1 Department of Molecular Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan
2 Graduate School of Life Sciences, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan
* Present address: Max-Planck-Institute for Molecular Cell Biology and Genetics, Pfotenhauerstr 108, D-01307 Dresden, Germany
Author for correspondence (e-mail: nakamura{at}idac.tohoku.ac.jp)
Accepted July 26, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Pax3, Pax7, En, Tectum, Chick
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pax3 and Pax7 belong to the same Pax subfamily, and widely expressed in the nervous system and somites (Jostes et al., 1990; Goulding et al., 1991). In the nervous system Pax3 and Pax7 are expressed in the dorsal part of the neural tube (Jostes et al., 1990; Goulding et al., 1991). They are expressed in the whole region of the tectum anlage (Kawakami et al., 1997). Pax3 mutant mice, Splotch, show exencephaly and defects in myogenesis and neural crest cell differentiation (Epstein et al., 1991; Franz, 1993; Tremblay, 1995; Conway et al., 1997). Pax7 mutant mice also show the defects in myogenesis and neural crest cell differentiation but no defects in the central nervous system (Mansouri et al., 1996). Pax3 and Pax7 share their expression domain broadly in the central nervous system, which may explain functional redundancy and rather mild defects in mutant mice. Pax3 and Pax7 double mutant mice show severe exencephaly, spina bifida and defects in commissural neurons in the spinal cord, and die by E11.0 (Mansouri and Gruss, 1998).
As Pax3 and Pax7 are expressed in the mesencephalic alar plate, the possibility that they are involved in regionalization of the tectum has been suggested (Kawakami et al., 1997; Nomura et al., 1998). A tectum could be induced ectopically in the diencephalic region by transplantation of the isthmus or by misexpression of En, Pax2/5 or Fgf8. Pax7 was always induced where the ectopic tectum structure was formed (Nomura et al., 1998; Araki and Nakamura, 1999). On the other hand when the tectum development was repressed by Shh misexpression, Pax7 expression was repressed in the dorsal mesencephalon (Watanabe and Nakamura, 2000). Therefore, we suspected that Pax3 and Pax7 would work to define the identity of the alar plate of the mesencephalon, and that Pax3 and Pax7 misexpression would induce an ectopic tectum. Pax3 overexpression was carried out in the transgenic mice, in which Pax3 expression was regulated by Hoxb4 promoter (Tremblay et al., 1996). In the transgenic mice, however, effects of Pax3 overexpression on the mesencephalon were not assessed as the Hoxb4 promoter does not assure expression in the mesencephalon. To explore the roles of Pax3/7 in tectum development, we carried out misexpression experiments of Pax3 and Pax7 in the diencephalon and ventral mesencephalon by in ovo electroporation. An ectopic tectum in the diencephalon and ventral mesencephalon was differentiated after Pax3 and Pax7 misexpression. A torus semicricularis, which derives from the caudal part of the mesencephalic alar plate and corresponds to the mammalian inferior corriculus, was also differentiated ectopically in the diencephalic region. Analysis of marker gene expression indicated fate change of the diencephalon occurred after sequential induction of Fgf8, En2 and Pax3/7. In normal development, Pax3 and Pax7 expression in the alar plate of the presumptive mesencephalon commences after Otx2, En and Pax2/5. Thus, results of misexpression experiments, together with normal expression patterns, suggests that Pax3 and Pax7 defines the alar plate of the mesencephalon subsequent to determination of the mesencephalon by Otx2, En and Pax2/5.
![]() |
MATERIAL AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In ovo electroporation
Fertilized chicken eggs from a local farm were incubated at 38°C. Pax3, Pax7 and Pax7-HA expression vector (3.0 µg/ml), ß-galactosidase expression vector (MiwZ) (a gift from Dr Kondoh) and the green fluorescence (GFP) expression vector (pEGFP-N1, Clontech) (0.5 µg/ml) were transfected to chick embryos by in ovo electroporation at stage 10 (Hamburger and Hamilton, 1951) as previously described (Funahashi et al., 1999). A GFP expression vector was co-transfected to check the efficiency of electroporation. Transfection to the ventral mesencephalon was carried out at stage 13.
Electroporation of this condition does not induce defects in the embryos (Funahashi et al., 1999; Nakamura et al., 2000).
