Department of Developmental Neurobiology, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
*Author for correspondence (e-mail: osumi{at}mail.cc.tohoku.ac.jp)
Accepted 18 December 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Neuronal specification, Progenitor domains, Hindbrain, Pax6, Small eye rat, Somatic motoneurones, Interneurones, Homeodomain proteins
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The role of Shh in the patterning of ventral neural tube has been well studied in the spinal cord. Several homeodomain (HD) proteins are expressed in specific domains of the ventricular zone of the developing spinal cord (Goulding et al., 1993; Ericson et al., 1997
; Pierani et al., 1999
). They are categorised into two classes: the genes whose expression is repressed by Shh (Pax6, Dbx2, Irx3, Dbx1 and Pax7) are known as class I, while those whose expression is induced by Shh (Nkx2.2 and Nkx6.1) are class II (Briscoe et al., 2000
). Misexpression and loss of functions of the HD protein genes can cause alteration of the neuronal subtypes (Briscoe et al., 1999
; Briscoe et al., 2000
; Sander et al., 2000
; Pierani et al., 2001
). These data suggest that the progenitor cell identity and the neuronal subtypes are regulated via distinct regionalization of the ventricular zone in the neural tube represented by the combinatorial expression of the HD proteins, i.e., the HD code.
Members of Pax family proteins are HD-containing transcription factors, and Pax6 is the most characterised member (reviewed by Gruss and Walther, 1992; Hill and Hanson, 1992
; van Heyningen, 1998
; Gehring and Ikeo, 1999
). During development, Pax6 is expressed in the dorsal forebrain, including a region that gives rise to the cortex, dorsal thalamus and pretectum, and functions in patterning the brain (Walther and Gruss, 1991
; Stoykova et al., 1997
; Pratt et al., 2000
; Osumi, 2001
). In the hindbrain and spinal cord, Pax6 is expressed in the ventral region and plays crucial roles in generation of ventral neurones. Five types of neurones that are respectively marked with expressions of specific transcriptional factors differentiate in the ventral hindbrain. They are, from ventral to dorsal, branchiomotor (BM) and somatic (SM) motoneurones, and V2, V1 and V0 interneurones (Fig. 1A). In the hindbrain of Pax6 homozygous mutant mice and rats, the SM neurones and V1 interneurones are missing, while the BM neurones increased in number (Osumi et al., 1997
; Ericson et al., 1997
; Burill et al., 1997
; Osumi and Nakafuku, 1998
; Sun et al., 1998
). However, in the Pax6 mutant spinal cord, SM neurones do develop and a small number of V1 interneurones appear at later stages. Therefore, how Pax6 functions in ventral neurone development is still enigmatic.
|
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemistry
Immunohistochemistry on frozen sections was performed as described previously (Osumi et al., 1997). Anti-Pax6 rabbit polyclonal antibody (Inoue et al., 2000
) was used at 1:1000 dilution. 40.2D6 anti-Islet1/2 (1:100) (Ericson et al., 1992
), 74.5A5 anti-Nkx2.2 (1:50) (Ericson et al., 1997
) and 81.5C10 anti-MNR2 (1:25) (Tanabe et al., 1998
) mouse monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank (University of Iowa), and used at the specified dilution. Although rat HB9 homologue has not been identified, the staining pattern with the anti-chick MNR2 monoclonal antibody was similar to previous reports for HB9 expression in the mouse embryo (Arber et al., 1999
; Thaler et al., 1999
). As amino acid sequence of MNR2 is very similar to that of HB9, it is possible that anti-chick MNR2 antibody recognised HB9 and/or HB9 related protein on the rat tissue. Anti-Lim3 rabbit polyclonal antibody was kindly provided by S. L. Pfaff and used at 1:5000 (Sharma et al., 1998
). Anti-GFP mouse monoclonal antibody was purchased from Clontech and used at 1:1,000. Antigen enhancement was performed according to the method described previously (Osumi et al., 1997
). As secondary antibodies, Cy3-conjugated affinity-purified donkey anti-rabbit IgG (1:600) and anti-mouse IgG (1:400) solutions, FITC-conjugated affinity-purified donkey anti-mouse IgG (1:200) and anti-rabbit IgG (1:200) solutions, and biotin-conjugated affinity-purified donkey anti-rabbit IgG (1:200) and anti-mouse IgG (1:200) solutions (Jackson Immunoresearch Laboratories, Chemicon International, respectively) were used. ABC kit (Vector Laboratories) and Metal enhanced DAB kit (Pierce) were used for detection with horseradish peroxidase.
