Cas was restricted to a separate group of NBs located at the VNC midline (Fig. 6a’, a”). In agreement with the transient overlap of Nub and Cas expression [3], co-localization of nub-46 enhancer activity and Cas in NBs was observed during late developmental time points (Fig. 6b–d). While the costaining of nub-46 enhancer activity and Cas shows correct temporal expression, additional work is needed to characterize the temporal window of the other NB enhancers.Fig. 6

Bottom Line: To facilitate data accessibility, the results of our analysis are catalogued in cisPatterns, an online database of the structure and function of these and other Drosophila enhancers.A subset of these enhancers share conserved elements including sequences that correspond to known TF DNA binding sites.Although comparative analysis of the nubbin and pdm-2 encoding sequences indicate that these two genes most likely arose from a duplication event, we found only partial evidence of sequence duplication between their enhancers, suggesting that after the putative duplication their cis-regulatory DNA diverged at a higher rate than their coding sequences.

View Article: PubMed Central - PubMed

Affiliation: The Neural Cell-Fate Determinants Section, NINDS, NIH, Bethesda, MD, USA. rossje@ninds.nih.gov.

ABSTRACT

Background: One of the major challenges in developmental biology is to understand the regulatory events that generate neuronal diversity. During Drosophila embryonic neural lineage development, cellular temporal identity is established in part by a transcription factor (TF) regulatory network that mediates a cascade of cellular identity decisions. Two of the regulators essential to this network are the POU-domain TFs Nubbin and Pdm-2, encoded by adjacent genes collectively known as pdm. The focus of this study is the discovery and characterization of cis-regulatory DNA that governs their expression.

Results: Phylogenetic footprinting analysis of a 125 kb genomic region that spans the pdm locus identified 116 conserved sequence clusters. To determine which of these regions function as cis-regulatory enhancers that regulate the dynamics of pdm gene expression, we tested each for in vivo enhancer activity during embryonic development and postembryonic neurogenesis. Our screen revealed 77 unique enhancers positioned throughout the noncoding region of the pdm locus. Many of these activated neural-specific gene expression during different developmental stages and many drove expression in overlapping patterns. Sequence comparisons of functionally related enhancers that activate overlapping expression patterns revealed that they share conserved elements that can be predictive of enhancer behavior. To facilitate data accessibility, the results of our analysis are catalogued in cisPatterns, an online database of the structure and function of these and other Drosophila enhancers.

Conclusions: These studies reveal a diversity of modular enhancers that most likely regulate pdm gene expression during embryonic and adult development, highlighting a high level of temporal and spatial expression specificity. In addition, we discovered clusters of functionally related enhancers throughout the pdm locus. A subset of these enhancers share conserved elements including sequences that correspond to known TF DNA binding sites. Although comparative analysis of the nubbin and pdm-2 encoding sequences indicate that these two genes most likely arose from a duplication event, we found only partial evidence of sequence duplication between their enhancers, suggesting that after the putative duplication their cis-regulatory DNA diverged at a higher rate than their coding sequences.

No MeSH data available.


Related in: MedlinePlus