In situ hybridization
Whole-mount in situ hybridization was performed as described by Bally-Cuif et al. (Bally-Cuif et al., 1995) or by Stern (Stern, 1998). In situ hybridization for sections was carried out as described by Ishii et al. (Ishii et al., 1997). Probes for Fgf8, Lim1 and Pax6 were described previously (Araki and Nakamura, 1999; Matsunaga et al., 2000). For Pax3 probe, partial fragment, isolated from cDNA library of E3 chick brain, was used. These fragments were inserted in pBluescript II SK (-) (Stratagene). After linearization, digoxigenin (DIG)-labeled antisense RNA was generated by T3 or T7 RNA polymerase (Stratagene) (Funahashi et al., 1999). For detection, alkaline phosphatase (ALP)-conjugated anti-DIG sheep-polyclonal antibody (Roche Molecular Biochemicals) was used, and 4-nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) (Roche Molecular Biochemicals) were used for the coloring. In some cases, NBT staining was washed out by incubating in dimethylformamide (DMF) at 55°C.
Immunohistochemistry
Anti-Pax6 rabbit polyclonal antibody (provided by Dr N. Osumi), anti-Pax7 monoclonal antibody (Developmental Studies Hybridoma Bank) (Kawakami et al., 1997), anti-En2 monoclonal antibody, 4D9 (American Type Culture Collection) (Patel et al., 1989) and anti-HA rabbit polyclonal antibody (Berkeley Antibody Company) were used as primary antibodies. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Jackson Immuno Research Laboratories) was used as the secondary antibody. For double staining on sections, Alexa488-conjugated anti-rabbit IgG antibody (Molecular Probes) and Cy3-conjugated anti-mouse IgG antibody (Jackson Immuno Research Laboratories) were used as secondary antibodies.
Histology
Embryos were fixed in 4% paraformaldehyde in PBS (phosphate buffered saline), embedded in Technovit (Kulter), serially sectioned at 5 µm, and stained with Hematoxylin and Eosin. Tiling images were automatically composed by MCID Image analyzer (Imaging Research Inc). ß-galactosidase activity was detected on whole-mount embryos as previously described (Katahira et al., 2000).
Tracing retinal fibers with HRP
For tracing of the retinal fibers 30% HRP solution, dissolved in PBS, was injected into the eye using a glass micropipette by air pressure. At 24 hours after injection, the brain was dissected, and the HRP-positive fibers were stained with p-cresol-diaminobenzidine method (Streit and Reubi, 1977). After observation of the fiber trajectory, the specimen was embedded in Technovit, and sagittal sections of 5 µm were prepared.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Lim1 is a good marker expressed in the pretectum (Mastick et al., 1997; Matsunaga et al., 2000). Lim1 expression remained in the p1 region after Pax7 misexpression, though expression domain a little narrowed, which indicates that most of the pretectal region retained even after Pax7 misexpression (n=5/5) (Fig. 2G-J).
At E6.5 (stage 27, 5 days after electroporation of Pax3 expression vector), morphological change was more remarkable (Fig. 2C,D). Histologically, the ectopic swelling was generated in the diencephalic region (Fig. 2E) (n=4/5). High power micrograph of this area clearly shows that the ectopic swelling consists of laminar structure that is characteristic of the tectum, though cytoarchitectonic differentiation in the ectopic tectum was behind of the tectum proper (Fig. 2F). In the tectum proper, three layers were prominent in addition to the neuroepithelial layer, and in the ectopic swelling two layers were discernible in addition to the neuroepithelial layer (Fig. 2F).
