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Plasticity of avian mesencephalic polarity revealed by trajectories of tectofugal axons
De~,elopmental Brain Research, 75 (1993) 39-44
39
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00
B R E S D 51675
Plasticity of avian mesencephalic polarity revealed by trajectories of tectofugal axons Toru Matsuno and Harukazu Nakamura Department of Biology, Kyoto Prefectural Unit~ersityof Medicine, Kita-Ku, Kyoto (Japan) (Accepted 6 April 1993)
Key words: Quail-chick chimera; Rostrocaudal polarity; Plasticity; Mesencephalon rotation; Circumferential axon
In normal E6 (sixth day of incubation) quail mesencephalons, circumferential axons originate in the tectum (the dorsal part of a mesencephalon), course in a ventral direction and split into three trajectories in the ventral region of the mesencephalon; two of them turned on the ipsilateral side of the mesencephalon, one rostrally and the other caudally, while the third crossed the ventral midline and turned caudally on the contralateral side. In this study, we examined whether the ventral part of the mesencephalon has plasticity in its rostrocaudal polarity formation. We transplanted quail mesencephalons at the 9 - 1 0 somite stage (E2)with reversed rostrocaudal orientation prior to axon outgrowth. In reversely transplanted mesencephalons, circumferential axons at E6 took the same trajectory pattern as that of normal embryos; one turned rostrally and the second caudally on the ipsilateral side, and the third one turned caudally on the contralateral side. These results indicate that the rostrocaudal polarity of the mesencephalon is not fixed at E2, and that it may develop u n d e r the influence of tissues surrounding the mesencephalon.
INTRODUCTION
The function of the nervous system depends on the correct network of neurons and their targets. For accurate connections, axons should be precisely guided to their appropriate targets. Avian tecta send efferent fibers. In developing tecta, young neurons send axons circumferentially to the ventral region of the mesencephalon by E3, then the axons are grouped together in fascicles and are called circumferential axons ~9 or tectobulbar fibers 5. In E6 chick brains, circumferential axons split into two trajectories in the ventral region of the mesencephalon and turn caudally 11. Recently, the development of rostrocaudal polarity of the tectum has received special attention 8 - 1014 • ' 16'21"22 . The development of the rostrocaudal polarity of the tectum is characterized by the gradient in the engrailed expression pattern 4'18, the gradient in the cytoarchitectonic development 12'2°, and finally the positional specification along the rostrocaudal axis which retinal fibers read to find their target 21'2~. Previous tectum rotation experiments have shown that the tectum polarity is
regulated to the host pattern after rotation of the rectum anlage at E21'~'~4'~6. It was shown that the dorsal part of the mesencephalon (tectal anlage) has plasticity in the establishment of its rostrocaudal polarity, but whether the ventral part of the mesencephalon has plasticity was not previously demonstrated. Since the direction of circumferential axons are determined at the ventral part of the mesencephalon, we checked the rostrocaudal polarity of the ventral part of the mesencephalon by the trajectory pattern of circumferential axons. For this purpose, we transplanted avian mesencephalons with reversed rostrocaudal orientation on E2, and observed in E6 embryos the direction of circumferential axons. We used quail mesencephalons as a graft and chick embryos as a host. Carbocyanine dye, DiI was used as an anterograde tracer of circumferential axons. MATERIALS AND METHODS Transplantations Fertilized chick (Gallus gallus domesticus) and quail (Coturnix coturnixjaponica) eggs obtained from local farms were incubated in a
Correspondence: T. Matsuno, D e p a r t m e n t of Biology, Kyoto Prefectural University of Medicine, 13 Nishitakatsukasa-cho, Taishogun, Kita-Ku, Kyoto 603, Japan. Fax: (81) (75) 465-7651.
40
R
Fig. 1. Schematic drawing of the transplantation. The chick (CH) mesencephalon (MS) was removed and the quail (Q) mesencephalon was transplanted into the host with reversed rostrocaudal orientation at the 9-10 somite stage. C, caudal; R, rostral; star, carbon particle as an orientation marker.
humidified atmosphere at 37.8°C for about 36 h (E2). All surgery was performed at the 9-10 somite stage (stage 10 as described by Hamburger and Hamilton7) in ovo. In all cases, grafts were prepared from quail embryos, and transplanted into host chick embryos. The entire mesencephalon with the epidermis and the notochord was excised from the host chick embryos, and the same portion of quail brain was transplanted as shown in Fig. 1. The rostrocaudal orientation of the graft was reversed. A carbon particle was placed at the original caudal portion of the graft to mark the orientation.
Anterograde labeling of circumferential axons At E5, a tiny crystal of DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, Molecular Probes) was inserted into the tectum of the chimera to label the axons in ovo. At E6, stage 28 as described by Hamburger and Hamilton7, the diencephalon, the mesencephalon and the metencephalon were excised together and cut open along the dorsal midline. Specimens were fixed for 1 h with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After fixation, they were whole-mounted with a solution of 9 parts glycerol and 1 part 0.1 M phosphate buffer (pH 7.4) containing 5% n-propyl gallate as described by Nakamura and O'Leary 17, and observed under an epifluorescence microscope. Circumferential axons of normal embryos were also observed as controls.
