- Email: [email protected]
Differential expression of trkC mRNA in the chicken embryo from gastrulation to development of secondary brain vesicles
Developmental Brain Research 116 Ž1999. 205–209 www.elsevier.comrlocaterbres
Short communication
Differential expression of trkC mRNA in the chicken embryo from gastrulation to development of secondary brain vesicles Paulette Bernd a
a,b,)
, Rong Li
a
Departments of Anatomy and Otolaryngology, State UniÕersity of New York, Health Science Center, Brooklyn, NY 11203, USA b Department of Cell Biology, State UniÕersity of New York, Health Science Center, Brooklyn, NY 11203, USA Accepted 1 June 1999
Abstract In situ hybridization revealed that mRNA for the neurotrophin receptor trkC first appears in the chicken embryo at stage 4 anterior to Hensen’s node. At stages 6 and 8, trkC mRNA was restricted to the neural plate. By stage 11, trkC mRNA was absent from much of the prosencephalon, the entire mesencephalon and rhombomeres 1 and 4. At stage 15, trkC mRNA expression was limited to rhombomeres 3 and 5, and, by stage 18, there was no apparent expression of trkC mRNA in the hindbrain. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Neural plate; Neurotrophin-3; Neurulation; Rhombencephalon; Rhombomeres
trkC is a receptor tyrosine kinase required for high affinity binding of neurotrophin-3 ŽNT-3. w10x, a member of the nerve growth factor family of neurotrophins. The studies included in this paper use whole-mount in situ hybridization to describe the initial appearance of trkC mRNA in whole chicken embryos during gastrulation, and the pattern of expression during neurulation and formation of the secondary brain vesicles. Whole-mount in situ hybridization and sectioning were performed according to Harland w6x, Izpisua-Belmonte et al. w7x and Stern et al. w15x. Plasmid preparation was done as described by Li and Bernd w11x. Chicken embryos from prestreak stages onward were examined and were staged according to Eyal-Giladi and Kochav w2x or Hamburger and Hamilton w5x. Identical results were obtained using riboprobes prepared exclusively from the tyrosine kinase domain Ždetecting mRNA for full-length trkC. or with riboprobes including the extracellular region Ždetecting mRNA for full-length and truncated trkC.. This suggests that if truncated trkC mRNA is present, it is co-localized with full-length trkC.
)
Corresponding author. [email protected]
Fax:
q 1-718-270-3732;
E-mail:
No trkC mRNA was detected in prestreak embryos; an example of a stage XI embryo is shown in Fig. 1A. trkC mRNA was first detected at stage 4 anterior to Hensen’s node ŽFig. 1B.. A cross section through this region revealed that trkC mRNA was restricted to the epiblast layer ŽFig. 1C.. Expression of trkC mRNA persisted in the region of the extending notochordal process during stages 4 q and 5 ŽFig. 1D and E.. As shown previously by this laboratory w11x, trkC mRNA was localized to the neural plate and neural folds at stages 6 through 8 ŽFig. 1F and G., with the exception of the midline. This was confirmed by cross sectioning ŽFig. 1J and K.; no staining was seen in the underlying mesoderm or endoderm, adjacent ectoderm or notochord. The distribution of trkC mRNA was homogeneous in the neural plate and neural folds at stages 6 through 8 Žexcluding the midline.. Differential expression of trkC mRNA became apparent at stage 9 ŽFig. 1H and L.. There were areas of lower staining intensity along the rostrocaudal axis ŽFig. 1H. and a slight increase in trkC mRNA expression in the dorsal region of the neural groove ŽFig. 1L.. By stage 11, areas of the neural primordium were devoid of trkC mRNA ŽFig. 1I.; the anterior prosencephalon still expressed trkC mRNA, but the remainder of the prosencephalon, the entire mesencephalon and part of the rhombencephalon
0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 9 . 0 0 0 8 2 - 6
206
P. Bernd, R. Li r DeÕelopmental Brain Research 116 (1999) 205–209
Žrhombomeres 1 and 4. were negative. A cross section revealed a greater disparity in staining between the dorsal and ventral regions of the neural tube, with trkC mRNA
expression being considerably higher dorsally ŽFig. 1M.. Note that at both stages 9 and 11, trkC mRNA expression in the neural tube extends caudally to the newly formed
P. Bernd, R. Li r DeÕelopmental Brain Research 116 (1999) 205–209
207
Fig. 2. Expression of trkC mRNA at stages 15 and 18. Expression of trkC mRNA following in situ hybridization of embryos at stages 15 ŽA, B, C, D, E. and 18 ŽF.. Dashed lines indicate the level at which cross-sections were taken. Hybridization was performed using the 2500 bp riboprobe described in Fig. 1. Abbreviations: ov, otic vesicle; r, rhombomere; V, trigeminal ganglion; VIIrVIII, combined facial and cochleovestibular ganglion; IX, petrosal ganglion; X, nodose ganglion.
somites; there is no trkC mRNA caudal to the somites ŽFig. 1H and I..
