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First published online 12 November 2008
doi: 10.1242/dev.023119

Development 135, 4113-4122 (2008)
Published by The Company of Biologists 2008
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Directing pathfinding along the dorsolateral path - the role of EDNRB2 and EphB2 in overcoming inhibition

Melissa L. Harris*, Ronelle Hall and Carol A. Erickson

University of California, Davis, Department of Molecular and Cellular Biology, One Shields Avenue, Davis, CA 95616, USA.


Figure 1
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  		Fig. 1. EphB2 and EDNRB2 expression. (A) Quantitative RT-PCR of quail NCCs reveals an increase in EphB2 expression in melanoblasts. (B-E') Dorsal views of chick embryos processed for in situ hybridization of EDNRB2. Horizontal lines in C-E correspond to transverse sections C'-E'. EDNRB2 is not present at stage 18 (B). At stage 19, EDNRB2 extends the length of the forelimb (C, between black arrows), is localized to the MSA (C'), and is only rarely observed dorsal to the neural tube (C''). At stage 20, EDNRB2 expression extends into the tailbud (D) and the DL path (D',D''). By stage 22, EDNRB2 is detected along the entire trunk (E), and over the dermamyotome (E'). EDNRB2 (red, pseudo-colored) colocalizes with the NC marker HNK-1 (E', green). fl, forelimb; hl, hindlimb; nt, neural tube; dm, dermamyotome.

 

Figure 2
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  		Fig. 2. Loss of EDNRB2 or EphB2 reduces DL migration. (A,B,G,H) Dorsal views of chick embryos electroporated with siSTRIKE-empty (A,G), EphB2-siSTRIKE (B) or EDNRB2-siSTRIKE (H) at stage 16 and assayed for melanoblast migration at stage 24. Dotted line marks the region of the dorsal neural tube. In siSTRIKE-empty embryos (A,G), GFP fluorescence is observed ventrally (diffuse, typical of neuronal cells) and laterally (punctuate, typical of melanoblasts) within the MSA (small arrow) and the DL path (large arrow). By contrast, embryos electroporated with EphB2-siSTRIKE (B) or EDNRB2-siSTRIKE (H) lack most dorsolaterally migrating GFP+ cells. (C-F) By immunohistochemistry, the EphB2 antibody detects cells of the neuroepithelium, ventral root and DRG, and is unaffected by the expression of siSTRIKE-empty (C,D). EphB2-siSTRIKE effectively knocks down EphB2 protein production (F) on the electroporated side of the neural tube (E). (I-L) In lateral views of the trunk, EDNRB2-siSTRIKE embryos (K,L) have a severe reduction in EDNRB2 expression (by in situ hybridization) in comparison to siSTRIKE-empty embryos (I,J). (M) Quantification of WRS+/GFP+ cells counted in transverse sections of siSTRIKE-empty, EphB2-siSTRIKE and EDNRB2-siSTRIKE embryos at stage 24. Data are presented as mean±s.d., with significance set at P≤0.05. Identical letters above the bars indicate those categories that, when compared, were significantly different (a, P=0.006; b, P=0.047; c, P=0.023; d, P=0.021). ant, anterior.

 

Figure 3
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  		Fig. 3 Loss of EDNRB2 does not lead to increased apoptosis. (A-H) Embryos electroporated with siSTRIKE-empty (A,B,E,F) or EDNRB2-siSTRIKE (C,D,G,H) exhibit qualitatively similar numbers of TUNEL-positive cells (indicated by arrows) along their trunks at stage 21 (A-D; dorsal views) and stage 24 (E-H, lateral views). ant, anterior.

 

Figure 4
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  		Fig. 4. Overexpression of EphB2 or EDNRB2 in the neurogenic crest leads to ectopic DL migration. (A-C) Dorsal views of stage-16 chick embryos co-electroporated at stage 12 with pBluescript/siSTRIKE-empty (A), EphB2-pMES/siSTRIKE-empty (B) or pCAGG-E2+/siSTRIKE-empty (C). In comparison to pBluescript/siSTRIKE-empty control embryos (A), in which cells are organized in segmental streams typical of ventral migration (arrows), GFP+ cells in EphB2-pMES and pCAGG-E2+/GFP reporter embryos (B,C) appear in a subectodermal, non-segmented wave typical of DL migration (arrowheads). (D,E) Transverse sections of EphB2-pMES/siSTRIKE-empty (D) and pCAGG-E2+/siSTRIKE-empty (E) embryos confirm neural crest cell migration (HNK1+, blue) in both the ventral (arrowheads) and DL pathways (arrows). Cells in the DL path are not recognized by the melanoblast marker WRS (red). ant, anterior; nt, neural tube.