Identification of grafts in hosts After observations of circumferential axons, specimens were dehydrated and embedded in paraffin, and serially sectioned. Sections were stained according to the Feulgen-Rossenbeck method to distinguish quail and chick cells 13. RESULTS
Normal circumferential axons I n E5 quail a n d E6 chick e m b r y o s , a n t e r o g r a d e l a b e l i n g by D i I s h o w e d t h a t c i r c u m f e r e n t i a l axons split into two t r a j e c t o r i e s , a n d b o t h t u r n e d c a u d a l l y as des c r i b e d by K r 6 g e r a n d Schwarz tl ( n o t i l l u s t r a t e d here). In E6 quail e m b r y o s , c i r c u m f e r e n t i a l axons c o u r s e d in a v e n t r a l d i r e c t i o n f o r m i n g thick fascicles in the t e c t u m . Since t h e a s c e n d i n g fiber b u n d l e was f o r m e d , c i r c u m f e r e n t i a l axons split into t h r e e d i f f e r e n t t r a j e c t o ries, T1, T2 a n d T3 (Fig. 2). T1 a n d T2 w e r e on the i p s i l a t e r a l side a n d t u r n e d r o s t r a l l y a n d caudally, respectively. T3 crossed t h e v e n t r a l m i d l i n e a n d t u r n e d a b r u p t l y in the c a u d a l d i r e c t i o n on the c o n t r a l a t e r a l side. T2 a n d T3 a r e t e c t o b u l b a r fibers ~ a n d T1 m a y b e axons a s c e n d i n g to the h i g h e r b r a i n centers.
E7 chick e m b r y o s c i r c u m f e r e n t i a l axons in o n e of four cases, a l t h o u g h o t h e r axons trated).
s h o w e d the s a m e nzqectorics ot as those in E6 quail embryos, but o n e axon in "I"3 t u r n e d rostraliy in T3 t u r n e d c a u d a l l y (not illus.
Circumferential axons of grafts and graft identification In all five E6 c h i m e r a s o b t a i n e d , in which the rost r o c a u d a l axis of the m e s e n c e p h a l o n was r o t a t e d 180 °, c i r c u m f e r e n t i a l axons s h o w e d the s a m e trajectory pattern as that in n o r m a l E6 quail brains. Thick fascicles c o u r s e d in a v e n t r a l d i r e c t i o n in the t e c t u m and split into t h r e e d i f f e r e n t t r a j e c t o r i e s (R1, R2 a n d R3 in Figs. 3 a n d 4) n e a r the v e n t r a l midline. RI c o u r s e d rostrally a n d R2 c o u r s e d c a u d a l l y on the i p s i l a t e r a l side of the m e s e n c e p h a l o n . R3 c r o s s e d the ventral m i d l i n e a n d a b r u p t l y t u r n e d c a u d a l l y on the c o n t r a l a t eral side. In s p e c i m e n No. 44, o n e axon ( i n d i c a t e d by an arrow in Fig. 4C) in R3 d e v i a t e d from the m a i n c o u r s e a n d t u r n e d rostrally. This kind of axon was also f o u n d in the control. In s p e c i m e n No. 31, R3 c o u r s e d to the ipsilateral side in the host m e t e n c e p h a l o n ( a r r o w in Fig. 4B). H i s t o l o g i c a l study s h o w e d that t h e e n t i r e m e s e n c e p h a l o n a n d the c a u d a l p a r t of the d i e n c e p h a l o n c o n s i s t e d o f quail ceils, while o t h e r p a r t s of the specim e n c o n s i s t e d of host chick cells in s p e c i m e n s Nos. 25, 31, 44 a n d 47. H o w e v e r , the w h o l e d i e n c e p h a l o n consisted o f host chick tissues in s p e c i m e n No. 3(1 (Figs. 3 a n d 4). DISCUSSION In m e s e n c e p h a l o n s of n o r m a l E6 quail a n d c h i m e r i c embryos, c i r c u m f e r e n t i a l axons split into t h r e e trajectories; two c o u r s e d on t h e i p s i l a t e r a l side of the m e s e n c e p h a l o n , o n e o f t h e s e two t u r n e d rostrally a n d the o t h e r t u r n e d caudally. T h e third o n e crossed t h e ventral m i d l i n e a n d t u r n e d c a u d a l l y on the c o n t r a l a t e r a l side (Figs. 2 - 4 ) . R o t a t i o n of the m e s e n c e p h a l o n r e v e r s e d its original r o s t r o c a u d a l a n d l e f t / r i g h t o r i e n t a t i o n s . Fig. 5 A s c h e m a t i c a l l y illustrates the E6 quail m e s e n c e p h a l o n a n d the n o r m a l t r a j e c t o r i e s o f c i r c u m f e r e n t i a l axons o f the right side. W h e n the m e s e n c e p h a l o n is r o t a t e d , the ' r i g h t ' c i r c u m f e r e n t i a l axons t a k e t r a j e c t o r i e s as shown in Fig. 5B if the t r a j e c t o r i e s of t h e axons are fixed b e f o r e surgery. H o w e v e r , c i r c u m f e r e n t i a l axons in rot a t e d m e s e n c e p h a l o n s t o o k t r a j e c t o r i e s as shown in Fig. 5C. This t r a j e c t o r y p a t t e r n was d i f f e r e n t from that in Fig. 5B, b u t was similar to the n o r m a l p a t t e r n of c i r c u m f e r e n t i a l axons on the left side. This indicates that the t r a j e c t o r i e s o f c i r c u m f e r e n t i a l axons were not
41 As a source of cues establishing the rostrocaudal polarity of the mesencephalon, we can eliminate mesoderm because the polarity of the grafts which included surrounding mesoderm and notochord was adjusted to
determined at E2, i.e. the rostrocaudal polarity recognized by circumferential axons is not fixed at E2, and polarity may be formed under the influence of tissues surrounding the mesencephalon.