As development proceeds, trkC mRNA appeared to be down regulated throughout the neural primordium, and by
Fig. 1. Expression of trkC mRNA from stages XI through 11. Expression of trkC mRNA following in situ hybridization of embryos at stages XI ŽA., 4 ŽB, C., 4 q ŽD., 5 ŽE., 6 ŽF, J., 8 ŽG, K., 9 ŽH, L. and 11 ŽI, M.. A dashed line indicates the level at which cross sections were taken. Hybridization was performed using an 800 bp riboprobe prepared exclusively from the tyrosine kinase domain ŽA, D, F, H. or a 2500 bp riboprobe including both the tyrosine kinase domain and the extracellular region ŽB, E, G, I; details in Ref. w11x.. Abbreviations: ao, area opaca; en, endoderm; ep, epiblast; hn, Hensen’s node; hy, hypoblast; ica, isolated cell aggregates; m, mesoderm; mes, mesencephalon; nne, nonneural ectoderm; no, notochord; nop, notochordal process; np, neural plate; ps, primitive streak; pro, prosencephalon; r, rhombomere; s, somite.
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P. Bernd, R. Li r DeÕelopmental Brain Research 116 (1999) 205–209
stage 15 was restricted to rhombomeres 3 and 5 ŽFig. 2A,B,C,D,E.. In addition, trkC mRNA could be seen in the sensory ganglion of cranial nerve V Žtrigeminal. and the combined ganglion of VII Žfacial. and VIII Žcochleovestibular.. The whole-mount is shown in three views Žposterior, posterolateral and lateral; Fig. 2A,B and C, respectively. in order to appreciate the relationship between the rhombomeres, cranial ganglia and otic vesicle. Note that rhombomere 3 is posterior to the ganglion of V, while rhombomere 5 is adjacent to the rostral half of the otic vesicle and the ganglion of VIIrVIII Žthe appearance of rhombomeres at different developmental stages in the chicken was reviewed by Lumsden w12x.. Note that the staining of rhombomere 3 traversed the depth of the hindbrain ŽFig. 2D., while that of rhombomere 5 was limited to cells closer to the pial surface of the hindbrain ŽFig. 2E.. By stage 18, no trkC mRNA was apparent in any rhombomeres ŽFig. 2F.; trkC mRNA expression was prominent in the sensory ganglia mentioned above as well as those of cranial nerves IX Žpetrosal. and X Žnodose.. Expression of trkC mRNA in the cranial ganglia of the chicken at stage 18 correlates with the results of Williams et al. w16x. Previously, the earliest expression of trkC mRNA in chickens was reported in the neural plate using in situ hybridization w11x and at embryonic day 2 Žapproximately stages 12 to 13. using RNase protection w16x. Functional studies revealed that NT-3 increased both neurite outgrowth and apoptosis in explants of the chicken neural plate w11x. Similar patterns of trkC mRNA expression were obtained with stage 1 to 6 quail embryos using the polymerase chain reaction ŽRT-PCR. w17x; however, in situ hybridization at stages 3 through 10 revealed the additional presence of trkC mRNA in the primitive streak, Hensen’s node and somites w18x. It is not known whether this inconsistency is due to a species difference or to technical differences Žthe current study was conducted using higher stringency and non-hydrolyzed riboprobes.. In contrast to our results in chicken and quail, Kahane and Kalcheim w9x did not detect a trkC signal in the chicken neural plate; Averbuch-Heller et al. w1x first detected trkC mRNA in the neural tube at stage 8 q . Our results are consistent with Kalcheim’s findings that the initial distribution was homogeneous in the transverse plane of the neural tube, and at later stages trkC mRNA expression increased in the dorsal neural tube and decreased in the rest of the tube. In contrast to our study, Averbuch-Heller et al. w1x reported isolated spots of trkC mRNA at the periphery of the neural tube and found that the dorsal neural tube remains positive through embryonic day 4 Žapproximately stage 26.. Williams et al. w16x also described expression of trkC mRNA in the chicken CNS at stage 18; however, unlike our results at earlier stages and those of Averbuch-Heller et al. w1x, they found that labeling was most intense ventrally. Neither Kahane and Kalcheim w9x nor Williams et al. w16x described any differ-
