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NOVA2-mediated RNA regulation is required for axonal pathfinding during development.

Saito Y, Miranda-Rottmann S, Ruggiu M, Park CY, Fak JJ, Zhong R, Duncan JS, Fabella BA, Junge HJ, Chen Z, Araya R, Fritzsch B, Hudspeth AJ, Darnell RB - Elife (2016)

Bottom Line: The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators.NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2.Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, The Rockefeller University, New York, United States.

ABSTRACT
The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

No MeSH data available.


Related in: MedlinePlus

Reduced efferent innervation and increased afferent innervation to the apex of the Nova2-/- mice cochlea.(A–C) Quantitation of efferent neuronal axons by colorimetric detection of the enzyme acetyl cholinesterase (AChE) in the cochlear apex of (B,B’) Nova2-/- and control (A,A’) wild-type mice. Representative images are shown. Green arrows indicate the 3 rows of OHC and red Arrow the row of IHC. These preparations were done in non-decalcified cochlea and only the apex could be preserved after dissection. (B) Plot showing quantification of the colorimetric signal. Heterozygote Nova2+/- mice were included in the plot to show that defect is dependent on the dose of NOVA2 expressed (n = 4). P15-P25 mice were used **p<0.01. (D,E) Cochlea whole mount and (D’,E’) Cryosections immunostaining in wild-type and Nova2-/- P20 mice. The images show apparent increased afferent innervation staining (NF200, red). Hair cells are labeled with a MYOVI antibody (green) and hair bundles with Phall. (magenta). (F–I) Afferent innervation to the IHC was quantified by counting individual ribbon synapses stained with CTBP2 antibody at P18. (F–H) wild-type (G–I) Nova2-/-, (F,G) segment 2, apex and (H,I) segment 4, apex (see Figure 10-O). (F’–I’) Respective amplified afferent IHC synapses. (J) A total of 30 wild-type and 44 Nova2-/- cells from 2 mice of each genotype were quantified. Each dot in the plot represents 2–5 averaged cells. The figure shows the approximate location of cells from segment 1–4 along the% length of the cochlea from apex to base on the lower x axes and the approximate frequency representation in the upper x axes. The number of dots (synapses) per cell was quantified by counterstaining with MYOVI which allows to outline the IHC perimeter. **p<0.01.DOI:http://dx.doi.org/10.7554/eLife.14371.029
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fig10s1: Reduced efferent innervation and increased afferent innervation to the apex of the Nova2-/- mice cochlea.(A–C) Quantitation of efferent neuronal axons by colorimetric detection of the enzyme acetyl cholinesterase (AChE) in the cochlear apex of (B,B’) Nova2-/- and control (A,A’) wild-type mice. Representative images are shown. Green arrows indicate the 3 rows of OHC and red Arrow the row of IHC. These preparations were done in non-decalcified cochlea and only the apex could be preserved after dissection. (B) Plot showing quantification of the colorimetric signal. Heterozygote Nova2+/- mice were included in the plot to show that defect is dependent on the dose of NOVA2 expressed (n = 4). P15-P25 mice were used **p<0.01. (D,E) Cochlea whole mount and (D’,E’) Cryosections immunostaining in wild-type and Nova2-/- P20 mice. The images show apparent increased afferent innervation staining (NF200, red). Hair cells are labeled with a MYOVI antibody (green) and hair bundles with Phall. (magenta). (F–I) Afferent innervation to the IHC was quantified by counting individual ribbon synapses stained with CTBP2 antibody at P18. (F–H) wild-type (G–I) Nova2-/-, (F,G) segment 2, apex and (H,I) segment 4, apex (see Figure 10-O). (F’–I’) Respective amplified afferent IHC synapses. (J) A total of 30 wild-type and 44 Nova2-/- cells from 2 mice of each genotype were quantified. Each dot in the plot represents 2–5 averaged cells. The figure shows the approximate location of cells from segment 1–4 along the% length of the cochlea from apex to base on the lower x axes and the approximate frequency representation in the upper x axes. The number of dots (synapses) per cell was quantified by counterstaining with MYOVI which allows to outline the IHC perimeter. **p<0.01.DOI:http://dx.doi.org/10.7554/eLife.14371.029

Mentions: These defects were also evident in apical cholinergic innervation (Dalian et al., 2001; Maison et al., 2003) after the onset of hearing (P15-P25; Figure 10—figure supplement 1A–C). At these stages, we were able to demonstrate that the loss of axonal innervation was functionally significant, as assessed by auditory brainstem responses (ABRs) and distortion products otoacustic emissions (DPOAEs) recordings (Figure 10M–N) In addition, an apparent increase in afferent innervation was found in Nova2-/- mice by neurofilament immunodetection (Figure 10A–L and Figure 10—figure supplement 1D–E). The difference was significant in the apex as determined by quantitation of IHC ribbon synapses (Meyer et al., 2009) (Figure 10—figure supplement 1F–J). Taken together, these data demonstrate a role for NOVA in cochlear innervation, efferent pathfinding, and normal hearing function in vivo.