In situ hybridisation
RT-PCR was performed to obtain rat cDNA clones for templates. Total RNA taken from the head of E13.5 SD rat embryos was purified by RNeasy column (Qiagen) and cDNA was synthesised using reverse transcriptase and oligo dT primer (Superscript preamplification system; Gibco BRL). Oligonucleotides used to amplify cDNAs were as follows: Chx10, 5'-AGCGCTGAGCAAGCCAAAT-3' and 5'-CTAAGCCATGTCCTCCAGCT-3'; Dbx1, 5'-TCTAGAATGATGTTCCCCGG-3' and 5'-CTAGGACACCGTGATTTCCT-3', according to previously described mouse sequences (Liu et al., 1994; Lu et al., 1994
). Amplification was performed with a thermal cycler (Mastercycler Gradient; Eppendolf) using Taq DNA polymerase (Promega) using the following protocol: denaturation for 5 minutes at 96°C, annealing for 1 minute at 62.4°C (Chx10) or 63.4°C (Dbx1), extension for 1 minute at 72°C, 35 cycles. These cDNA fragments, including the open reading frames, were cloned into pBluescript II SK () (Stratagene) and sequenced to confirm they were rat counterparts. Rat En1, Nkx2.2, Shh and Pax6 cDNAs were used previously (Matsuo et al., 1993
; Osumi and Nakafuku, 1998
), and rat Islet2 cDNA was a kind gift from S. Pfaff (Tsuchida et al., 1994
). Rat Smoothened cDNA (Stone et al., 1996
) was kindly provided by A. Rosenthal. To synthesise other probes, mouse cDNA clones were used. Evx1 cDNA (Bastian and Gruss, 1990
) was provided by M. Goulding, Nkx6.1 cDNA (Qiu et al., 1998
) by J. Rubenstein, Irx3 cDNA (Bosse et al., 1997
) by P. Gruss, Dbx2 cDNA (Shoji et al., 1996
) by N. Takahashi, Gli1 and Gli2 cDNAs (Ding et al., 1999
) by H. Sasaki, and Patched1 cDNA (Goodrich et al., 1996
) by M. Scott. Digoxigenin-labelled antisense riboprobes were generated with T3 or T7 RNA polymerase (Promega). In situ hybridisation on frozen sections was performed as described previously (Ishii et al., 2000
). In some cases, immunohistochemistry was performed on the same sections after in situ hybridisation.
Electroporation into cultured rat embryos
The method used for electroporation into cultured mammalian embryos was described previously (Osumi and Inoue, 2001). Chamber-type electrodes (8x20 mm electrodes and 20 mm distance between electrodes) were used in this study. To construct Pax6 expression plasmid, blunted SpeI-KpnI fragment of mouse Pax6 cDNA (a kind gift from P. Gruss) (Walther and Gruss, 1991
) was inserted into blunted HindIII-KpnI site of pCAX expression plasmid containing the cytomegalovirus enhancer and chicken ß-actin promoter (pCAX and pCAX-GFP plasmids were kindly provided by the late K. Umesono). At E10.75 stage, the uterus was dissected out from anaesthetised rSey2 heterozygous females, and littermate embryos were dissected out with their placenta and yolk sac intact. After a 2 hour preculture, the embryos were transferred into the chamber-type electrodes and DNA solution of pCAX-mPax6 and pCAX-GFP (9:1) dissolved in PBS at 5 mg/ml was injected into the hindbrain. Immediately, square pulses (50 mseconds, 70 V, five times) were sent using an electroporator (CUY21; NEPPA GENE), and the embryos were further cultured. At this point, we could not distinguish homozygous embryos from external features. Twelve hours later when the cultured embryos developed to the stage corresponding to E11.5, the yolk sac was opened and homozygous embryos were identified based on morphological defects in the brain and eyes. In electroporation of the Pax6 mutant at early E11.5 (22-somite stage), littermate embryos were dissected with the yolk sac opened, and homozygous embryos that were identified from the external features were used for electroporation. Both the wild-type and homozygous embryos were precultured for 2 hours, and electroporated with square pulses (50 mseconds, 70-90 V, five times). The cultured embryos were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight at 4°C. Selected embryos in which GFP fluorescence was seen only in one side of the neural tube were processed for further analyses as described above. In total, 10 wild-type and eight homozygous mutant embryos at early E11.5, and three mutant embryos at E10.75 were used for analysis.