D ~----,,-
i(
C
DI
~
~
V
~---"" D
I I I I I
'
_
\
"
"_ T i l l ] "
DE
\
CA
Fig. 2. Circumferential axons of a normal E6 quail mesencephalon. A: the dorsal midline of diencephalon (DE), mesencephalon (MS) and metencephalon (MT) was cut open and whole-mounted. Circumferential axons (CA) stained with Dil crystal (DI) were split into three trajectories (T1-T3). B: the photomosaic framed in (A). C, caudal; D, dorsal; M, ventral midline of the brain; R, rostral; V, ventral. Bars = 1 mm for (A) and 0.2 m m for (B).
D ~-----~ V " ~
42
D
R
C
25 : DE t
~
MS "R1
I I'
~ '
IR 3
DI CA
Right
Left
A
Fig. 3. Circumferential axons of a chimeric brain at E6 (specimen No. 25), A: flattened display of the chimeric brain. Circumferential axons (CA) split into three trajectories (R1-R3) and took the trajectory pattern similar to that in normal E6 quails. B: the photomosaic framed in (A). C: micrograph near the boundary of chick (CH) and quail (Q) tissues. Quail nuclei include condensed heterochromatin. In this specimen, the entire mesencephalon (MS) and the caudal part of the diencephalon (DE) consisted of quail tissues. C, caudal; D, dorsal; DI, DiI crystal; M, ventral midline of the brain; MT, metencephalon; R, rostral; V, ventral; hatched areas, host chick tissues. Bars = i mm for (A), 0.2 mm for (B)and 20 /xm for (C).
43
30
B
R3 MS
Fig. 4. Camera lucida drawing of circumferential axons in four other chimeric brains at E6. The entire mesencephalon and the caudal part of thc diencephalon consisted of quail tissues except in specimen No. 30. R3 in specimen No. 31 coursed into the ipsilateral side in the metencephalon (arrow in B). One axon in R3 turned rostrally in specimen No. 44 (arrow in C). Symbols are the same as those in Fig. 3. Bar = 1 ram.
the polarity of the host. Martinez et al. 15 and Bally-Cuif et al. 2 grafted the met-mesencephalic segment of the vertebrate to the E2 chick prosencephalon, and showed that the segment induced phenotypic changes of the host diencephalon. Itasaki et a[. 9 transplanted tectal primordium (mesencephalon) into the chick E2 prosen-
I,T3
/
k_J
Fig. 5. Schematic drawings of mesencephalons and circumferential axons. A: normal trajectories (T1-T3) of circumferential axons in the right half of the tectum. B: hypothetical trajectories (T1-T3) in the rotated mesencephalon where trajectories of circumferential axons were determined before rotation. C: results of our experiments in rotated mesencephalons. The pattern of trajectories (R1-R3) was different from that in (B). C, caudal; DI, Dil crystal; R, rostral.
cephalon and found that the mes-diencephalon junction influenced the formation of the polarity of the grafted tectum. These findings indicated that polarity of the mesencephalon was formed under cues emanating from the mes-metencephalon a n d / o r mes-diencephalon junctions. The R3 trajectory in chimeras shared common characteristics with T3 in normal embryos, i.e. R3 and T3 crossed the ventral midline of the mesencephalon and were the longest among the trajectories. Therefore, R3 may correspond to T3. In E6 quail embryos, the T1 trajectory was formed last among the trajectories, and was the shortest. R1 was the shortest in E6 chimeras, therefore, R1 may correspond to T1. These findings suggest that rostrocaudal polarity recognized by circumferential axons in rotated mesencephalons develops in conformity with that of the hosts. In specimen No. 31, the R3 trajectory coursed to the ipsilateral side in the host metencephalon (Fig. 4B). This may have arisen from an incomplete g r a f t / h o s t junction, and the axons may have turned to the ipsilateral side for a smooth passage. In avian tecta which differentiate from the dorsal part of the mesencephalon, the development of rostrocaudal polarity includes: the engrailed gene expression pattern 4"Is, the cytoarchitectonic development Iz'2° and retinotectal projection map formation 3. Previous experiments have shown that these were all regulated to the host pattern after tectum rotation at E21"s''~'~4'16. The present study showed that the trajectories of efferent
44
fibers in the tectum are also adjusted to the host pattern after the rotation of the mesencephalons at E2. Since axonal direction is determined at the ventral part of the mesencephalon after the axons leave the rectum, the results of the present study indicate that the ventral part of the mesencephalon has plasticity around the 10 somite stage (E2) with respect to the establishment of rostrocaudal polarity. Yaginuma and Oppenheim 24 rotated three segments of chick neural tube (future spinal cord) around the rostrocaudal axis, and observed that axons of the dorsolateral border cells coursed to the contralateral side of the spinal cord and turned rostrally, similar to control axons. They discussed the possibility that longitudinal fibers originating from normal segments may have provided axons of the dorsolateral border cells with rostral cues. Wilson et al. 23 demonstrated that a simple grid of tracts and commissures in the brain of zebrafish formed an initial axon scaffold, and suggested that the scaffold plays a crucial role in guiding additional axons. In our observations, trajectories T3 and R3 turned suddenly at a right angle and grew rostrally in a straight line. One possible interpretation of our results is that molecular cues distributed in the mesencephalon were reorganized because of the change of the fate of the inverted graft, and scaffold formation coincided with the host type. The circumferential axons were then guided by this scaffold. It is alternatively conceivable that the rostrocaudal polarity in the ventral region of the mesencephalon was formed by a gradient of chemotropic factors emanating from the diencephalon a n d / o r metencephalon, attracting circumferential axons to their appropriate directions. Guthrie and Lumsden 6 transplanted a segment of chick rhombomeres with reversed rostrocaudal orientation, and showed that the majority of motor neurons grew to the appropriate rostral exit point. They suggested that the motor neurons were attracted to the exit point. Acknowledgements. We thank Dr. Nobue ltasaki for her discussion. This study was supported by a Grant-in-Aid for Scientific Research 04454130 from the Japanese Ministry of Education, Science and Culture, by the Life Science Foundation of Japan, by the Brain Science Foundation and the Mitsubishi Foundation.
