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Neuropilins define distinct populations of neural crest cells.

Lumb R, Wiszniak S, Kabbara S, Scherer M, Harvey N, Schwarz Q - Neural Dev (2014)

Bottom Line: Taken together, our results demonstrate that Nrp1 and Nrp2 are expressed in different populations of NCCs, and that Nrp2-expressing NCCs are strongly biased towards a sensory fate.In the trunk, Nrp2-expressing NCCs specifically give rise to sensory ganglia, whereas Nrp1-expressing NCCs likely give rise to both sensory and sympathetic ganglia.Our findings therefore suggest that neuropilins play an essential role in coordinating NCC migration with fate specification.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Biology, University of South Australia and SA Pathology, Frome Road, Adelaide 5000, Australia. quenten.schwarz@health.sa.gov.au.

ABSTRACT

Background: Neural crest cells (NCCs) are a transient embryonic cell type that give rise to a wide spectrum of derivatives, including neurons and glia of the sensory and autonomic nervous system, melanocytes and connective tissues in the head. Lineage-tracing and functional studies have shown that trunk NCCs migrate along two distinct paths that correlate with different developmental fates. Thus, NCCs migrating ventrally through the anterior somite form sympathetic and sensory ganglia, whereas NCCs migrating dorsolaterally form melanocytes. Although the mechanisms promoting migration along the dorsolateral path are well defined, the molecules providing positional identity to sympathetic and sensory-fated NCCs that migrate along the same ventral path are ill defined. Neuropilins (Nrp1 and Nrp2) are transmembrane glycoproteins that are essential for NCC migration. Nrp1 and Nrp2 knockout mice have disparate phenotypes, suggesting that these receptors may play a role in sorting NCCs biased towards sensory and sympathetic fates to appropriate locations.

Results: Here we have combined in situ hybridisation, immunohistochemistry and lineage-tracing analyses to demonstrate that neuropilins are expressed in a non-overlapping pattern within NCCs. Whereas Nrp1 is expressed in NCCs emigrating from hindbrain rhombomere 4 (r4) and within trunk NCCs giving rise to sympathetic and sensory ganglia, Nrp2 is preferentially expressed in NCCs emigrating from r2 and in trunk NCCs giving rise to sensory ganglia. By generating a tamoxifen-inducible lineage-tracing system, we further demonstrate that Nrp2-expressing NCCs specifically populate sensory ganglia including the trigeminal ganglia (V) in the head and the dorsal root ganglia in the trunk.

Conclusions: Taken together, our results demonstrate that Nrp1 and Nrp2 are expressed in different populations of NCCs, and that Nrp2-expressing NCCs are strongly biased towards a sensory fate. In the trunk, Nrp2-expressing NCCs specifically give rise to sensory ganglia, whereas Nrp1-expressing NCCs likely give rise to both sensory and sympathetic ganglia. Our findings therefore suggest that neuropilins play an essential role in coordinating NCC migration with fate specification.

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Generation of Nrp2-CreERT2/Kikume lineage-tracing mice. (A) The Nrp2- CreERT2/Kikume transgenic construct consisted of a BAC (RP24-250G22) encompassing 62 kb upstream of the Nrp2 start codon and 4 kb downsteam of the 3′ UTR. A cassette consisting of the sequences for CreERT2, IRES ,and Kikume tagged in-frame to the FLAG tag was inserted in place of the start codon of Nrp2. (B-D) Whole mount X-gal staining of E10.0 Nrp2-CreERT2/Kikume X R26R embryos injected with tamoxifen (TM) at E9.0 and E9.5 (arrows). (B,C) In the head, X-gal staining was present in NCCs condensing into the anlagen of the Vth cranial ganglia (dashed circle) and not the VII-VIIIth cranial ganglia. (B, D) In the trunk, X-gal staining was present within the anterior somite. (E-H) Whole mount embryos were sectioned and counterstained with p75. (E-F) Transverse sections through r2 showed that LacZ expression occurred within NCCs (arrow). (G-H) Longitudinal sections through the trunk show that LacZ expression occurred within migrating trunk NCCs (arrow). e, eye; h, heart; nt, neural tube; ov, otic vesicle; *, collection of embryos. Scale bars = A, 10 kb; B, 500 μm; D, 100 μm.
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Fig6: Generation of Nrp2-CreERT2/Kikume lineage-tracing mice. (A) The Nrp2- CreERT2/Kikume transgenic construct consisted of a BAC (RP24-250G22) encompassing 62 kb upstream of the Nrp2 start codon and 4 kb downsteam of the 3′ UTR. A cassette consisting of the sequences for CreERT2, IRES ,and Kikume tagged in-frame to the FLAG tag was inserted in place of the start codon of Nrp2. (B-D) Whole mount X-gal staining of E10.0 Nrp2-CreERT2/Kikume X R26R embryos injected with tamoxifen (TM) at E9.0 and E9.5 (arrows). (B,C) In the head, X-gal staining was present in NCCs condensing into the anlagen of the Vth cranial ganglia (dashed circle) and not the VII-VIIIth cranial ganglia. (B, D) In the trunk, X-gal staining was present within the anterior somite. (E-H) Whole mount embryos were sectioned and counterstained with p75. (E-F) Transverse sections through r2 showed that LacZ expression occurred within NCCs (arrow). (G-H) Longitudinal sections through the trunk show that LacZ expression occurred within migrating trunk NCCs (arrow). e, eye; h, heart; nt, neural tube; ov, otic vesicle; *, collection of embryos. Scale bars = A, 10 kb; B, 500 μm; D, 100 μm.