Assay for cell death and cell proliferation
Cell death was assayed quantitatively by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labelling (TUNEL) as described previously (Wakamatsu et al., 1998) with minor modification. The embryos were fixed with 4% PFA in PBS overnight at 4°C. Frozen sections were treated with 1 mg/ml proteinase K/PBS for 5 minutes at 37°C, and refixed with 4% PFA in PBS for 10 minutes. Sections were incubated with TdT buffer containing boitin-14-ATP (Gibco BRL) and terminal transferase (Roche Molecular Systems) for 1 hour at 37°C. Labelled cells were detected with avidin-Cy3 (1:200, Jackson).
Cell proliferation was assayed quantitatively by pulse-labelling with bromodeoxyuridine (BrdU) of cultured mammalian embryos (Ishii et al., 2000) with minor modification. The E12.5 wild-type and homozygous embryos were precultured for 1 hour, followed by the addition of BrdU solution to the culture medium. The embryos were exposed to BrdU for just 20 minutes and then fixed immediately. Detection of BrdU was performed according to Marusich et al. (Marusich et al., 1994
) using anti-BrdU antibody (1:50; Becton Dickinson) and Cy3-conjugated affinity-purified donkey anti-mouse IgG (1:400, Jackson) antibody. Sections were counterstained with DAPI.
In situ hybridisation for Dbx1 probe was performed on adjacent sections to determine the area for cell count. For count of BrdU-, and TUNEL-positive cells, 10 sections at the r7 level from three Pax6 homozygous and three wild-type rat embryos were analysed.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Next, we investigated differentiation of other interneurones, including V2 (Chx10-positive) and V0 (Evx1-positive) populations, in the Pax6 homozygous embryos. Chx10 transcripts were detected from E12.0 in the hindbrain of wild-type and Pax6 homozygous embryos (Fig. 1A,B,D,H). It has previously been reported that cells expressing Chx10 protein are reduced at r7 in the mouse Pax6 homozygous embryo (Ericson et al., 1997). In the rat Pax6 homozygous embryo, however, we did observe Chx10-positive cells at E12.0-13.5, and the domain of Chx10 expression expanded somewhat dorsally (bracket in Fig. 1H). The onset of Evx1 expression was later than that of En1 and Chx10; Evx1-positive cells emerged from E12.5 and afterwards in the hindbrain of the wild type (Fig. 1A,F). In the Pax6 mutant, Evx1-positive cells were not observed until E13.5 at r7 level, while they were found to expand somewhat ventrally at r3-6 levels on E12.75-E13.0 (Fig. 1B,J). Thus, differentiation of the ventral interneurones was impaired in the Pax6 mutant hindbrain.