NOVA2-mediated RNA regulation is required for axonal pathfinding during development.

Saito Y, Miranda-Rottmann S, Ruggiu M, Park CY, Fak JJ, Zhong R, Duncan JS, Fabella BA, Junge HJ, Chen Z, Araya R, Fritzsch B, Hudspeth AJ, Darnell RB - Elife (2016)

Reduced efferent innervation and increased afferent innervation to the apex of the Nova2-/- mice cochlea.(A–C) Quantitation of efferent neuronal axons by colorimetric detection of the enzyme acetyl cholinesterase (AChE) in the cochlear apex of (B,B’) Nova2-/- and control (A,A’) wild-type mice. Representative images are shown. Green arrows indicate the 3 rows of OHC and red Arrow the row of IHC. These preparations were done in non-decalcified cochlea and only the apex could be preserved after dissection. (B) Plot showing quantification of the colorimetric signal. Heterozygote Nova2+/- mice were included in the plot to show that defect is dependent on the dose of NOVA2 expressed (n = 4). P15-P25 mice were used **p<0.01. (D,E) Cochlea whole mount and (D’,E’) Cryosections immunostaining in wild-type and Nova2-/- P20 mice. The images show apparent increased afferent innervation staining (NF200, red). Hair cells are labeled with a MYOVI antibody (green) and hair bundles with Phall. (magenta). (F–I) Afferent innervation to the IHC was quantified by counting individual ribbon synapses stained with CTBP2 antibody at P18. (F–H) wild-type (G–I) Nova2-/-, (F,G) segment 2, apex and (H,I) segment 4, apex (see Figure 10-O). (F’–I’) Respective amplified afferent IHC synapses. (J) A total of 30 wild-type and 44 Nova2-/- cells from 2 mice of each genotype were quantified. Each dot in the plot represents 2–5 averaged cells. The figure shows the approximate location of cells from segment 1–4 along the% length of the cochlea from apex to base on the lower x axes and the approximate frequency representation in the upper x axes. The number of dots (synapses) per cell was quantified by counterstaining with MYOVI which allows to outline the IHC perimeter. **p<0.01.DOI:http://dx.doi.org/10.7554/eLife.14371.029
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Related In: Results  -  Collection

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Show All Figures
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fig10s1: Reduced efferent innervation and increased afferent innervation to the apex of the Nova2-/- mice cochlea.(A–C) Quantitation of efferent neuronal axons by colorimetric detection of the enzyme acetyl cholinesterase (AChE) in the cochlear apex of (B,B’) Nova2-/- and control (A,A’) wild-type mice. Representative images are shown. Green arrows indicate the 3 rows of OHC and red Arrow the row of IHC. These preparations were done in non-decalcified cochlea and only the apex could be preserved after dissection. (B) Plot showing quantification of the colorimetric signal. Heterozygote Nova2+/- mice were included in the plot to show that defect is dependent on the dose of NOVA2 expressed (n = 4). P15-P25 mice were used **p<0.01. (D,E) Cochlea whole mount and (D’,E’) Cryosections immunostaining in wild-type and Nova2-/- P20 mice. The images show apparent increased afferent innervation staining (NF200, red). Hair cells are labeled with a MYOVI antibody (green) and hair bundles with Phall. (magenta). (F–I) Afferent innervation to the IHC was quantified by counting individual ribbon synapses stained with CTBP2 antibody at P18. (F–H) wild-type (G–I) Nova2-/-, (F,G) segment 2, apex and (H,I) segment 4, apex (see Figure 10-O). (F’–I’) Respective amplified afferent IHC synapses. (J) A total of 30 wild-type and 44 Nova2-/- cells from 2 mice of each genotype were quantified. Each dot in the plot represents 2–5 averaged cells. The figure shows the approximate location of cells from segment 1–4 along the% length of the cochlea from apex to base on the lower x axes and the approximate frequency representation in the upper x axes. The number of dots (synapses) per cell was quantified by counterstaining with MYOVI which allows to outline the IHC perimeter. **p<0.01.DOI:http://dx.doi.org/10.7554/eLife.14371.029
Mentions: These defects were also evident in apical cholinergic innervation (Dalian et al., 2001; Maison et al., 2003) after the onset of hearing (P15-P25; Figure 10—figure supplement 1A–C). At these stages, we were able to demonstrate that the loss of axonal innervation was functionally significant, as assessed by auditory brainstem responses (ABRs) and distortion products otoacustic emissions (DPOAEs) recordings (Figure 10M–N) In addition, an apparent increase in afferent innervation was found in Nova2-/- mice by neurofilament immunodetection (Figure 10A–L and Figure 10—figure supplement 1D–E). The difference was significant in the apex as determined by quantitation of IHC ribbon synapses (Meyer et al., 2009) (Figure 10—figure supplement 1F–J). Taken together, these data demonstrate a role for NOVA in cochlear innervation, efferent pathfinding, and normal hearing function in vivo.

Bottom Line: The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators.NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2.Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, The Rockefeller University, New York, United States.

ABSTRACT
The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

No MeSH data available.


Related in: MedlinePlus