Mentions: To identify the fate of neuropilin-expressing NCCs and to determine if Nrp2-expressing trunk NCCs are biased towards the sensory neuronal lineage, we generated a tamoxifen-inducible Nrp2 lineage-tracing mouse model. A Nrp2-CreERT2/Kikume transgenic construct was generated by replacing the ATG start codon of the Nrp2 gene with the entire CreERT2 IRES Kikume sequence followed by a polyadenylation signal sequence, thus placing CreERT2 IRES Kikume under control of the endogenous regulatory elements contained in the Nrp2 gene locus (Figure 6A). The engineered construct was used to generate a transgenic founder mouse that was identified by PCR using primers specific for CreERT2. The offspring from the founder line appeared grossly normal, and the transgene was transmitted as predicted by Mendelian ratios.Figure 6


Neuropilins define distinct populations of neural crest cells.

Lumb R, Wiszniak S, Kabbara S, Scherer M, Harvey N, Schwarz Q - Neural Dev (2014)

Generation of Nrp2-CreERT2/Kikume lineage-tracing mice. (A) The Nrp2- CreERT2/Kikume transgenic construct consisted of a BAC (RP24-250G22) encompassing 62 kb upstream of the Nrp2 start codon and 4 kb downsteam of the 3′ UTR. A cassette consisting of the sequences for CreERT2, IRES ,and Kikume tagged in-frame to the FLAG tag was inserted in place of the start codon of Nrp2. (B-D) Whole mount X-gal staining of E10.0 Nrp2-CreERT2/Kikume X R26R embryos injected with tamoxifen (TM) at E9.0 and E9.5 (arrows). (B,C) In the head, X-gal staining was present in NCCs condensing into the anlagen of the Vth cranial ganglia (dashed circle) and not the VII-VIIIth cranial ganglia. (B, D) In the trunk, X-gal staining was present within the anterior somite. (E-H) Whole mount embryos were sectioned and counterstained with p75. (E-F) Transverse sections through r2 showed that LacZ expression occurred within NCCs (arrow). (G-H) Longitudinal sections through the trunk show that LacZ expression occurred within migrating trunk NCCs (arrow). e, eye; h, heart; nt, neural tube; ov, otic vesicle; *, collection of embryos. Scale bars = A, 10 kb; B, 500 μm; D, 100 μm.
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Fig6: Generation of Nrp2-CreERT2/Kikume lineage-tracing mice. (A) The Nrp2- CreERT2/Kikume transgenic construct consisted of a BAC (RP24-250G22) encompassing 62 kb upstream of the Nrp2 start codon and 4 kb downsteam of the 3′ UTR. A cassette consisting of the sequences for CreERT2, IRES ,and Kikume tagged in-frame to the FLAG tag was inserted in place of the start codon of Nrp2. (B-D) Whole mount X-gal staining of E10.0 Nrp2-CreERT2/Kikume X R26R embryos injected with tamoxifen (TM) at E9.0 and E9.5 (arrows). (B,C) In the head, X-gal staining was present in NCCs condensing into the anlagen of the Vth cranial ganglia (dashed circle) and not the VII-VIIIth cranial ganglia. (B, D) In the trunk, X-gal staining was present within the anterior somite. (E-H) Whole mount embryos were sectioned and counterstained with p75. (E-F) Transverse sections through r2 showed that LacZ expression occurred within NCCs (arrow). (G-H) Longitudinal sections through the trunk show that LacZ expression occurred within migrating trunk NCCs (arrow). e, eye; h, heart; nt, neural tube; ov, otic vesicle; *, collection of embryos. Scale bars = A, 10 kb; B, 500 μm; D, 100 μm.
Mentions: To identify the fate of neuropilin-expressing NCCs and to determine if Nrp2-expressing trunk NCCs are biased towards the sensory neuronal lineage, we generated a tamoxifen-inducible Nrp2 lineage-tracing mouse model. A Nrp2-CreERT2/Kikume transgenic construct was generated by replacing the ATG start codon of the Nrp2 gene with the entire CreERT2 IRES Kikume sequence followed by a polyadenylation signal sequence, thus placing CreERT2 IRES Kikume under control of the endogenous regulatory elements contained in the Nrp2 gene locus (Figure 6A). The engineered construct was used to generate a transgenic founder mouse that was identified by PCR using primers specific for CreERT2. The offspring from the founder line appeared grossly normal, and the transgene was transmitted as predicted by Mendelian ratios.Figure 6