To examine if the ectopic tectal swelling can receive retinal fibers, we looked at fiber trajectory of retinal axons at E13.5 after Pax7 misexpression. Fiber tracing with HRP showed that most of retinal fibers innervated the ectopic tectum (n=3/3) (Fig. 2K,L). Sagittal sections of this specimen (Fig. 2M-R) show that the ectopic swelling contains well differentiated tectal structure though cytoarchitectonic differentiation was behind of normal tectum. According to LaVail and Cowan (LaVail and Cowan, 1971), the tectum of E12-14 has 12 layers. In the ectopic tectum (Fig. 2P), cell-dense layers of vi and viii are conspicuous; SO (stratum opticum) was easily recognized because retinal fibers had been labeled as shown in Fig. 2L. As the tectum proper at the experimental side was deprived of retinal fibers (Fig. 2L), superficial layers were poorly differentiated (Fig. 2O) as shown by Kelly and Cowan (Kelly and Cowan, 1972). The torus semicircularis (Fig. 2Q,R) that corresponds to the mammalian inferior colliculus (Puelles et al., 1994) was also formed ectopically. Taken together, whole the derivatives of the mesencephalic alar plate were differentiated ectopically. Pretectal nuclei persisted between the tectum proper and the ectopic tectum (Fig. 2N). The pattern of fiber trajectory and histology indicated that ectopic swelling acquired the character of the tectum.
Pax7 represses Pax6 expression and induces ectopic tectum by activating the gene cascade for tectum development
Next we examined effects on marker gene expression after Pax7 misexpression. The results of timecourse analysis are summarized in Table 1.
|
|
Repression of Otx2 by Pax7 was not recognized at 24 hours after electroporation (n=3/3). Ectopic expressions of En1 and Pax2 was not detected in the diencephalic region (data not shown). Pax5 induction was very weakly detected at 48 hours after electroporation, but not detected before 48 hours after electroporation (data not shown).
Next we looked at inter-relationship between Pax3 and Pax7. Pax3 was induced ectopically in the diencephalon by 18 hours after electroporation of Pax7 (Table 1; Fig. 4A-E). Induction was very weak at first, and higher magnification indicates that the induction is cell autonomous (Fig. 4F-I). By 96 hours after electroporation, when the ectopic swelling became conspicuous, Pax3 expression became strong and covered the whole ectopic swelling (Fig. 4J, N). Serial sections showed that Pax3 was expressed in the ectopic swelling, where En2 had been ectopically induced and Pax6 was repressed (Fig. 4K,L,O-Q). Pax7 also covered whole the ectopic swelling (Fig. 4M,Q). To distinguish induced and exogenous Pax7 expression, we misexpressed HA-tagged Pax7. The ectopic swelling was not stained immunocytochemically by anti-HA antibody at 96 hours after electroporation (Fig. 4M). As expression vector adopted in the present study assures transient expression, it is conceivable that Pax7 expression by introduced expression vector disappeared by this stage. Thus, Pax7 expression seen in the ectopic swelling was not exogenous one, but expression from the host Pax7 gene. Timecourse analysis shows that Pax3 was weakly induced by Pax7 at 18 and 24 hours after electroporation, then Pax3 expression nearly disappeared by 48 hours after electroporation. Then by 96 hours after electroporation, Pax3 expression covered whole the ectopic swelling. These results indicates that there are two modes in Pax3 induction by Pax7 in the ectopic swelling; one is direct induction by Pax7, and the other is indirect and may be induced after the feedback loop of tectum organizing molecules had been turned on.
|
At E6.5, the tectum and the tegmentum can be distinguished histologically; the tectum shows specific laminar structure as shown previously. After Pax7 misexpression, tectal structure extended ventrally (n=3/6) (Fig. 5A-C). Next we checked effects on marker gene expression for the tectum and tegmentum. Induction of Pax3 in the ventral mesencephalon was observed at 24 hours after electroporation of Pax7 (n=3/3) (Fig. 5G,H). Higher power micrographs indicate that induction of Pax3 by Pax7 is cell autonomous (Fig. 5H',I'). Pax3 misexpression also expanded the tectal domain ventrally (Fig. 5D-F).