ential staining within the secondary brain vesicles. The discrepancies between our findings and those of Kalcheim and Williams may be due to technical differences, such as lower stringency resulting in detection of a related kinase or higher stringency resulting in lower sensitivity. In addition, these studies were not done using whole-mount in situ hybridization and it may be more difficult to detect rostrocaudal differential expression in serial sections. The restriction of trkC mRNA to rhombomeres 3 and 5 is striking because there is segmental migration of the chicken hindbrain neural crest resulting in a lack of neural crest cells in the mesenchyme lateral to rhombomeres 3 and 5 w8,13,14x. While there is no disagreement that neural crest cells are generated from rhombomeres 3 and 5, two divergent explanations have been given for the apparent lack of neural crest cells lateral to these rhombomeres. The first is that neural crest cells generated from rhombomeres 3 and 5 die and fail to migrate, as evidenced by increased levels of apoptosis in the dorsal midline over rhombomeres 3 and 5 w8,13x. The second explanation is that neural crest cells migrate from rhombomeres 3 and 5 but deviate rostrally and caudally as they do so, thereby failing to enter the regions adjacent to those rhombomeres w14x. Neural crest cell migration appears to be related to gene expression in the individual rhombomeres. For example, if rhombomeres 3 and 5 are maintained as isolated units in vitro, neural crest cells do not undergo apoptosis and there is migration of neural crest cells away from the explant w4x. This appears to be due to downregulation of Bmp-4 in rhombomeres 3 and 5 upon isolation w4x, because addition of Bmp-4 to isolated explants prevented neural crest cell migration by increasing apoptosis in this population w3x. It is intriguing to speculate that the persistent expression of trkC mRNA in rhombomeres 3 and 5 is also somehow related to the segmental migration of hindbrain neural crest cells. This seem plausible since the formation and migration of neural crest cells generated from rhombomeres 3 and 5 occurs between stages 9 and 11 w8,13x, coincidental with the shift from homogeneous to differential expression of trkC mRNA. Acknowledgements The authors are indebted to Dr. Claudio Stern for his guidance and support throughout this study. They are also grateful to Drs. Tom Large and Luis Parada for providing plasmids containing chicken trkC cDNA. We also thank the members of the Stern laboratory for their advice and help, and Mr. Vincent Garofalo for photographic and computer work. This work was supported by grants from the Dysautonomia Foundation. References w1x L. Averbuch-Heller, M. Pruginin, N. Kahane, P. Tsoulfas, L. Parada, A. Rosenthal, C. Kalcheim, Neurotrophin 3 stimulates the differenti-
P. Bernd, R. Li r DeÕelopmental Brain Research 116 (1999) 205–209
w2x
w3x
w4x
w5x w6x
w7x
w8x w9x
w10x
ation of motoneurons from avian neural tube progenitor cells, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 3247–3251. H. Eyal-Giladi, S. Kochav, From cleavage to primitive streak formation: A complementary normal table and a new look at the first stages of the development of the chick. I. General morphology, Dev. Biol. 49 Ž1976. 321–337. A. Graham, P. Francis-West, P. Brickell, A. Lumsden, The signaling molecule BMP4 mediates apoptosis in the rhombencephalic neural crest, Nature 372 Ž1994. 684–686. A. Graham, I. Heyman, A. Lumsden, Even-numbered rhombomeres control the apoptotic elimination of neural crest cells from odd-numbered rhombomeres in the chick hindbrain, Development 119 Ž1993. 233–245. V. Hamburger, H.L. Hamilton, A series of normal stages in the development of the chick embryo, J. Morphol. 88 Ž1951. 49–92. R.M. Harland, In situ hybridization: an improved whole-mount method for xenopus embryos, Methods Cell Biol. 36 Ž1991. 685– 695. J.C. Izpisua-Belmonte, E.M. De Robertis, K.G. Storey, C.D. Stern, The homeobox gene goosecoid and the origin of organizer cells in the early chick blastoderm, Cell 74 Ž1993. 645–659. P. Jeffs, K. Jaques, M. Osmond, Cell death in cranial neural crest development, Anat. Embryol. 185 Ž1992. 583–588. N. Kahane, C. Kalcheim, Expression of trkC receptor mRNA during development of the avian nervous system, J. Neurobiol. 25 Ž1994. 571–584. F. Lamballe, R. Klein, M. Barbacid, trkC, a new member of the trk