We have previously demonstrated that mutation in Pax6 gene results in loss of SM and increase of BM neurones in the rat hindbrain (Osumi et al., 1997). The same defect is seen in the homozygote of small eye mouse mutant (Ericson et al., 1997
; Burrill et al., 1997
). However, the SM neurone defect is less severe at the cervical level of the spinal cord. Thus, we re-examined the expression of Islet2 with special reference to embryonic stages and AP levels. At r5 and r7 levels in the wild-type rat hindbrain, Islet2 expression was first detected at E11.5 and maintained at E13.5 (Fig. 1A). In the Pax6 homozygous embryos, Islet2 transcripts were not detected until E12.5, as reported previously (Fig. 1B) (Osumi et al., 1997
). However, we found a few cells expressing Islet2 at r7 level during E12.75-13.0 in the Pax6 mutant embryos (Fig. 1Q). We further checked whether the expression of HB9/MNR2, another marker for SM neurones, transiently emerged in the Pax6 homozygous embryos. In the wild-type rat embryo, HB9/MNR2 immunoreactivity was observed from E11.5 (Fig. 1A). In the Pax6 mutant, cells expressing HB9/MNR2 were not seen in the E12.5 mutant hindbrain, but a few cells expressing HB9/MNR2 were observed at r7 level of E12.75-13.0 (Fig. 1R). Such Islet2 and HB9/MNR2-positive cells were no longer detected at E13.5 in the mutant (Fig. 1B). As for another marker of SM neurones, Lim3, it was expressed in most of Islet2-positive cells of E11.5-13.0 wild-type hindbrain (data not shown) (Varela-Echavarría et al., 1996
). However, Islet2-positive cells transiently observed in E12.75-13.0 mutant did not express Lim3 (data not shown).
In summary, emergence of SM neurones and V1 interneurones in the hindbrain, although transiently, suggests that Pax6 is not directly required for induction of these cell types.
Progenitor domain formation is perturbed in the Pax6 mutant
Transient emergence of small number of SM neurones and V1 interneurones prompted us to assume that neuronal progenitor domains for these populations become narrower in the Pax6 mutant. Therefore, we compared the expression patterns of the HD protein genes in the hindbrain at E11.5-12.5 between the wild type and Pax6 mutant. We found that the expression patterns of Nkx2.2, Nkx6.1, Irx3, Dbx1 and Dbx2 (Fig. 2A-E) in the wild-type rat embryos were the same as reported in the mouse (Lu et al., 1992; Shoji et al., 1996
; Bosse et al., 1997
; Ericson et al., 1997
), while all those in the Pax6 mutant rat were markedly different (Fig. 2F-J). The Nkx2.2 domain, which faces the Pax6 domain, expanded dorsally (white arrowhead in Fig. 2F) as previously reported (Ericson et al., 1997
; Osumi and Nakafuku, 1998
). The dorsal boundary of Nkx6.1 expression was slightly blurred (double green arrowhead in Fig. 2G). Irx3 expression markedly expanded ventrally into Nkx2.2 domain (red arrowhead in Fig. 2H). The ventral boundary of Dbx2 expression was also blurred (double green arrowhead in Fig. 2I). Cells expressing Dbx1 were scattered ventrally (red arrowhead in Fig. 2J) and decreased in number when stained cells were counted in five sections from three embryos for both the wild type and mutant; 183.6±7.7 cells/section were positive for Dbx1 in the wild-type hindbrain, while 101.8±7.9 cells/section were positive in the mutant (t-test; P<0.001).
|
Altered expression of HD code genes by misexpression of exogenous Pax6
As HD code gene expressions were altered in the loss-of-function condition of Pax6, we next examined the effects of Pax6 gain-of-function on the expression of the HD code genes. Pax6 expression vector (pCAX-mPax6) and GFP expression vector (pCAX-GFP) were co-transfected into the hindbrain of wild-type and Pax6 mutant embryos by electroporation at 22-somite stage (early E11.5) in the rat embryos. These electroporated embryos were cultured for 26-30 hours up to the stage corresponding to 35- to 36-somite stage (E12.5; see Fig. 3A). We analysed embryos transfected on the right side of the hindbrain excluding the floor plate (Fig. 3C-H). In this electroporation, the expression of Shh was not affected (Fig. 3E,H).