|
Lim1 is expressed in the ventral mesencephalon at E4.0, and is thought as a good marker for the tegmentum (Watanabe and Nakamura, 2000). At E4.0 (60 hours after electroporation), Lim1 expression in the ventral mesencephalon made two stripes on the control side; broad lateral stripe and narrow medial stripe (Fig. 5M). On the experimental side, broader stripe was completely missing although narrow stripe remained and the position of narrow stripe did not shift ventrally (n=2/5) (Fig. 5M). On the control ventral side, Pax6 expression band is discernible (Agarwala, 2001). The Pax6 expression band disappeared after Pax3 misexpression (n=3/4) and after Pax7 misexpression (n=4/4, Fig. 5N).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pax3/7 induced ectopic tectum in the diencephalon and ventral mesencephalon
The present study has shown that Pax3 and Pax7 induced ectopic swelling in the diencephalon, and that the swelling had the laminar structure characteristic of the tectum and the torus semicircularis. The ectopically differentiated tectal structure could receive retinal fibers. As torus semicircularis was also differentiated, it was concluded that the alar plate of the presumptive diencephalon changed its fate to the mesencephalic alar plate by Pax3 or Pax7 misexpression. As for dorsoventral axis, the tectal swelling expanded ventrally (see Fig. 5C). One may raise a question if this ventral expansion of the tectum is caused by transformation of the dorsal tegmentum or by overgrowth of the tectum. If the former is the case, dorsal part of the tegmentum may be lost, and if the latter is the case, the tegmental structure is condensed relatively. We paid attention to the Lim1 and Pax6 expression in the ventral mesencephalon. Lim1 is expressed in two stripes; broad lateral stripe and narrow medial stripe (see Fig. 5M). Pax6 is expressed in a band of arch, as suggested by Agarwala et al. (Agarwala et al., 2001). After Pax7 misexpression broader stripe of Lim1 expression was completely missing although narrow stripe remained almost intact. Arch of Pax6 expression was also disappeared (see Fig. 5N). The results indicate that dorsal tegmentum region was missing but ventral tegmentum region was almost intact. Thus, we concluded that ventral expansion of the tectal swelling may not be due to increasing or decreasing of cell proliferation but to fate change of the tegmentum to the tectum.
In normal development, Pax3 and Pax7 are expressed almost in an overlapping manner in the mesencephalic alar plate, and Pax3 and Pax7 misexpression caused very similar phenotype. These results indicate functional redundancy of Pax3 and Pax7 in tectum development as suggested previously from the phenotype of mutant mice (Epstein et al., 1991; Mansouri et al., 1996; Mansouri et al., 1998). Our misexpression study showed that in the dorsal mesencephalon and p1 region, Pax3 and Pax7 repressed each others expression. At the same time, some regulation mechanism to balance total expression level of Pax3 and Pax7 by downregulating or by upregulating each others expression may exist. In Pax3 mutant mice Pax7 was upregulated in the somite and neural tube (Borycki et al., 1999).
Pax7 repressed Pax6 expression in the alar plate of the p2 region, which may mean that Pax7 repressed diencephalic property to cause re-specification of gene expression cascade toward mesencephalic development. In the p1 region, Pax6 expression was not affected so much, and most of it differentiated according to its original developmental program, that is, p1 differentiated into the diencephalic pretectum. This difference may be attributed to that both Pax3/7 and Pax6 are expressed normally in the p1 region. Pax3/7 and Pax6 repress each others expression (Matsunaga, et al., 2000) (present study), but in p1 region there may be some mechanism for each of them to escape from others repressive effect. As a consequence, forced expression of these genes would not exert strong effect, and would result in separation of ectopically differentiated tectum from the tectum proper at the p1 region.
Role of Pax3/7 in tectum development
The ectopically introduced Pax7 may turn off the gene expression cascade toward the diencephalic differentiation, by repressing Pax6 expression. At the same time, or rather earlier induction of Fgf8 expression took place. Fgf8 was broadly induced, and subsequently En2 expression was induced in the p2 region, not in the p1 region. Fgf8 and Otx2 are mutually repressive in their expression (Liu et al., 1999; Martinez et al., 1999; Katahira et al., 2000), but weak signal of Fgf8 does not affect Otx2 expression (Sato et al., 2001). In the present study, Otx2 expression was not affected by ectopically induced Fgf8 so that ectopic swelling in the p2 region may have differentiated into the tectum. If Fgf8 signal is strong enough to repress Otx2 expression in the diencephalon, cerebellum would ectopically differentiate (Martinez et al., 1999; Sato et al., 2001). Fate change to the tectum occurred in the region where ectopic En was induced, that is, p2 region differentiated into the tectum, but p1 region kept its original fate after Pax3/7 misexpression. Expression of Lim1, pretectum marker gene, and the persistence of the pretectal nuclei also indicated that the pretectum still remained. The results of the present study, those of En2 misexpression (Araki and Nakamura, 1999) and those in En1/2 double mutant mice that show complete deletion of the mesencephalon (Liu and Joyner, 2001) all indicate that the neural tissue should express En to differentiate into the tectum.
