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A key role for poly(ADP-ribose) polymerase 3 in ectodermal specification and neural crest development.

Rouleau M, Saxena V, Rodrigue A, Paquet ER, Gagnon A, Hendzel MJ, Masson JY, Ekker M, Poirier GG - PLoS ONE (2011)

Bottom Line: We have used several in vitro and in vivo approaches to examine the possible functions of PARP3 as a transcriptional regulator, a function suggested from its previously reported association with several Polycomb group (PcG) proteins.It delays pigmentation and severely impedes the development of the median fin fold and tail bud.Our findings demonstrate that Parp3 is crucial in the early stages of zebrafish development, possibly by exerting its transcriptional regulatory functions as early as during the specification of the neural plate border.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Laboratory, CHUQ Research Center, Centre Hospitalier de l'Université Laval, Québec, Canada.

ABSTRACT

Background: The PARP family member poly(ADP-ribose) polymerase 3 (PARP3) is structurally related to the well characterized PARP1 that orchestrates cellular responses to DNA strand breaks and cell death by the synthesis of poly(ADP-ribose). In contrast to PARP1 and PARP2, the functions of PARP3 are undefined. Here, we reveal critical functions for PARP3 during vertebrate development.

Principal findings: We have used several in vitro and in vivo approaches to examine the possible functions of PARP3 as a transcriptional regulator, a function suggested from its previously reported association with several Polycomb group (PcG) proteins. We demonstrate that PARP3 gene occupancy in the human neuroblastoma cell line SK-N-SH occurs preferentially with developmental genes regulating cell fate specification, tissue patterning, craniofacial development and neurogenesis. Addressing the significance of this association during zebrafish development, we show that morpholino oligonucleotide-directed inhibition of parp3 expression in zebrafish impairs the expression of the neural crest cell specifier sox9a and of dlx3b/dlx4b, the formation of cranial sensory placodes, inner ears and pectoral fins. It delays pigmentation and severely impedes the development of the median fin fold and tail bud.

Conclusion: Our findings demonstrate that Parp3 is crucial in the early stages of zebrafish development, possibly by exerting its transcriptional regulatory functions as early as during the specification of the neural plate border.

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Related in: MedlinePlus

Developmental perturbations in zebrafish embryos with impaired parp3 expression.A. Immunoblot analysis (upper panel) of zebrafish Parp3 in wild type (WT) and parp3 morphants (MO) using an antibody raised against human PARP3. A whole cell extract of human SK-N-SH cells is shown as a control. The protein bands corresponding to PARP3 are indicated by “>”. The faster migrating band corresponds to a non-related protein that cross-reacts with the antibody. The Western blot membrane was stained with Ponceau S as a protein loading control (lower panel). B. Enlarged lateral views of the head regions of wild type and parp3 morphants injected with 4 ng MO1. The inner ears (small arrow) and the pectoral fins (large arrow) in the wt embryos are not formed in the parp3 morphants. C. Enlarged lateral views of the tail of wild type and parp3 morphants injected with 4 ng MO1. The median fin fold (arrow) is less developed in the morphants and has a more granular aspect. Effects are more pronounced on the dorsal side (arrow). D. Zebrafish embryos 48hrs after injection of increasing amounts of the parp3-specific morpholino oligonucleotide MO1 at the one-cell stage (ng amounts indicated in the lower right corner). The short length of morphant embryos, their curved tail and their reduced pigmentation is increasingly severe with increasing amounts of injected MO1. Lateral views with anterior to the right and dorsal to the top. Scale bars represent 10 µm.
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pone-0015834-g001: Developmental perturbations in zebrafish embryos with impaired parp3 expression.A. Immunoblot analysis (upper panel) of zebrafish Parp3 in wild type (WT) and parp3 morphants (MO) using an antibody raised against human PARP3. A whole cell extract of human SK-N-SH cells is shown as a control. The protein bands corresponding to PARP3 are indicated by “>”. The faster migrating band corresponds to a non-related protein that cross-reacts with the antibody. The Western blot membrane was stained with Ponceau S as a protein loading control (lower panel). B. Enlarged lateral views of the head regions of wild type and parp3 morphants injected with 4 ng MO1. The inner ears (small arrow) and the pectoral fins (large arrow) in the wt embryos are not formed in the parp3 morphants. C. Enlarged lateral views of the tail of wild type and parp3 morphants injected with 4 ng MO1. The median fin fold (arrow) is less developed in the morphants and has a more granular aspect. Effects are more pronounced on the dorsal side (arrow). D. Zebrafish embryos 48hrs after injection of increasing amounts of the parp3-specific morpholino oligonucleotide MO1 at the one-cell stage (ng amounts indicated in the lower right corner). The short length of morphant embryos, their curved tail and their reduced pigmentation is increasingly severe with increasing amounts of injected MO1. Lateral views with anterior to the right and dorsal to the top. Scale bars represent 10 µm.