REFERENCES 1 Alvarado-Mallart, R.M., Martinez, S. and Lance-Jones, C.C., Pluripotentiality of the 2-day-old avian germinative neuroepithelium, Dev. Biol., 139 (19901 75-88. 2 Bally-Cuif, L., Alvarado-Mallart, R.M., Darnell, D.K. and Wassef, M., Relationship between Writ-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon, Development, 115 (1992) 999-1009.
3 Crossland, W.J. and Uchwat, C.J., lopographic pn.~lcctions of the retina and optic tectum upon the ventral lateral geniculatc nucleus in the chick, J. Comp. Neurol., 185 (19791 87- 106 4 Gardner, C.A., Darnell, D.K.. Poole, S.J., Ordahl, (LP and Barald, K.F., Expression of an engrailed-like gone during development of the early embryonic chick nervous system, .I. Neurosci. Rea., 21 (1988) 426-437. 5 Goldberg, S., Studies on the mechanics of development of the visual pathways in the chick embryo, Dec. Biol., 36 (1974) 24-43. 6 Guthrie, S. and Lumsden, A., Motor neuron pathfinding followlug rhombomerc reversals in the chick embryo hindbrain, Det~elopment, 114 (1992) 663-673. 7 ltamburger, V. and Hamilton, ll.L, A series o! normal stages in the development of the chick embryo, Z MorphoL, 88 (1951) 49-92. 8 lchijo, H., Fujita, S. Matsuno, T. and Nakamura, H., Rotation of the tectal primordium reveals plasticity of target recognition in retinotectal projection, Development, 110 (1990)331-342. 9 ltasaki, N., Ichijo, H., Hama, C., Matsuno, T. and Nakamura, H., Establishment of rostrocaudal polarity in tectal primordium: engrailed expression and subsequent tectal polarity, Development, 113 (19911 1133-1144. 1(1 Itasaki, N. and Nakamura, lq., Rostrocaudal polarity of the rectum in birds: correlation of en gradient and topographic order in retinotectal projection, Neuron, 8 (1992) 787-798. 11 Kr6ger, S. and Schwarz, U., The avian tectobulbar tract: development, explant culture, and effects of antibodies on the pattern of neurite outgrowth, J. Neurosci., 10 (1990) 3118-3134. 12 LaVail, J.H. and Cowan, W.M., The development of the chick optic rectum. I. Normal morphology and cytoarchitectonic development, Brain Res., 28 (1971) 391-419. 13 Le Douarin, N.M., A biological cell labeling technique and its use in experimental embryology, Dec. Biol., 30 (1973) 217-222. 14 Martinez, S. and Alvarado-Mallart, R.M., Expression of the homeobox chick-en gene in chick/quail chimeras with inverted mes-metencephalic grafts, Dec. Biol., 139 (1990) 432-436. 15 Martinez, S., Wassef, M. and Alvarado-Mallart, R.M., Induction of a mesencephalic phenotype in the 2-day-old chick prosencephalon is preceded by the early expression of the homeobox gene en, Neuron. 6 (1991) 971-981. 16 Matsuno, T., lchijo, H. and Nakamura, H., Regulation of the rostrocaudal axis of the optic tectum: histological study after rostrocaudal rotation in quail-chick chimeras, Dec Brain Res., 58 ( 1991 ) 265-270. 17 Nakamura, H. and O'Leary, D.D.M., Inaccuracies in initial growth and arborization of chick retinotectal axons followed by course corrections and axon remodeling to develop topographic order, J. Neurosci., 9 (1989) 3776-3795. 18 Patel, N.H., Martin-Blanco, E., Coleman, K.G., Poole, S.J., Ellis, M.C, Kornberg, T.B. and Goodman, C.S., Expression of engrailed proteins in arthropods, annelids, and chordates, ('~,//, 58 (1989) 955 968. 19 Puelles, L. and Bendala, M.C., Differentiation of neuroblasts in the chick optic tectum up to eight days of incubation: a Golgi study, Neuroscience, 3 (1978) 307-325. 20 Senut, M.C. and Alvarado-Mallart, R.M., Development of the retinotectal system in normal quail embryos: cytoarchitectonic development and optic fiber innervation, Dev. Brain Res., 29 (1986) 123-140. 21 Walter, J., Henke-Fahle, S. and Bonhoeffer, F., Avoidance of posterior tectal membranes by temporal retinal axons, Det~elopment. 101 (19871 909-913. 22 Walter, J., Kern-Veits, B., Huf, J., Stolze, B. and Bonhoeffer, F., Recognition of position-specific properties of tectal cell membranes by retinal axons in vitro, Development, 101 (1987) 685-696. 23 Wilson, S.W., Ross, LS., Parrett, T. and Easter Jr., S.S., The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio, Development, 108 (19901 121 145. 24 Yaginuma, H. and Oppenheim, R.W., An experimental analysis of in vivo guidance cues used by axons of spinal interneurons in the chick embryo: evidence for chemotropism and related guidance mechanisms..L Neurosci., 11 (19911 2598-2613.