The results of the present study indicate that the tissue should express Pax3 and Pax7 followed by En expression to differentiate into the tectum. Exogenously introduced Pax3 or Pax7 might not be directly involved in the transformation of presumptive diencephalic alar plate to the mesencephalic property. Introduced Pax3/7 may first turn off the gene expression program towards diencephalon, then may induce Fgf8 and En expression. At first, En expression was found on the entire ectopic swelling, but then, confined to the rostral border of the swelling (see Fig. 4J-Q), which may indicate that the rostrocaudal polarity of the ectopic tectum was established in a mirror image to the tectum proper. Following En expression, Pax3 and Pax7 expression covered whole the ectopic swelling. The results of transfection experiments with HA-tagged Pax7 expression vector suggested that expressions of Pax3 and Pax7, covering whole the ectopic swelling, were not expressed from the transfected expression vector, but from the embryonic gene induced sequentially after En expression (see Fig. 4L,M). These results together with the chronological order of expression of these genes in normal development indicate that Pax3 and Pax7 may take part in mesencephalic development after the En expression.
It was suggested that the brain vesicle should express Otx2, En and Pax2 to acquire the property to differentiate into the tectum (Nakamura, 2001). We would like to add Pax3 and Pax7, expressed in the mesencephalic alar plate, for the tectum development. En, Otx2 and Pax2 are expressed or once expressed in both the alar plate and the basal plate of the mesencephalon. In this scenario, addition of Pax3 or Pax7 expression at the basal plate may be enough to change its fate to the tectum. This assumption well explains the results that the morphological change was detectable earlier in the basal plate of the mesencephalon than in the diencephalon after Pax3 and Pax7 misexpression
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agarwala, S., Sanders, T. A. and Ragsdale, C. W. (2001). Sonic hedgehog control of size and shape in midbrain pattern formation. Science 291, 2147-2150.
Alvarado-Mallart, R.-M. (1993). Fate and potentialities of the avian mesencephalic/metencephalic neuroepithelium. J. Neurobiol. 24, 1341-1355.[Medline]
Araki, I. and Nakamura, H. (1999). Engrailed defines the position of dorsal di-mesencephalic boundary by repressing diencephalic fate. Development 126, 5127-5135.
Bally-Cuif, L., Cholley, B. and Wassef, M. (1995). Involvement of Wnt-1 in the formation of the mes/metencephalic boundary. Mech. Dev. 53, 23-34.[Medline]
Borycki, A. G., Li, J., Jin, F., Emerson, C. P. and Epstein, J. A. (1999). Pax3 functions in cell survival and in pax7 regulation. Development 126, 1665-1674.
Conway, S. J., Henderson, D. J. and Copp, A. J. (1997). Pax3 is required for cardiac neural crest migration in the mouse: evidence from the splotch (Sp2H) mutant. Development 124, 505-514.
Crossley, P. H., Martinez, S. and Martin, G. R. (1996). Midbrain development induced by FGF8 in the chick embryo. Nature 380, 66-68.[Medline]
Epstein, D. J., Vekemans, M. and Gruss, P. (1991). Splotch (Sp2H), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Pax-3. Cell 67, 767-774.[Medline]
Franz, T., Kothary, R., Surani, M. A., Halata, Z. and Grim, M. (1993). The Splotch mutation interferes with muscle development in the limbs. Anat. Embryol. 187, 153-160.[Medline]
Funahashi, J.-I., Okafuji, T., Ohuchi, H., Noji, S., Tanaka, H. and Nakamura, H. (1999). Role of Pax-5 in the regulation of a mid-hindbrain organizers activity. Dev. Growth Differ. 41, 59-72.[Medline]
Goulding, M. D., Chalepakis, G., Deutsch, U., Erselius, J. R. and Gruss, P. (1991). Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J. 10, 1135-1147.[Abstract]
Grindley, J. C., Hargett, L. K., Hill, R. E., Ross, A. and Hogan, B. L. (1997). Disruption of PAX6 function in mice homozygous for the Pax6Sey-1Neu mutation produces abnormalities in the early development and regionalization of the diencephalon. Mech. Dev. 64, 111-126.[Medline]
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92.