 

Figure 5
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  		Fig. 5. Overexpression of EDNRB2 in melanoblasts leads to precocious DL migration. (A,B) Dorsal views of stage-19 chick embryos co-electroporated at stage 15 with pBluescript/siSTRIKE-empty (A) or pCAGG-E2+/siSTRIKE-empty (B). At stage 19, GFP+ cells are seen just dorsal to the neural tube in pBluescript/siSTRIKE-empty control embryos (A). In pCAGG-E2+/GFP reporter embryos, GFP+ cells precociously invade the DL path (B, arrows). (C) Transverse section of a stage-19 pCAGG-E2+/GFP reporter embryo in which MITF+ (red) melanoblasts expressing pCAGG-E2+ (green) are observed along the DL path (arrows), whereas those that do not express pCAGG-E2+ remain clustered in the MSA (arrowhead). (D) Quantification of MITF+/GFP+ cells in transverse sections confirms the precocious DL migration of melanoblasts in pCAGG-E2+-treated embryos. Data are presented as mean±s.d., with significance (*) set at P≤0.05. ant, anterior; dm, dermamyotome; nt, neural tube.

 

Figure 6
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  		Fig. 6. EDNRB2 acts as a chemoattractant and can rescue loss of EphB2. (A,B) Melanoblasts placed in the top well of a trans-filter migration chamber migrate to the bottom side of the filter in response to 1 and 5 nM ET3; however, this effect is diminished at 10 nM (A). Melanoblasts only migrate to the bottom side of the filter when in the presence of a chemotactic gradient of ET3. When ET3 is in the top well, migration into the bottom well does not vary from that of control wells, showing that ET3 does not produce a chemokinetic effect (B). (C-F) Dorsal views of chick embryos electroporated along the trunk with EphB2-siSTRIKE or EphB2-siSTRIKE/pCAGG-E2+ at stage 16 and assayed at stage 24. Embryos electroporated with EphB2-siSTRIKE (C) have reduced numbers of GFP+ cells that migrate into the DL path; however, this loss can be rescued by the overexpression of pCAGG-E2+ (D, compare with Fig. 2A). Overexpression of EDNRB2 in combination with EphB2 knockdown does not rescue the EphB2 phenotype by affecting EphB2 protein levels (E,F). (G) Dorsal view of a chick embryo electroporated along the trunk with EDNRB2-siSTRIKE/EphB2-pMES at stage 16 and assayed at stage 24. EphB2 overexpression mitigated EDNRB2 knockdown in two out of the six embryos analyzed. In these embryos, GFP+ cells appear more rounded than in embryos electroporated with EphB2-siSTRIKE/pCAGG-E2+. ant, anterior; nt, neural tube.

 

Figure 7
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  		Fig. 7. Absence of EDNRB2 and PNA-binding molecules at the vagal axial level. (A,B) In situ hybridization reveals a complete lack of EDNRB2 at the vagal axial level of a stage-11 chick embryo (A). Conversely, EDNRB is observed in the head mesenchyme, in rhombomere 4, over the dorsal neural tube and extending laterally from the first three somites (B). (B') In a transverse section, EDNRB is localized to the region dorsal to the neural tube and the DL path. (C,D) A transverse section of a stage-13 embryo exhibits high levels of PNA-binding in the sclerotome and low levels in the lateral DL pathway, and is absent from the dorsal portion of the DL pathway (C). HNK1+ NCCs (arrows) migrate into regions of the DL and ventral pathways that are not occupied by PNA glycoconjugates (D). r, rhombencephalon; s, somite; nt, neural tube; dm, dermamyotome.

 

Figure 8
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  		Fig. 8. Vagal neural crest cells do not migrate dorsolaterally at the trunk axial level; neuronal cells migrate dorsolaterally at the vagal axial level. (A,C) Schematics depicting the heterotopic/chronic transplantation of quail neural tube (donor) into a chick host. (B,B') Transverse section of a quail/chick chimera in which vagal NCCs are transplanted into a trunk environment. QCPN+ (red nuclei) NCCs (HNK1+, white) have migrated into the ventral pathway (arrows), but none are observed along the DL pathway. High magnification of the ventral path is shown in B'. (D,D') Transverse section of a quail/chick chimera in which neuronal cells are transplanted into a vagal environment. QCPN+ (red nuclei) NCCs (HNK1+, green) are observed on the DL pathway (arrows). High magnification of the dorsolateral path is shown in D'.

 

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© The Company of Biologists Ltd 2008