39
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00
B R E S D 51675
Plasticity of avian mesencephalic polarity revealed by trajectories of tectofugal axons Toru Matsuno and Harukazu Nakamura Department of Biology, Kyoto Prefectural Unit~ersityof Medicine, Kita-Ku, Kyoto (Japan) (Accepted 6 April 1993)
Key words: Quail-chick chimera; Rostrocaudal polarity; Plasticity; Mesencephalon rotation; Circumferential axon
In normal E6 (sixth day of incubation) quail mesencephalons, circumferential axons originate in the tectum (the dorsal part of a mesencephalon), course in a ventral direction and split into three trajectories in the ventral region of the mesencephalon; two of them turned on the ipsilateral side of the mesencephalon, one rostrally and the other caudally, while the third crossed the ventral midline and turned caudally on the contralateral side. In this study, we examined whether the ventral part of the mesencephalon has plasticity in its rostrocaudal polarity formation. We transplanted quail mesencephalons at the 9 - 1 0 somite stage (E2)with reversed rostrocaudal orientation prior to axon outgrowth. In reversely transplanted mesencephalons, circumferential axons at E6 took the same trajectory pattern as that of normal embryos; one turned rostrally and the second caudally on the ipsilateral side, and the third one turned caudally on the contralateral side. These results indicate that the rostrocaudal polarity of the mesencephalon is not fixed at E2, and that it may develop u n d e r the influence of tissues surrounding the mesencephalon.
INTRODUCTION
The function of the nervous system depends on the correct network of neurons and their targets. For accurate connections, axons should be precisely guided to their appropriate targets. Avian tecta send efferent fibers. In developing tecta, young neurons send axons circumferentially to the ventral region of the mesencephalon by E3, then the axons are grouped together in fascicles and are called circumferential axons ~9 or tectobulbar fibers 5. In E6 chick brains, circumferential axons split into two trajectories in the ventral region of the mesencephalon and turn caudally 11. Recently, the development of rostrocaudal polarity of the tectum has received special attention 8 - 1014 • ' 16'21"22 . The development of the rostrocaudal polarity of the tectum is characterized by the gradient in the engrailed expression pattern 4'18, the gradient in the cytoarchitectonic development 12'2°, and finally the positional specification along the rostrocaudal axis which retinal fibers read to find their target 21'2~. Previous tectum rotation experiments have shown that the tectum polarity is
regulated to the host pattern after rotation of the rectum anlage at E21'~'~4'~6. It was shown that the dorsal part of the mesencephalon (tectal anlage) has plasticity in the establishment of its rostrocaudal polarity, but whether the ventral part of the mesencephalon has plasticity was not previously demonstrated. Since the direction of circumferential axons are determined at the ventral part of the mesencephalon, we checked the rostrocaudal polarity of the ventral part of the mesencephalon by the trajectory pattern of circumferential axons. For this purpose, we transplanted avian mesencephalons with reversed rostrocaudal orientation on E2, and observed in E6 embryos the direction of circumferential axons. We used quail mesencephalons as a graft and chick embryos as a host. Carbocyanine dye, DiI was used as an anterograde tracer of circumferential axons. MATERIALS AND METHODS Transplantations Fertilized chick (Gallus gallus domesticus) and quail (Coturnix coturnixjaponica) eggs obtained from local farms were incubated in a
Correspondence: T. Matsuno, D e p a r t m e n t of Biology, Kyoto Prefectural University of Medicine, 13 Nishitakatsukasa-cho, Taishogun, Kita-Ku, Kyoto 603, Japan. Fax: (81) (75) 465-7651.
40
R
Fig. 1. Schematic drawing of the transplantation. The chick (CH) mesencephalon (MS) was removed and the quail (Q) mesencephalon was transplanted into the host with reversed rostrocaudal orientation at the 9-10 somite stage. C, caudal; R, rostral; star, carbon particle as an orientation marker.
humidified atmosphere at 37.8°C for about 36 h (E2). All surgery was performed at the 9-10 somite stage (stage 10 as described by Hamburger and Hamilton7) in ovo. In all cases, grafts were prepared from quail embryos, and transplanted into host chick embryos. The entire mesencephalon with the epidermis and the notochord was excised from the host chick embryos, and the same portion of quail brain was transplanted as shown in Fig. 1. The rostrocaudal orientation of the graft was reversed. A carbon particle was placed at the original caudal portion of the graft to mark the orientation.
Anterograde labeling of circumferential axons At E5, a tiny crystal of DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, Molecular Probes) was inserted into the tectum of the chimera to label the axons in ovo. At E6, stage 28 as described by Hamburger and Hamilton7, the diencephalon, the mesencephalon and the metencephalon were excised together and cut open along the dorsal midline. Specimens were fixed for 1 h with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After fixation, they were whole-mounted with a solution of 9 parts glycerol and 1 part 0.1 M phosphate buffer (pH 7.4) containing 5% n-propyl gallate as described by Nakamura and O'Leary 17, and observed under an epifluorescence microscope. Circumferential axons of normal embryos were also observed as controls.