|
|
|
As differentiation to SM neurones started in the wild-type hindbrain at E11.5 (Fig. 1A), electroporation at early E11.5 might be too late to rescue Islet2 and HB9/MNR2 expression. Therefore, we next electroporated exogenous Pax6 into the hindbrain of E10.75 Pax6 mutant embryo, and analysed the results after 42 hours culture (corresponding to E12.5). Although the efficiency of gene transfer at E10.75 was very low (Fig. 5K), in the area where Pax6 was electroporated (Fig. 5K,L) a small number of Islet2-positive and HB9/MNR2-positive cells were detected at the ventral hindbrain (Fig. 5M-P). It was at E12.75-E13.0 that small populations of cells expressing Islet2 and HB9/MNR2 were observed at r7 in the Pax6 homozygous embryos (Fig. 1Q,R). Thus, the induction of Islet2 and HB9/MNR2 expression was likely to be caused by exogenous Pax6 expression. The induction of Islet2 and HB9/MNR2 was only seen in a small number of cells (compare Fig. 5N,P with Fig. 1Q,R). Importantly, expression of these markers were never observed ectopically out of the positions where SM neurones normally exist, again suggesting that Pax6 function is not sufficient to induce these neurones.
In summary, these results of the overexpression experiments together with the loss-of-function data suggest that Pax6 plays a crucial role in establishing V1 and SM progenitor domains and in subsequent differentiation of V1 and SM neurones.
Expression of Shh signalling molecules in the Pax6 mutant hindbrain
What kind of mechanism is involved in progenitor domain formation? Expression of HD proteins in the spinal cord is influenced by graded action of Shh (Briscoe et al., 2000). Moreover, in the telencephalon of Pax6 mutant mice, the domain expressing Shh expanded dorsally compared to that of the wild type (Stoykova et al., 2000
). However, the expression patterns of Shh were not altered in the hindbrain and spinal cord of Pax6 mutant mice and rats (Fig. 6A,G) (Ericson et al., 1997
; Osumi and Nakafuku, 1998
). To explore whether altered expression of HD code genes in the Pax6 mutant hindbrain is due to changes in Shh signalling, we examined the expression of Shh receptor Patched1 (Ptc1) and Gli family genes, both of which are direct targets of Shh signal (Goodrich et al., 1996
; Hynes et al., 1997
; Lee et al., 1997
; Sasaki et al., 1999
).
|
Expressions of Gli1 and Gli2 were detected at high level in the SM progenitor domain (bracket in Fig. 6D,E), and the ventral limits of the domains are adjacent to the dorsal limit of Nkx2.2 domain (black arrowhead in Fig. 6B,D,E). The dorsal limit of Gli1 and Gli2 domains corresponded to that of Dbx1 domain (red arrowhead in Fig. 6D-F). In the hindbrain of Pax6 mutant, Gli1 and Gli2 were similarly expressed in the region between dorsal limits of Nkx2.2 and Dbx1 (green and red arrowheads, respectively, in Fig. 6H,J-L), but the high levels of Gli1 and Gli2 expression in the ventral region decreased. We also examined the expression patterns of a co-receptor of Shh, Smoothened (Smo) (Stone et al., 1996). In the hindbrain, Smo was expressed in the entire ventricular zone within the hindbrain, and the expression pattern was not altered in the Pax6 mutant (data not shown). Thus, altered expressions of Shh signalling molecules were restricted to the progenitor domain of SM neurones in the Pax6 mutant hindbrain, while expression patterns of these molecules were unchanged in the dorsal regions, including the progenitor domains for V2, V1 and V0 interneurones.
Cell death and cell proliferation in the Pax6 mutant hindbrain
The impaired progenitor domain formation could be due to altered cell kinetics in the Pax6 mutant. That is, the unstable narrow V1 and SM progenitor domains in Pax6 mutant may be resulted from the change in cell death/proliferation in these and flanking domains. Thus, we investigated the relationship between cell death/proliferation and individual progenitor domains.
To detect apoptotic cells, we performed TUNEL staining at E12.5 stage (Fig. 7A,C) and counted the number of TUNEL-positive cells per section within the ventral region, including Dbx1 domain. The mean number of TUNEL-positive cells in the wild-type hindbrain (9.4±1.1/section, ±s.e.m., n=10) was not significantly different from that in the Pax6 mutant (12.3±1.3/section, n=10, t-test; P>0.1, Fig. 7E). Moreover, apoptotic cells were observed in random regardless of the progenitor domains.
