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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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Nrp1 and Nrp2 expression defines distinct cranial NCC populations along the anteroposterior axis. (A-D) Whole mount immunofluorescence analysis of Nrp1 and Nrp2 on E9.0 Wnt1Cre; Z/EG embryos in which all NCCs were labelled by GFP expression (B). In addition to staining of NCCs, both Nrp1 and Nrp2 were present in blood vessels within and around the r2 and r4 streams. (C-D) Antibody staining showed that Nrp1 expression was restricted to the r4 stream of NCCs (closed arrowhead) and Nrp2 was restricted to the r2 stream of NCCs (open arrowhead). (E-H) Longitudinal sections through the head confirmed that Nrp1 expression was indeed within NCCs of the r4 stream and not in the r2 stream (delta), while Nrp2 staining was within NCCs of the r2 stream and not in the r4 stream (closed arrowhead). Nrp1 and Nrp2 antibodies also labelled the major blood vessels (bv). e, eye; ov, otic vesicle. Scale bar = 100 μm.
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Fig2: Nrp1 and Nrp2 expression defines distinct cranial NCC populations along the anteroposterior axis. (A-D) Whole mount immunofluorescence analysis of Nrp1 and Nrp2 on E9.0 Wnt1Cre; Z/EG embryos in which all NCCs were labelled by GFP expression (B). In addition to staining of NCCs, both Nrp1 and Nrp2 were present in blood vessels within and around the r2 and r4 streams. (C-D) Antibody staining showed that Nrp1 expression was restricted to the r4 stream of NCCs (closed arrowhead) and Nrp2 was restricted to the r2 stream of NCCs (open arrowhead). (E-H) Longitudinal sections through the head confirmed that Nrp1 expression was indeed within NCCs of the r4 stream and not in the r2 stream (delta), while Nrp2 staining was within NCCs of the r2 stream and not in the r4 stream (closed arrowhead). Nrp1 and Nrp2 antibodies also labelled the major blood vessels (bv). e, eye; ov, otic vesicle. Scale bar = 100 μm.

Mentions: To compare expression of Nrp1 and Nrp2 in NCCs, we immunostained embryos generated by crossing Wnt1Cre[35] mice with Z/EG[36] reporter mice to specifically label all NCCs and their derivatives with green fluorescent protein (GFP). Whole mount immunofluorescence staining clearly demonstrated that Nrp1 and Nrp2 are expressed in a reciprocal pattern, replicating the mRNA expression seen in our previous in situ hybridisation analysis (Figure 2A-D). Immunostaining also identified expression of Nrp1 and Nrp2 in blood vessels around the r2 and r4 migrating NCCs (Figure 2A-D). Longitudinal sections through the head confirmed this restricted expression along the anteroposterior axis, as well as expression within the developing vasculature (Figure 2E-H). The membrane localisation of neuropilins is consistent with their role as cell surface receptors for guidance molecules of the SEMA3 and VEGF families [30]. Taken together, our in situ hybridisation, immunohistochemical, and lineage-tracing analyses confirm that neuropilins are expressed in different populations of cranial NCCs that are defined by their position along the anteroposterior axis. These restricted expression profiles therefore explain the disparate phenotypes of neuropilin knockout mice in which Nrp1 has ectopically placed neurons of the facio-acoustic cranial ganglia (VII-VIII) and Nrp2 has ectopically placed neurons of the trigeminal ganglia (V) [31, 32]. Future work addressing how the expression of neuropilins is controlled should provide essential clues to the mechanisms dividing cranial NCCs into distinct populations.Figure 2


Neuropilins define distinct populations of neural crest cells.

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

Nrp1 and Nrp2 expression defines distinct cranial NCC populations along the anteroposterior axis. (A-D) Whole mount immunofluorescence analysis of Nrp1 and Nrp2 on E9.0 Wnt1Cre; Z/EG embryos in which all NCCs were labelled by GFP expression (B). In addition to staining of NCCs, both Nrp1 and Nrp2 were present in blood vessels within and around the r2 and r4 streams. (C-D) Antibody staining showed that Nrp1 expression was restricted to the r4 stream of NCCs (closed arrowhead) and Nrp2 was restricted to the r2 stream of NCCs (open arrowhead). (E-H) Longitudinal sections through the head confirmed that Nrp1 expression was indeed within NCCs of the r4 stream and not in the r2 stream (delta), while Nrp2 staining was within NCCs of the r2 stream and not in the r4 stream (closed arrowhead). Nrp1 and Nrp2 antibodies also labelled the major blood vessels (bv). e, eye; ov, otic vesicle. Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4233049&req=5

Fig2: Nrp1 and Nrp2 expression defines distinct cranial NCC populations along the anteroposterior axis. (A-D) Whole mount immunofluorescence analysis of Nrp1 and Nrp2 on E9.0 Wnt1Cre; Z/EG embryos in which all NCCs were labelled by GFP expression (B). In addition to staining of NCCs, both Nrp1 and Nrp2 were present in blood vessels within and around the r2 and r4 streams. (C-D) Antibody staining showed that Nrp1 expression was restricted to the r4 stream of NCCs (closed arrowhead) and Nrp2 was restricted to the r2 stream of NCCs (open arrowhead). (E-H) Longitudinal sections through the head confirmed that Nrp1 expression was indeed within NCCs of the r4 stream and not in the r2 stream (delta), while Nrp2 staining was within NCCs of the r2 stream and not in the r4 stream (closed arrowhead). Nrp1 and Nrp2 antibodies also labelled the major blood vessels (bv). e, eye; ov, otic vesicle. Scale bar = 100 μm.
Mentions: To compare expression of Nrp1 and Nrp2 in NCCs, we immunostained embryos generated by crossing Wnt1Cre[35] mice with Z/EG[36] reporter mice to specifically label all NCCs and their derivatives with green fluorescent protein (GFP). Whole mount immunofluorescence staining clearly demonstrated that Nrp1 and Nrp2 are expressed in a reciprocal pattern, replicating the mRNA expression seen in our previous in situ hybridisation analysis (Figure 2A-D). Immunostaining also identified expression of Nrp1 and Nrp2 in blood vessels around the r2 and r4 migrating NCCs (Figure 2A-D). Longitudinal sections through the head confirmed this restricted expression along the anteroposterior axis, as well as expression within the developing vasculature (Figure 2E-H). The membrane localisation of neuropilins is consistent with their role as cell surface receptors for guidance molecules of the SEMA3 and VEGF families [30]. Taken together, our in situ hybridisation, immunohistochemical, and lineage-tracing analyses confirm that neuropilins are expressed in different populations of cranial NCCs that are defined by their position along the anteroposterior axis. These restricted expression profiles therefore explain the disparate phenotypes of neuropilin knockout mice in which Nrp1 has ectopically placed neurons of the facio-acoustic cranial ganglia (VII-VIII) and Nrp2 has ectopically placed neurons of the trigeminal ganglia (V) [31, 32]. Future work addressing how the expression of neuropilins is controlled should provide essential clues to the mechanisms dividing cranial NCCs into distinct populations.Figure 2

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