Identification of grafts in hosts After observations of circumferential axons, specimens were dehydrated and embedded in paraffin, and serially sectioned. Sections were stained according to the Feulgen-Rossenbeck method to distinguish quail and chick cells 13. RESULTS
Normal circumferential axons I n E5 quail a n d E6 chick e m b r y o s , a n t e r o g r a d e l a b e l i n g by D i I s h o w e d t h a t c i r c u m f e r e n t i a l axons split into two t r a j e c t o r i e s , a n d b o t h t u r n e d c a u d a l l y as des c r i b e d by K r 6 g e r a n d Schwarz tl ( n o t i l l u s t r a t e d here). In E6 quail e m b r y o s , c i r c u m f e r e n t i a l axons c o u r s e d in a v e n t r a l d i r e c t i o n f o r m i n g thick fascicles in the t e c t u m . Since t h e a s c e n d i n g fiber b u n d l e was f o r m e d , c i r c u m f e r e n t i a l axons split into t h r e e d i f f e r e n t t r a j e c t o ries, T1, T2 a n d T3 (Fig. 2). T1 a n d T2 w e r e on the i p s i l a t e r a l side a n d t u r n e d r o s t r a l l y a n d caudally, respectively. T3 crossed t h e v e n t r a l m i d l i n e a n d t u r n e d a b r u p t l y in the c a u d a l d i r e c t i o n on the c o n t r a l a t e r a l side. T2 a n d T3 a r e t e c t o b u l b a r fibers ~ a n d T1 m a y b e axons a s c e n d i n g to the h i g h e r b r a i n centers.
E7 chick e m b r y o s c i r c u m f e r e n t i a l axons in o n e of four cases, a l t h o u g h o t h e r axons trated).
s h o w e d the s a m e nzqectorics ot as those in E6 quail embryos, but o n e axon in "I"3 t u r n e d rostraliy in T3 t u r n e d c a u d a l l y (not illus.
Circumferential axons of grafts and graft identification In all five E6 c h i m e r a s o b t a i n e d , in which the rost r o c a u d a l axis of the m e s e n c e p h a l o n was r o t a t e d 180 °, c i r c u m f e r e n t i a l axons s h o w e d the s a m e trajectory pattern as that in n o r m a l E6 quail brains. Thick fascicles c o u r s e d in a v e n t r a l d i r e c t i o n in the t e c t u m and split into t h r e e d i f f e r e n t t r a j e c t o r i e s (R1, R2 a n d R3 in Figs. 3 a n d 4) n e a r the v e n t r a l midline. RI c o u r s e d rostrally a n d R2 c o u r s e d c a u d a l l y on the i p s i l a t e r a l side of the m e s e n c e p h a l o n . R3 c r o s s e d the ventral m i d l i n e a n d a b r u p t l y t u r n e d c a u d a l l y on the c o n t r a l a t eral side. In s p e c i m e n No. 44, o n e axon ( i n d i c a t e d by an arrow in Fig. 4C) in R3 d e v i a t e d from the m a i n c o u r s e a n d t u r n e d rostrally. This kind of axon was also f o u n d in the control. In s p e c i m e n No. 31, R3 c o u r s e d to the ipsilateral side in the host m e t e n c e p h a l o n ( a r r o w in Fig. 4B). H i s t o l o g i c a l study s h o w e d that t h e e n t i r e m e s e n c e p h a l o n a n d the c a u d a l p a r t of the d i e n c e p h a l o n c o n s i s t e d o f quail ceils, while o t h e r p a r t s of the specim e n c o n s i s t e d of host chick cells in s p e c i m e n s Nos. 25, 31, 44 a n d 47. H o w e v e r , the w h o l e d i e n c e p h a l o n consisted o f host chick tissues in s p e c i m e n No. 3(1 (Figs. 3 a n d 4). DISCUSSION In m e s e n c e p h a l o n s of n o r m a l E6 quail a n d c h i m e r i c embryos, c i r c u m f e r e n t i a l axons split into t h r e e trajectories; two c o u r s e d on t h e i p s i l a t e r a l side of the m e s e n c e p h a l o n , o n e o f t h e s e two t u r n e d rostrally a n d the o t h e r t u r n e d caudally. T h e third o n e crossed t h e ventral m i d l i n e a n d t u r n e d c a u d a l l y on the c o n t r a l a t e r a l side (Figs. 2 - 4 ) . R o t a t i o n of the m e s e n c e p h a l o n r e v e r s e d its original r o s t r o c a u d a l a n d l e f t / r i g h t o r i e n t a t i o n s . Fig. 5 A s c h e m a t i c a l l y illustrates the E6 quail m e s e n c e p h a l o n a n d the n o r m a l t r a j e c t o r i e s o f c i r c u m f e r e n t i a l axons o f the right side. W h e n the m e s e n c e p h a l o n is r o t a t e d , the ' r i g h t ' c i r c u m f e r e n t i a l axons t a k e t r a j e c t o r i e s as shown in Fig. 5B if the t r a j e c t o r i e s of t h e axons are fixed b e f o r e surgery. H o w e v e r , c i r c u m f e r e n t i a l axons in rot a t e d m e s e n c e p h a l o n s t o o k t r a j e c t o r i e s as shown in Fig. 5C. This t r a j e c t o r y p a t t e r n was d i f f e r e n t from that in Fig. 5B, b u t was similar to the n o r m a l p a t t e r n of c i r c u m f e r e n t i a l axons on the left side. This indicates that the t r a j e c t o r i e s o f c i r c u m f e r e n t i a l axons were not
41 As a source of cues establishing the rostrocaudal polarity of the mesencephalon, we can eliminate mesoderm because the polarity of the grafts which included surrounding mesoderm and notochord was adjusted to
determined at E2, i.e. the rostrocaudal polarity recognized by circumferential axons is not fixed at E2, and polarity may be formed under the influence of tissues surrounding the mesencephalon.