w11x
w12x w13x
w14x
w15x
w16x
w17x
w18x
209
family of tyrosine protein kinases, is a receptor for neurotrophin-3, Cell 66 Ž1991. 967–979. R. Li, P. Bernd, Neurotrophin-3 increases neurite outgrowth and apoptosis in explants of the chicken neural plate, Dev. Neurosci. 21 Ž1999. 12–21. A. Lumsden, The cellular basis of segmentation in the developing hindbrain, Trends Neurosci. 13 Ž1990. 329–335. A. Lumsden, N. Sprawson, A. Graham, Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo, Development 113 Ž1991. 1281–1291. J. Sechrist, G.N. Serbedzija, T. Scherson, S.E. Fraser, M. BronnerFraser, Segmental migration of the hindbrain neural crest does not arise from its segmental generation, Development 118 Ž1993. 691– 703. C.D. Stern, R.T. Yu, A. Kakizuka, C.R. Kintner, L.S. Mathews, W.W. Vale, R.M. Evans, K. Umesono, Activin and its receptors during gastrulation and the later phases of mesoderm development in the chick embryo, Dev. Biol. 172 Ž1995. 192–205. R. Williams, A. Backstrom, T. Ebendal, F. Hallbook, Molecular cloning and cellular localization of trkC in the chicken embryo, Dev. Brain Res. 75 Ž1993. 235–252. L. Yao, D. Zhang, P. Bernd, The onset of neurotrophin and trk mRNA expression in early embryonic tissues of the quail, Dev. Biol. 165 Ž1994. 727–730. D. Zhang, L. Yao, P. Bernd, Expression of neurotrophin trk and p75 receptors in quail embryos undergoing gastrulation and neurulation, Dev. Dynam. 205 Ž1996. 150–161.
Short communication
Differential expression of trkC mRNA in the chicken embryo from gastrulation to development of secondary brain vesicles Paulette Bernd a
a,b,)
, Rong Li
a
Departments of Anatomy and Otolaryngology, State UniÕersity of New York, Health Science Center, Brooklyn, NY 11203, USA b Department of Cell Biology, State UniÕersity of New York, Health Science Center, Brooklyn, NY 11203, USA Accepted 1 June 1999
Abstract In situ hybridization revealed that mRNA for the neurotrophin receptor trkC first appears in the chicken embryo at stage 4 anterior to Hensen’s node. At stages 6 and 8, trkC mRNA was restricted to the neural plate. By stage 11, trkC mRNA was absent from much of the prosencephalon, the entire mesencephalon and rhombomeres 1 and 4. At stage 15, trkC mRNA expression was limited to rhombomeres 3 and 5, and, by stage 18, there was no apparent expression of trkC mRNA in the hindbrain. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Neural plate; Neurotrophin-3; Neurulation; Rhombencephalon; Rhombomeres
trkC is a receptor tyrosine kinase required for high affinity binding of neurotrophin-3 ŽNT-3. w10x, a member of the nerve growth factor family of neurotrophins. The studies included in this paper use whole-mount in situ hybridization to describe the initial appearance of trkC mRNA in whole chicken embryos during gastrulation, and the pattern of expression during neurulation and formation of the secondary brain vesicles. Whole-mount in situ hybridization and sectioning were performed according to Harland w6x, Izpisua-Belmonte et al. w7x and Stern et al. w15x. Plasmid preparation was done as described by Li and Bernd w11x. Chicken embryos from prestreak stages onward were examined and were staged according to Eyal-Giladi and Kochav w2x or Hamburger and Hamilton w5x. Identical results were obtained using riboprobes prepared exclusively from the tyrosine kinase domain Ždetecting mRNA for full-length trkC. or with riboprobes including the extracellular region Ždetecting mRNA for full-length and truncated trkC.. This suggests that if truncated trkC mRNA is present, it is co-localized with full-length trkC.