|
These results indicate that the disturbance of the progenitor domains in the Pax6 mutant hindbrain is not attributed to change of cell death or proliferation in the specific progenitor domains.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The most important finding in this study is that loss of Pax6 function leads to failure in formation of the correct progenitor domains within the ventricular zone. As illustrated in Fig. 2L, the expression boundaries of all HD protein genes are blurred and shifted in the Pax6 mutant. Expression of Nkx2.2 and Dbx2 expands dorsally, while that of Nkx6.1, Irx3 and Dbx1 shifts ventrally. The altered expression patterns of the HD code genes in the Pax6 mutant explain very well why a small number of V1 interneurones and SM neurones emerge; the progenitor domains for V1 interneurones and SM neurones, which are defined by the expression boundaries of Dbx1/Dbx2 and Irx3/Nkx2.2, respectively, are formed as extremely narrow domains in the Pax6 mutant. Emergence of these V1 and SM neurones may be transient (only for about 10 hours) because these expression boundaries are not firmly maintained and such neurones will be diminished soon after. By contrast, progenitor domains for BM neurones, V2 interneurones and V0 interneurones became expanded. This is consistent with the observation that the number of V2 interneurones increased in the mutant rat, which differs from the results in the Pax6 mutant mice reported by Ericson et al. (Ericson et al., 1997
).
If Pax6 is required for establishment of the progenitor domains in a correct manner, is it sufficient for progenitor domain formation? To answer this question, we performed overexpression of Pax6 by electroporation into cultured rat embryos, and indeed rescued development of SM neurones and V1 interneurones in correct positions. The numbers of these rescued neurones were less than in normal development. The reason for this partial rescue may be that exogenous Pax6 cannot fully re-establish the progenitor domains for SM neurones and V1 interneurones at the stage of electroporation. Irx3, the gene reported to repress SM fate in the chick spinal cord (Briscoe et al., 2000), was already expanded ventrally at the time of electroporation. Alternatively, exogenous Pax6 could not cause a complete repression of the expanded expression of Nkx2.2. Taking these loss-of-function and gain-of-function studies together, we conclude that Pax6 seems to regulate formation of the precursor domains in the hindbrain, thereby specify the fates of ventral neurones (Fig. 8).
|
The expression of HD code proteins is regulated by graded action of Shh released from the floor plate (Ericson et al., 1997; Briscoe et al., 2000
). Therefore, we examined the expression patterns of the genes that are known to be direct targets of Shh signalling. We found that in the Pax6 mutant, strong expression of Ptc1, Gli1 and Gli2 was eliminated in the ventral region corresponding to the SM precursor domain. Altered expression of Shh signalling molecules in the ventral region may suggest that establishment of SM progenitor domain may require Pax6 function to respond properly to Shh signal. However, this might conversely be resulted from the perturbed progenitor domain formation.
The next possibility is different cell kinetics in individual progenitor domains. Previous studies have demonstrated that Pax6 regulates cell proliferation in development of the diencephalon, telencephalon and retina (Caric et al., 1997; Warren and Price, 1997
; Götz et al., 1998
; Marquardt et al., 2001
). The frequency and positions of dead cells were not different in the hindbrains of wild type and Pax6 mutants. Although BrdU incorporation was slightly higher in the mutant, there was no relationship with specific progenitor domains. Therefore, it seems that overall disruption of progenitor domain boundaries in the mutant hindbrain is not explained by the alteration of cell death and proliferation.
Another possibility is that cell motility is accelerated in the neuroepithelium of the Pax6 mutant. Notably, cell tracing analyses in chick embryos have revealed a widespread dispersal of neuroepithelial cells in the early stages, but such cell mixing becomes less obvious in later development when progenitor domains are established (Clarke et al., 1998; Erskine et al., 1998
). There are accumulating data to suggest that Wnt signal regulates cell motility (Heisenberg et al., 2000
; Jönsson and Anderson, 2001
), and expression of Wnt7b is actually diminished in the Pax6 mutant hindbrain (Osumi et al., 1997
). It has also been reported that Pax6 controls R-cadherin expression in the developing neocortex (Stoykova et al., 1997
) (T. Inoue and N. O., unpublished). We have also found that the motility of neuroepithelial cells in the ventral telencephalon, where cadherin-6 is normally expressed, seems to be increased in cadherin-6 deficient mice (Inoue et al., 2001
). Pax6 also controls granule cell migration in the cerebellum by modulating cytoskeletal components (Yamasaki et al., 2001
). Collectively, Pax6 may regulate, directly or indirectly, certain cell adhesion molecule(s) and/or cytoskeletal molecule(s) expressed in the neuroepithelium, thereby functioning in the establishment of the rigid precursor domains in the hindbrain.