D ~----,,-
i(
C
DI
~
~
V
~---"" D
I I I I I
'
_
\
"
"_ T i l l ] "
DE
\
CA
Fig. 2. Circumferential axons of a normal E6 quail mesencephalon. A: the dorsal midline of diencephalon (DE), mesencephalon (MS) and metencephalon (MT) was cut open and whole-mounted. Circumferential axons (CA) stained with Dil crystal (DI) were split into three trajectories (T1-T3). B: the photomosaic framed in (A). C, caudal; D, dorsal; M, ventral midline of the brain; R, rostral; V, ventral. Bars = 1 mm for (A) and 0.2 m m for (B).
D ~-----~ V " ~
42
D
R
C
25 : DE t
~
MS "R1
I I'
~ '
IR 3
DI CA
Right
Left
A
Fig. 3. Circumferential axons of a chimeric brain at E6 (specimen No. 25), A: flattened display of the chimeric brain. Circumferential axons (CA) split into three trajectories (R1-R3) and took the trajectory pattern similar to that in normal E6 quails. B: the photomosaic framed in (A). C: micrograph near the boundary of chick (CH) and quail (Q) tissues. Quail nuclei include condensed heterochromatin. In this specimen, the entire mesencephalon (MS) and the caudal part of the diencephalon (DE) consisted of quail tissues. C, caudal; D, dorsal; DI, DiI crystal; M, ventral midline of the brain; MT, metencephalon; R, rostral; V, ventral; hatched areas, host chick tissues. Bars = i mm for (A), 0.2 mm for (B)and 20 /xm for (C).
43
30
B
R3 MS
Fig. 4. Camera lucida drawing of circumferential axons in four other chimeric brains at E6. The entire mesencephalon and the caudal part of thc diencephalon consisted of quail tissues except in specimen No. 30. R3 in specimen No. 31 coursed into the ipsilateral side in the metencephalon (arrow in B). One axon in R3 turned rostrally in specimen No. 44 (arrow in C). Symbols are the same as those in Fig. 3. Bar = 1 ram.
the polarity of the host. Martinez et al. 15 and Bally-Cuif et al. 2 grafted the met-mesencephalic segment of the vertebrate to the E2 chick prosencephalon, and showed that the segment induced phenotypic changes of the host diencephalon. Itasaki et a[. 9 transplanted tectal primordium (mesencephalon) into the chick E2 prosen-
I,T3
/
k_J
Fig. 5. Schematic drawings of mesencephalons and circumferential axons. A: normal trajectories (T1-T3) of circumferential axons in the right half of the tectum. B: hypothetical trajectories (T1-T3) in the rotated mesencephalon where trajectories of circumferential axons were determined before rotation. C: results of our experiments in rotated mesencephalons. The pattern of trajectories (R1-R3) was different from that in (B). C, caudal; DI, Dil crystal; R, rostral.
cephalon and found that the mes-diencephalon junction influenced the formation of the polarity of the grafted tectum. These findings indicated that polarity of the mesencephalon was formed under cues emanating from the mes-metencephalon a n d / o r mes-diencephalon junctions. The R3 trajectory in chimeras shared common characteristics with T3 in normal embryos, i.e. R3 and T3 crossed the ventral midline of the mesencephalon and were the longest among the trajectories. Therefore, R3 may correspond to T3. In E6 quail embryos, the T1 trajectory was formed last among the trajectories, and was the shortest. R1 was the shortest in E6 chimeras, therefore, R1 may correspond to T1. These findings suggest that rostrocaudal polarity recognized by circumferential axons in rotated mesencephalons develops in conformity with that of the hosts. In specimen No. 31, the R3 trajectory coursed to the ipsilateral side in the host metencephalon (Fig. 4B). This may have arisen from an incomplete g r a f t / h o s t junction, and the axons may have turned to the ipsilateral side for a smooth passage. In avian tecta which differentiate from the dorsal part of the mesencephalon, the development of rostrocaudal polarity includes: the engrailed gene expression pattern 4"Is, the cytoarchitectonic development Iz'2° and retinotectal projection map formation 3. Previous experiments have shown that these were all regulated to the host pattern after tectum rotation at E21"s''~'~4'16. The present study showed that the trajectories of efferent
44
fibers in the tectum are also adjusted to the host pattern after the rotation of the mesencephalons at E2. Since axonal direction is determined at the ventral part of the mesencephalon after the axons leave the rectum, the results of the present study indicate that the ventral part of the mesencephalon has plasticity around the 10 somite stage (E2) with respect to the establishment of rostrocaudal polarity. Yaginuma and Oppenheim 24 rotated three segments of chick neural tube (future spinal cord) around the rostrocaudal axis, and observed that axons of the dorsolateral border cells coursed to the contralateral side of the spinal cord and turned rostrally, similar to control axons. They discussed the possibility that longitudinal fibers originating from normal segments may have provided axons of the dorsolateral border cells with rostral cues. Wilson et al. 23 demonstrated that a simple grid of tracts and commissures in the brain of zebrafish formed an initial axon scaffold, and suggested that the scaffold plays a crucial role in guiding additional axons. In our observations, trajectories T3 and R3 turned suddenly at a right angle and grew rostrally in a straight line. One possible interpretation of our results is that molecular cues distributed in the mesencephalon were reorganized because of the change of the fate of the inverted graft, and scaffold formation coincided with the host type. The circumferential axons were then guided by this scaffold. It is alternatively conceivable that the rostrocaudal polarity in the ventral region of the mesencephalon was formed by a gradient of chemotropic factors emanating from the diencephalon a n d / o r metencephalon, attracting circumferential axons to their appropriate directions. Guthrie and Lumsden 6 transplanted a segment of chick rhombomeres with reversed rostrocaudal orientation, and showed that the majority of motor neurons grew to the appropriate rostral exit point. They suggested that the motor neurons were attracted to the exit point. Acknowledgements. We thank Dr. Nobue ltasaki for her discussion. This study was supported by a Grant-in-Aid for Scientific Research 04454130 from the Japanese Ministry of Education, Science and Culture, by the Life Science Foundation of Japan, by the Brain Science Foundation and the Mitsubishi Foundation.