)
Corresponding author. [email protected]
Fax:
q 1-718-270-3732;
E-mail:
No trkC mRNA was detected in prestreak embryos; an example of a stage XI embryo is shown in Fig. 1A. trkC mRNA was first detected at stage 4 anterior to Hensen’s node ŽFig. 1B.. A cross section through this region revealed that trkC mRNA was restricted to the epiblast layer ŽFig. 1C.. Expression of trkC mRNA persisted in the region of the extending notochordal process during stages 4 q and 5 ŽFig. 1D and E.. As shown previously by this laboratory w11x, trkC mRNA was localized to the neural plate and neural folds at stages 6 through 8 ŽFig. 1F and G., with the exception of the midline. This was confirmed by cross sectioning ŽFig. 1J and K.; no staining was seen in the underlying mesoderm or endoderm, adjacent ectoderm or notochord. The distribution of trkC mRNA was homogeneous in the neural plate and neural folds at stages 6 through 8 Žexcluding the midline.. Differential expression of trkC mRNA became apparent at stage 9 ŽFig. 1H and L.. There were areas of lower staining intensity along the rostrocaudal axis ŽFig. 1H. and a slight increase in trkC mRNA expression in the dorsal region of the neural groove ŽFig. 1L.. By stage 11, areas of the neural primordium were devoid of trkC mRNA ŽFig. 1I.; the anterior prosencephalon still expressed trkC mRNA, but the remainder of the prosencephalon, the entire mesencephalon and part of the rhombencephalon
0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 9 . 0 0 0 8 2 - 6
206
P. Bernd, R. Li r DeÕelopmental Brain Research 116 (1999) 205–209
Žrhombomeres 1 and 4. were negative. A cross section revealed a greater disparity in staining between the dorsal and ventral regions of the neural tube, with trkC mRNA
expression being considerably higher dorsally ŽFig. 1M.. Note that at both stages 9 and 11, trkC mRNA expression in the neural tube extends caudally to the newly formed
P. Bernd, R. Li r DeÕelopmental Brain Research 116 (1999) 205–209
207
Fig. 2. Expression of trkC mRNA at stages 15 and 18. Expression of trkC mRNA following in situ hybridization of embryos at stages 15 ŽA, B, C, D, E. and 18 ŽF.. Dashed lines indicate the level at which cross-sections were taken. Hybridization was performed using the 2500 bp riboprobe described in Fig. 1. Abbreviations: ov, otic vesicle; r, rhombomere; V, trigeminal ganglion; VIIrVIII, combined facial and cochleovestibular ganglion; IX, petrosal ganglion; X, nodose ganglion.
somites; there is no trkC mRNA caudal to the somites ŽFig. 1H and I..
As development proceeds, trkC mRNA appeared to be down regulated throughout the neural primordium, and by
Fig. 1. Expression of trkC mRNA from stages XI through 11. Expression of trkC mRNA following in situ hybridization of embryos at stages XI ŽA., 4 ŽB, C., 4 q ŽD., 5 ŽE., 6 ŽF, J., 8 ŽG, K., 9 ŽH, L. and 11 ŽI, M.. A dashed line indicates the level at which cross sections were taken. Hybridization was performed using an 800 bp riboprobe prepared exclusively from the tyrosine kinase domain ŽA, D, F, H. or a 2500 bp riboprobe including both the tyrosine kinase domain and the extracellular region ŽB, E, G, I; details in Ref. w11x.. Abbreviations: ao, area opaca; en, endoderm; ep, epiblast; hn, Hensen’s node; hy, hypoblast; ica, isolated cell aggregates; m, mesoderm; mes, mesencephalon; nne, nonneural ectoderm; no, notochord; nop, notochordal process; np, neural plate; ps, primitive streak; pro, prosencephalon; r, rhombomere; s, somite.