Ishii, Y., Fukuda, K., Saiga, H., Matsushita, S., and Yasugi, S. (1997). Early specification of intestinal epithelium in the chicken embryo: a study on the localization and regulation of CdxA expression. Dev. Growth Differ. 39, 643-653.[Medline]
Jostes, B., Walther, C., and Gruss, P. (1990). The murine paired box gene, Pax7, is expressed specifically during the development of the nervous and muscular system. Mech. Dev. 33, 27-37.[Medline]
Joyner, A. L. (1996). Engrailed, Wnt and Pax genes regulate midbrainhindbrain development. Trends Genet. 12, 15-20.[Medline]
Katahira, T., Sato, T., Sugiyama, S., Okafuji, T., Araki, I., Funahashi, J.-I., and Nakamura, H. (2000). Interaction between Otx2 and Gbx2 defines the organizing center for the optic tectum. Mech. Dev. 91, 43-52.[Medline]
Kawakami, A., Kimura-Kawakami, M., Nomura, T. and Fujisawa, H. (1997). Distributions of PAX6 and PAX7 proteins suggest their involvement in both early and late phases of chick brain development. Mech. Dev. 66, 119-130.[Medline]
Kelly, J. P. and Cowan, W. M. (1972). Studies on the development of the chick optic tectum. III. Effects of early eye removal. Brain Res. 42, 263-288.[Medline]
LaVail, J. H. and Cowan, W. M. (1971). The development of the chick optic tectum. I. Normal morphology and cytoarchitectonic development. Brain Res. 28, 391-419.[Medline]
Liu, A., Losos, K. and Joyner, A. L. (1999). FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate. Development 126, 4827-4838.
Liu, A. and Joyner, A. L. (2001). EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse mid/hindbrain region. Development 128, 181-191.
Lun, K. and Brand, M. (1998). A series of no isthmus (noi) alleles of the zebrafish pax2.1 gene reveals multiple signaling events in development of the midbrain-hindbrain boundary. Development 125, 3049-3062.
Mansouri, A., Stoykova, A., Torres, M. and Gruss, P. (1996). Dysgenesis of cephalic neural crest derivatives in Pax7-/- mutant mice. Development 122, 831-838.
Mansouri, A. and Gruss, P. (1998). Pax3 and Pax7 are expressed in commissural neurons and restrict ventral neuronal identity in the spinal cord. Mech. Dev. 78, 171-178.[Medline]
Marin, F. and Puelles, L. (1994). Patterning of the embryonic avian midbrain after experimental inversions: a polarizing activity from the isthmus. Dev. Biol. 163, 19-37.[Medline]
Martinez, S., Wassef, M. and Alvarado-Mallart, R. M. (1991). 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, 971-981.[Medline]
Martinez, S., Marin, F., Nieto, M. A. and Puelles, L. (1995). Induction of ectopic engrailed expression and fate change in avian rhombomeres: intersegmental boundaries as barriers. Mech. Dev. 51, 289-303.[Medline]
Martinez, S., Crossley, P. H., Cobos, I., Rubenstein, J. L. R. and Martin, G. R. (1999). FGF8 induces formation of an ectopic isthmic organizer and isthmocerebellar development via a repressive effect on Otx2 expression. Development 126, 1189-1200.
Mastick, G. S., Davis, N. M., Andrew, G. L. and Easter, S. S., Jr (1997). Pax-6 functions in boundary formation and axon guidance in the embryonic mouse forebrain. Development 124, 1985-1997.
Matsunaga, E., Araki, I. and Nakamura, H. (2000). Pax6 defines the di-mesencephalic boundary by repressing En1 and Pax2. Development 127, 2357-2365.