Mentions: To investigate the biological functions of PARP3, we exploited the rapid and well characterized development schedule of the zebrafish. The zebrafish genome comprises a single parp3 gene orthologous to the human PARP3 gene. The human gene however encodes two PARP3 isoforms due to alternative splicing of the PARP3 transcripts. A long PARP3 isoform, expressed at low levels, comprises a 7 amino acid extension on its N-terminal side that is absent in the short and predominant isoform [5]. Based on an analysis of EST sequences, the zebrafish genome, similar to the mouse genome, appears to code only for the short isoform (Fig. S1A). Overall, zebrafish Parp3 shares 71% sequence similarity with the human PARP3 (short) sequence. The N-terminal domain, that lacks any similarity with known domains, is less well conserved (48% similarity) than the putative nucleic acid binding WGR domain (77% similarity) and the PARP catalytic domain (76% similarity). The catalytic core H-Y-E amino acid triad, critical for NAD+ binding and PARP activity, is conserved (Fig. S1A) [18]. Furthermore, antibodies raised against human PARP3 recognize zebrafish Parp3 (Fig. 1A). These observations support the notion that zebrafish Parp3 is highly related to human PARP3 at the amino acid level and that PARP3 is an evolutionarily conserved protein in multicellular organisms.


A key role for poly(ADP-ribose) polymerase 3 in ectodermal specification and neural crest development.

Rouleau M, Saxena V, Rodrigue A, Paquet ER, Gagnon A, Hendzel MJ, Masson JY, Ekker M, Poirier GG - PLoS ONE (2011)

Developmental perturbations in zebrafish embryos with impaired parp3 expression.A. Immunoblot analysis (upper panel) of zebrafish Parp3 in wild type (WT) and parp3 morphants (MO) using an antibody raised against human PARP3. A whole cell extract of human SK-N-SH cells is shown as a control. The protein bands corresponding to PARP3 are indicated by “>”. The faster migrating band corresponds to a non-related protein that cross-reacts with the antibody. The Western blot membrane was stained with Ponceau S as a protein loading control (lower panel). B. Enlarged lateral views of the head regions of wild type and parp3 morphants injected with 4 ng MO1. The inner ears (small arrow) and the pectoral fins (large arrow) in the wt embryos are not formed in the parp3 morphants. C. Enlarged lateral views of the tail of wild type and parp3 morphants injected with 4 ng MO1. The median fin fold (arrow) is less developed in the morphants and has a more granular aspect. Effects are more pronounced on the dorsal side (arrow). D. Zebrafish embryos 48hrs after injection of increasing amounts of the parp3-specific morpholino oligonucleotide MO1 at the one-cell stage (ng amounts indicated in the lower right corner). The short length of morphant embryos, their curved tail and their reduced pigmentation is increasingly severe with increasing amounts of injected MO1. Lateral views with anterior to the right and dorsal to the top. Scale bars represent 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3022025&req=5