REFERENCES 1 Alvarado-Mallart, R.M., Martinez, S. and Lance-Jones, C.C., Pluripotentiality of the 2-day-old avian germinative neuroepithelium, Dev. Biol., 139 (19901 75-88. 2 Bally-Cuif, L., Alvarado-Mallart, R.M., Darnell, D.K. and Wassef, M., Relationship between Writ-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon, Development, 115 (1992) 999-1009.
3 Crossland, W.J. and Uchwat, C.J., lopographic pn.~lcctions of the retina and optic tectum upon the ventral lateral geniculatc nucleus in the chick, J. Comp. Neurol., 185 (19791 87- 106 4 Gardner, C.A., Darnell, D.K.. Poole, S.J., Ordahl, (LP and Barald, K.F., Expression of an engrailed-like gone during development of the early embryonic chick nervous system, .I. Neurosci. Rea., 21 (1988) 426-437. 5 Goldberg, S., Studies on the mechanics of development of the visual pathways in the chick embryo, Dec. Biol., 36 (1974) 24-43. 6 Guthrie, S. and Lumsden, A., Motor neuron pathfinding followlug rhombomerc reversals in the chick embryo hindbrain, Det~elopment, 114 (1992) 663-673. 7 ltamburger, V. and Hamilton, ll.L, A series o! normal stages in the development of the chick embryo, Z MorphoL, 88 (1951) 49-92. 8 lchijo, H., Fujita, S. Matsuno, T. and Nakamura, H., Rotation of the tectal primordium reveals plasticity of target recognition in retinotectal projection, Development, 110 (1990)331-342. 9 ltasaki, N., Ichijo, H., Hama, C., Matsuno, T. and Nakamura, H., Establishment of rostrocaudal polarity in tectal primordium: engrailed expression and subsequent tectal polarity, Development, 113 (19911 1133-1144. 1(1 Itasaki, N. and Nakamura, lq., Rostrocaudal polarity of the rectum in birds: correlation of en gradient and topographic order in retinotectal projection, Neuron, 8 (1992) 787-798. 11 Kr6ger, S. and Schwarz, U., The avian tectobulbar tract: development, explant culture, and effects of antibodies on the pattern of neurite outgrowth, J. Neurosci., 10 (1990) 3118-3134. 12 LaVail, J.H. and Cowan, W.M., The development of the chick optic rectum. I. Normal morphology and cytoarchitectonic development, Brain Res., 28 (1971) 391-419. 13 Le Douarin, N.M., A biological cell labeling technique and its use in experimental embryology, Dec. Biol., 30 (1973) 217-222. 14 Martinez, S. and Alvarado-Mallart, R.M., Expression of the homeobox chick-en gene in chick/quail chimeras with inverted mes-metencephalic grafts, Dec. Biol., 139 (1990) 432-436. 15 Martinez, S., Wassef, M. and Alvarado-Mallart, R.M., Induction of a mesencephalic phenotype in the 2-day-old chick prosencephalon is preceded by the early expression of the homeobox gene en, Neuron. 6 (1991) 971-981. 16 Matsuno, T., lchijo, H. and Nakamura, H., Regulation of the rostrocaudal axis of the optic tectum: histological study after rostrocaudal rotation in quail-chick chimeras, Dec Brain Res., 58 ( 1991 ) 265-270. 17 Nakamura, H. and O'Leary, D.D.M., Inaccuracies in initial growth and arborization of chick retinotectal axons followed by course corrections and axon remodeling to develop topographic order, J. Neurosci., 9 (1989) 3776-3795. 18 Patel, N.H., Martin-Blanco, E., Coleman, K.G., Poole, S.J., Ellis, M.C, Kornberg, T.B. and Goodman, C.S., Expression of engrailed proteins in arthropods, annelids, and chordates, ('~,//, 58 (1989) 955 968. 19 Puelles, L. and Bendala, M.C., Differentiation of neuroblasts in the chick optic tectum up to eight days of incubation: a Golgi study, Neuroscience, 3 (1978) 307-325. 20 Senut, M.C. and Alvarado-Mallart, R.M., Development of the retinotectal system in normal quail embryos: cytoarchitectonic development and optic fiber innervation, Dev. Brain Res., 29 (1986) 123-140. 21 Walter, J., Henke-Fahle, S. and Bonhoeffer, F., Avoidance of posterior tectal membranes by temporal retinal axons, Det~elopment. 101 (19871 909-913. 22 Walter, J., Kern-Veits, B., Huf, J., Stolze, B. and Bonhoeffer, F., Recognition of position-specific properties of tectal cell membranes by retinal axons in vitro, Development, 101 (1987) 685-696. 23 Wilson, S.W., Ross, LS., Parrett, T. and Easter Jr., S.S., The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio, Development, 108 (19901 121 145. 24 Yaginuma, H. and Oppenheim, R.W., An experimental analysis of in vivo guidance cues used by axons of spinal interneurons in the chick embryo: evidence for chemotropism and related guidance mechanisms..L Neurosci., 11 (19911 2598-2613.