Bottom Line: Taken together, our results demonstrate that Nrp1 and Nrp2 are expressed in different populations of NCCs, and that Nrp2-expressing NCCs are strongly biased towards a sensory fate.In the trunk, Nrp2-expressing NCCs specifically give rise to sensory ganglia, whereas Nrp1-expressing NCCs likely give rise to both sensory and sympathetic ganglia.Our findings therefore suggest that neuropilins play an essential role in coordinating NCC migration with fate specification.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Biology, University of South Australia and SA Pathology, Frome Road, Adelaide 5000, Australia. quenten.schwarz@health.sa.gov.au.

ABSTRACT

Background: Neural crest cells (NCCs) are a transient embryonic cell type that give rise to a wide spectrum of derivatives, including neurons and glia of the sensory and autonomic nervous system, melanocytes and connective tissues in the head. Lineage-tracing and functional studies have shown that trunk NCCs migrate along two distinct paths that correlate with different developmental fates. Thus, NCCs migrating ventrally through the anterior somite form sympathetic and sensory ganglia, whereas NCCs migrating dorsolaterally form melanocytes. Although the mechanisms promoting migration along the dorsolateral path are well defined, the molecules providing positional identity to sympathetic and sensory-fated NCCs that migrate along the same ventral path are ill defined. Neuropilins (Nrp1 and Nrp2) are transmembrane glycoproteins that are essential for NCC migration. Nrp1 and Nrp2 knockout mice have disparate phenotypes, suggesting that these receptors may play a role in sorting NCCs biased towards sensory and sympathetic fates to appropriate locations.

Results: Here we have combined in situ hybridisation, immunohistochemistry and lineage-tracing analyses to demonstrate that neuropilins are expressed in a non-overlapping pattern within NCCs. Whereas Nrp1 is expressed in NCCs emigrating from hindbrain rhombomere 4 (r4) and within trunk NCCs giving rise to sympathetic and sensory ganglia, Nrp2 is preferentially expressed in NCCs emigrating from r2 and in trunk NCCs giving rise to sensory ganglia. By generating a tamoxifen-inducible lineage-tracing system, we further demonstrate that Nrp2-expressing NCCs specifically populate sensory ganglia including the trigeminal ganglia (V) in the head and the dorsal root ganglia in the trunk.

Conclusions: Taken together, our results demonstrate that Nrp1 and Nrp2 are expressed in different populations of NCCs, and that Nrp2-expressing NCCs are strongly biased towards a sensory fate. In the trunk, Nrp2-expressing NCCs specifically give rise to sensory ganglia, whereas Nrp1-expressing NCCs likely give rise to both sensory and sympathetic ganglia. Our findings therefore suggest that neuropilins play an essential role in coordinating NCC migration with fate specification.

Show MeSH
Related in: MedlinePlus