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P. Bernd, R. Li r DeÕelopmental Brain Research 116 (1999) 205–209
stage 15 was restricted to rhombomeres 3 and 5 ŽFig. 2A,B,C,D,E.. In addition, trkC mRNA could be seen in the sensory ganglion of cranial nerve V Žtrigeminal. and the combined ganglion of VII Žfacial. and VIII Žcochleovestibular.. The whole-mount is shown in three views Žposterior, posterolateral and lateral; Fig. 2A,B and C, respectively. in order to appreciate the relationship between the rhombomeres, cranial ganglia and otic vesicle. Note that rhombomere 3 is posterior to the ganglion of V, while rhombomere 5 is adjacent to the rostral half of the otic vesicle and the ganglion of VIIrVIII Žthe appearance of rhombomeres at different developmental stages in the chicken was reviewed by Lumsden w12x.. Note that the staining of rhombomere 3 traversed the depth of the hindbrain ŽFig. 2D., while that of rhombomere 5 was limited to cells closer to the pial surface of the hindbrain ŽFig. 2E.. By stage 18, no trkC mRNA was apparent in any rhombomeres ŽFig. 2F.; trkC mRNA expression was prominent in the sensory ganglia mentioned above as well as those of cranial nerves IX Žpetrosal. and X Žnodose.. Expression of trkC mRNA in the cranial ganglia of the chicken at stage 18 correlates with the results of Williams et al. w16x. Previously, the earliest expression of trkC mRNA in chickens was reported in the neural plate using in situ hybridization w11x and at embryonic day 2 Žapproximately stages 12 to 13. using RNase protection w16x. Functional studies revealed that NT-3 increased both neurite outgrowth and apoptosis in explants of the chicken neural plate w11x. Similar patterns of trkC mRNA expression were obtained with stage 1 to 6 quail embryos using the polymerase chain reaction ŽRT-PCR. w17x; however, in situ hybridization at stages 3 through 10 revealed the additional presence of trkC mRNA in the primitive streak, Hensen’s node and somites w18x. It is not known whether this inconsistency is due to a species difference or to technical differences Žthe current study was conducted using higher stringency and non-hydrolyzed riboprobes.. In contrast to our results in chicken and quail, Kahane and Kalcheim w9x did not detect a trkC signal in the chicken neural plate; Averbuch-Heller et al. w1x first detected trkC mRNA in the neural tube at stage 8 q . Our results are consistent with Kalcheim’s findings that the initial distribution was homogeneous in the transverse plane of the neural tube, and at later stages trkC mRNA expression increased in the dorsal neural tube and decreased in the rest of the tube. In contrast to our study, Averbuch-Heller et al. w1x reported isolated spots of trkC mRNA at the periphery of the neural tube and found that the dorsal neural tube remains positive through embryonic day 4 Žapproximately stage 26.. Williams et al. w16x also described expression of trkC mRNA in the chicken CNS at stage 18; however, unlike our results at earlier stages and those of Averbuch-Heller et al. w1x, they found that labeling was most intense ventrally. Neither Kahane and Kalcheim w9x nor Williams et al. w16x described any differ-
ential staining within the secondary brain vesicles. The discrepancies between our findings and those of Kalcheim and Williams may be due to technical differences, such as lower stringency resulting in detection of a related kinase or higher stringency resulting in lower sensitivity. In addition, these studies were not done using whole-mount in situ hybridization and it may be more difficult to detect rostrocaudal differential expression in serial sections. The restriction of trkC mRNA to rhombomeres 3 and 5 is striking because there is segmental migration of the chicken hindbrain neural crest resulting in a lack of neural crest cells in the mesenchyme lateral to rhombomeres 3 and 5 w8,13,14x. While there is no disagreement that neural crest cells are generated from rhombomeres 3 and 5, two divergent explanations have been given for the apparent lack of neural crest cells lateral to these rhombomeres. The first is that neural crest cells generated from rhombomeres 3 and 5 die and fail to migrate, as evidenced by increased levels of apoptosis in the dorsal midline over rhombomeres 3 and 5 w8,13x. The second explanation is that neural crest cells migrate from rhombomeres 3 and 5 but deviate rostrally and caudally as they do so, thereby failing to enter the regions adjacent to those rhombomeres w14x. Neural crest cell migration appears