Nakamura, H., Watanabe, Y. and Funahashi, J-I. (2000). Misexpression of the genes in the brain vesicles by in ovo electroporation. Dev. Growth Differ. 42, 199-201.[Medline]
Nakamura, H. (2001). Regionalisation of the optic tectum: combinations of gene expression that define the tectum. Trends Neurosci. 24, 32-39[Medline]
Nomura, T., Kawakami, A., and Fujisawa, H. (1998). Correlation between tectum formation and expression of two PAX family genes, PAX7 and PAX6, in avian brains. Dev. Growth Differ. 40, 485-495.[Medline]
Okafuji, T., Funahashi, J.-I., and Nakamura, H. (1999). Roles of Pax-2 in initiation of the chick tectal development. Brain Res. Dev. Brain Res. 116, 41-49.[Medline]
Patel, N. H., Martin-Blanco, E., Coleman, K. G., Poole, S. J., Ellis, M. C. Kornberg, T. B. and Goodman, C. S. (1989). Expression of engrailed proteins in arthropods, annelids, and chordates. Cell 58, 955-968.[Medline]
Puelles, L., Robles, C., Martinez de la Torre, M. and Martinez, S. (1994). New subdivision schema for the avian torus semicircularis: neurochemical maps in the chick. J. Comp. Neurol. 340, 98-125.[Medline]
Ristoratore, F., Carl, M., Deschet, K., Richard-Parpaillon, L., Boujard, D., Wittbrodt, J., Chourrout, D., Bourrat, F. and Joly, J. S. (1999). The midbrain-hindbrain boundary genetic cascade is activated ectopically in the diencephalon in response to the widespread expression of one of its components, the medaka gene Ol-eng2. Development 126, 3769-3779.
Rubenstein, J. L., Martinez, S., Shimamura, K. and Puelles, L. (1994). The embryonic vertebrate forebrain: the prosomeric model. Science 266, 578-580.[Medline]
Sato, T., Araki, I. and Nakamura, H. (2001). Inductive signal and tissue responsiveness to define the tectum and the cerebellum. Development 128, 2461-2469.
Shamim, H., Mahmood, R., Logan, C., Doherty, P., Lumsden, A. and Mason, I. (1999). Sequential roles for Fgf4, En1 and Fgf8 in specification and regionalisation of the midbrain. Development 126, 945-959.
Song, D. L., Chalepakis, G., Gruss, P. and Joyner, A. L. (1996). Two Pax-binding sites are required for early embryonic brain expression of an Engrailed-2 transgene. Development 122, 627-635.
Stern, C. D. (1998). Detection of multiple gene products simultaneously by in situ hybridization and immunohistochemistry in whole mounts of avian embryos. Curr. Top. Dev. Biol. 36, 223-243.[Medline]
Stoykova, A., Fritsch, R., Walther, C. and Gruss, P. (1996). Forebrain patterning defects in Small eye mutant mice. Development 122, 3453-3465.
Stoykova, A., Götz, M., Gruss, P. and Price, J. (1997). Pax6-dependent regulation of adhesive patterning, R-cadherin expression and boundary formation in developing forebrain. Development 124, 3765-3777.
Streit, P. and Reubi, J. C. (1977). A new and sensitive staining method for axonally transported horseradish peroxidase (HRP) in the pigeon visual system. Brain Res. 126, 530-537.[Medline]
Suemori, H., Kadokawa, Y., Goto, K., Araki, I., Kondoh, H. and Nakatsuji, N. (1990). A mouse embryonic stem cell line showing pluripotency of differentiation in early embryos and ubiquitous beta-galactosidase expression. Cell. Differ. Dev. 29, 181-186.[Medline]
Sugiyama, S., Funahashi, J.-I. and Nakamura, H. (2000). Antagonizing activity of chick Grg4 against tectum-organizing activity. Dev. Biol. 221, 168-180.[Medline]
Tremblay, P., Kessel, M. and Gruss, P. (1995). A transgenic neuroanatomical marker identifies cranial neural crest deficiencies associated with the Pax3 mutant Splotch. Dev. Biol. 171, 317-329.[Medline]
Tremblay, P., Pituello, F. and Gruss, P. (1996). Inhibition of floor plate differentiation by Pax3; evidence from ectopic expression in transgenic mice. Development 122, 2555-2567.
Walther, C. and Gruss, P. (1991). Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 113, 1435-1449.[Abstract]
Warren, N. and Price, D. J. (1997). Roles of Pax-6 in murine diencephalic development. Development 124, 1573-1582.
Watanabe, Y. and Nakamura, H. (2000). Control of chick tectum territory along dorsoventral axis by Sonic hedgehog. Development 127, 1131-1140.