pone-0015834-g001: Developmental perturbations in zebrafish embryos with impaired parp3 expression.A. Immunoblot analysis (upper panel) of zebrafish Parp3 in wild type (WT) and parp3 morphants (MO) using an antibody raised against human PARP3. A whole cell extract of human SK-N-SH cells is shown as a control. The protein bands corresponding to PARP3 are indicated by “>”. The faster migrating band corresponds to a non-related protein that cross-reacts with the antibody. The Western blot membrane was stained with Ponceau S as a protein loading control (lower panel). B. Enlarged lateral views of the head regions of wild type and parp3 morphants injected with 4 ng MO1. The inner ears (small arrow) and the pectoral fins (large arrow) in the wt embryos are not formed in the parp3 morphants. C. Enlarged lateral views of the tail of wild type and parp3 morphants injected with 4 ng MO1. The median fin fold (arrow) is less developed in the morphants and has a more granular aspect. Effects are more pronounced on the dorsal side (arrow). D. Zebrafish embryos 48hrs after injection of increasing amounts of the parp3-specific morpholino oligonucleotide MO1 at the one-cell stage (ng amounts indicated in the lower right corner). The short length of morphant embryos, their curved tail and their reduced pigmentation is increasingly severe with increasing amounts of injected MO1. Lateral views with anterior to the right and dorsal to the top. Scale bars represent 10 µm.
Mentions: To investigate the biological functions of PARP3, we exploited the rapid and well characterized development schedule of the zebrafish. The zebrafish genome comprises a single parp3 gene orthologous to the human PARP3 gene. The human gene however encodes two PARP3 isoforms due to alternative splicing of the PARP3 transcripts. A long PARP3 isoform, expressed at low levels, comprises a 7 amino acid extension on its N-terminal side that is absent in the short and predominant isoform [5]. Based on an analysis of EST sequences, the zebrafish genome, similar to the mouse genome, appears to code only for the short isoform (Fig. S1A). Overall, zebrafish Parp3 shares 71% sequence similarity with the human PARP3 (short) sequence. The N-terminal domain, that lacks any similarity with known domains, is less well conserved (48% similarity) than the putative nucleic acid binding WGR domain (77% similarity) and the PARP catalytic domain (76% similarity). The catalytic core H-Y-E amino acid triad, critical for NAD+ binding and PARP activity, is conserved (Fig. S1A) [18]. Furthermore, antibodies raised against human PARP3 recognize zebrafish Parp3 (Fig. 1A). These observations support the notion that zebrafish Parp3 is highly related to human PARP3 at the amino acid level and that PARP3 is an evolutionarily conserved protein in multicellular organisms.

Bottom Line: We have used several in vitro and in vivo approaches to examine the possible functions of PARP3 as a transcriptional regulator, a function suggested from its previously reported association with several Polycomb group (PcG) proteins.It delays pigmentation and severely impedes the development of the median fin fold and tail bud.Our findings demonstrate that Parp3 is crucial in the early stages of zebrafish development, possibly by exerting its transcriptional regulatory functions as early as during the specification of the neural plate border.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Laboratory, CHUQ Research Center, Centre Hospitalier de l'Université Laval, Québec, Canada.

ABSTRACT

Background: The PARP family member poly(ADP-ribose) polymerase 3 (PARP3) is structurally related to the well characterized PARP1 that orchestrates cellular responses to DNA strand breaks and cell death by the synthesis of poly(ADP-ribose). In contrast to PARP1 and PARP2, the functions of PARP3 are undefined. Here, we reveal critical functions for PARP3 during vertebrate development.

Principal findings: We have used several in vitro and in vivo approaches to examine the possible functions of PARP3 as a transcriptional regulator, a function suggested from its previously reported association with several Polycomb group (PcG) proteins. We demonstrate that PARP3 gene occupancy in the human neuroblastoma cell line SK-N-SH occurs preferentially with developmental genes regulating cell fate specification, tissue patterning, craniofacial development and neurogenesis. Addressing the significance of this association during zebrafish development, we show that morpholino oligonucleotide-directed inhibition of parp3 expression in zebrafish impairs the expression of the neural crest cell specifier sox9a and of dlx3b/dlx4b, the formation of cranial sensory placodes, inner ears and pectoral fins. It delays pigmentation and severely impedes the development of the median fin fold and tail bud.

Conclusion: Our findings demonstrate that Parp3 is crucial in the early stages of zebrafish development, possibly by exerting its transcriptional regulatory functions as early as during the specification of the neural plate border.

Show MeSH
Related in: MedlinePlus