to be related to gene expression in the individual rhombomeres. For example, if rhombomeres 3 and 5 are maintained as isolated units in vitro, neural crest cells do not undergo apoptosis and there is migration of neural crest cells away from the explant w4x. This appears to be due to downregulation of Bmp-4 in rhombomeres 3 and 5 upon isolation w4x, because addition of Bmp-4 to isolated explants prevented neural crest cell migration by increasing apoptosis in this population w3x. It is intriguing to speculate that the persistent expression of trkC mRNA in rhombomeres 3 and 5 is also somehow related to the segmental migration of hindbrain neural crest cells. This seem plausible since the formation and migration of neural crest cells generated from rhombomeres 3 and 5 occurs between stages 9 and 11 w8,13x, coincidental with the shift from homogeneous to differential expression of trkC mRNA. Acknowledgements The authors are indebted to Dr. Claudio Stern for his guidance and support throughout this study. They are also grateful to Drs. Tom Large and Luis Parada for providing plasmids containing chicken trkC cDNA. We also thank the members of the Stern laboratory for their advice and help, and Mr. Vincent Garofalo for photographic and computer work. This work was supported by grants from the Dysautonomia Foundation. References w1x L. Averbuch-Heller, M. Pruginin, N. Kahane, P. Tsoulfas, L. Parada, A. Rosenthal, C. Kalcheim, Neurotrophin 3 stimulates the differenti-
P. Bernd, R. Li r DeÕelopmental Brain Research 116 (1999) 205–209
w2x
w3x
w4x
w5x w6x
w7x
w8x w9x
w10x
ation of motoneurons from avian neural tube progenitor cells, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 3247–3251. H. Eyal-Giladi, S. Kochav, From cleavage to primitive streak formation: A complementary normal table and a new look at the first stages of the development of the chick. I. General morphology, Dev. Biol. 49 Ž1976. 321–337. A. Graham, P. Francis-West, P. Brickell, A. Lumsden, The signaling molecule BMP4 mediates apoptosis in the rhombencephalic neural crest, Nature 372 Ž1994. 684–686. A. Graham, I. Heyman, A. Lumsden, Even-numbered rhombomeres control the apoptotic elimination of neural crest cells from odd-numbered rhombomeres in the chick hindbrain, Development 119 Ž1993. 233–245. V. Hamburger, H.L. Hamilton, A series of normal stages in the development of the chick embryo, J. Morphol. 88 Ž1951. 49–92. R.M. Harland, In situ hybridization: an improved whole-mount method for xenopus embryos, Methods Cell Biol. 36 Ž1991. 685– 695. J.C. Izpisua-Belmonte, E.M. De Robertis, K.G. Storey, C.D. Stern, The homeobox gene goosecoid and the origin of organizer cells in the early chick blastoderm, Cell 74 Ž1993. 645–659. P. Jeffs, K. Jaques, M. Osmond, Cell death in cranial neural crest development, Anat. Embryol. 185 Ž1992. 583–588. N. Kahane, C. Kalcheim, Expression of trkC receptor mRNA during development of the avian nervous system, J. Neurobiol. 25 Ž1994. 571–584. F. Lamballe, R. Klein, M. Barbacid, trkC, a new member of the trk
w11x
w12x w13x
w14x
w15x
w16x
w17x
w18x
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family of tyrosine protein kinases, is a receptor for neurotrophin-3, Cell 66 Ž1991. 967–979. R. Li, P. Bernd, Neurotrophin-3 increases neurite outgrowth and apoptosis in explants of the chicken neural plate, Dev. Neurosci. 21 Ž1999. 12–21. A. Lumsden, The cellular basis of segmentation in the developing hindbrain, Trends Neurosci. 13 Ž1990. 329–335. A. Lumsden, N. Sprawson, A. Graham, Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo, Development 113 Ž1991. 1281–1291. J. Sechrist, G.N. Serbedzija, T. Scherson, S.E. Fraser, M. BronnerFraser, Segmental migration of the hindbrain neural crest does not arise from its segmental generation, Development 118 Ž1993. 691– 703. C.D. Stern, R.T. Yu, A. Kakizuka, C.R. Kintner, L.S. Mathews, W.W. Vale, R.M. Evans, K. Umesono, Activin and its receptors during gastrulation and the later phases of mesoderm development in the chick embryo, Dev. Biol. 172 Ž1995. 192–205. R. Williams, A. Backstrom, T. Ebendal, F. Hallbook, Molecular cloning and cellular localization of trkC in the chicken embryo, Dev. Brain Res. 75 Ž1993. 235–252. L. Yao, D. Zhang, P. Bernd, The onset of neurotrophin and trk mRNA expression in early embryonic tissues of the quail, Dev. Biol. 165 Ž1994. 727–730. D. Zhang, L. Yao, P. Bernd, Expression of neurotrophin trk and p75 receptors in quail embryos undergoing gastrulation and neurulation, Dev. Dynam. 205 Ž1996. 150–161.