Dose-dependent effect of Pax6 in specification of ventral neurones
The progenitor domain for SM neurones corresponds to the region where Pax6 expression is low (Ericson et al., 1997) (Fig. 2A). Overexpression of Pax6 in the Pax6low SM progenitor domain in the early E11.5 wild-type hindbrain repressed the production of Islet2-positive SM neurones. By contrast, exogenous Pax6 in the E10.75 mutant embryos induced Islet2- and HB9/MNR2-positive cells. In the E10.75 hindbrain, Pax6 protein distribution is not seen in a gradient pattern (data not shown). Therefore, it is likely that differentiation of SM neurones is dependent on temporary different doses of Pax6. In fact, it has been reported that Pax6 influences eye formation and development of the diencephalic dorsal midline secretory radial glia in a dose-dependent manner (Schedl et al., 1996
; Estivill-Torrús et al., 2001
). Therefore, another interesting issue to investigate would be how much Pax6 is required for precise specification of SM neurones and perhaps other types of neurones.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arber, S., Han, B., Mendelsohn, M., Smith, M., Jessell, T. M. and Sockanathan, S. (1999). Requirement for the homeobox gene Hb9 in the consolidation of motor neuron identity. Neuron 4, 659-674.
Bastian, H. and Gruss, P. (1990). A murine even-skipped homologue, Evx 1, is expressed during early embryogenesis and neurogenesis in a biphasic manner. EMBO J. 9, 1839-1852.[Abstract]
Bosse, A., Zulch, A., Becker, M. B., Torres, M., Gomez-Skarmeta, J. L., Modolell, J. and Gruss, P. (1997). Identification of the vertebrate Iroquois homeobox gene family with overlapping expression during early development of the nervous system. Mech. Dev. 69, 169-181.[Medline]
Briscoe, J., Sussel, L., Serup, P., Hartigan-OConnor, D., Jessell, T. M., Rubenstein, J. L. and Ericson, J. (1999). Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling. Nature 398, 622-627.[Medline]
Briscoe, J., Pierani, A., Jessell, T. M. and Ericson, J. (2000). A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101, 435-445.[Medline]
Burrill, J. D., Moran, L., Goulding, M. D. and Saueressig, H. (1997). PAX2 is expressed in multiple spinal cord interneurons, including a population of EN1+ interneurons that require PAX6 for their development. Development 124, 4493-4503.
Caric, D., Gooday, D., Hill, R. E., McConnell, S. K. and Price, D. J. (1997). Determination of the migratory capacity of embryonic cortical cells lacking the transcription factor Pax-6. Development 124, 5087-5096.
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383, 407-413.[Medline]
Clarke, J. D., Erskine, L. and Lumsden, A. (1998). Differential progenitor dispersal and the spatial origin of early neurons can explain the predominance of single-phenotype clones in the chick hindbrain. Dev. Dyn. 212, 14-26.[Medline]
Ding, Q., Fukami, S. I., Meng, X., Nishizaki, Y., Zhang, X., Sasaki, H., Dlugosz, A., Nakafuku, M. and Hui, C. C. (1999). Mouse suppressor of fused is a negative regulator of sonic hedgehog signaling and alters the subcellular distribution of Gli1. Curr. Biol. 9, 1119-1122.[Medline]
Ericson, J., Thor, S., Edlund, T., Jessell, T. M. and Yamada, T. (1992). Early stages of motor neuron differentiation revealed by expression of homeobox gene Islet-1. Science 256, 1555-1560.[Medline]
Ericson, J., Morton, S., Kawakami, A., Roelink, H. and Jessell, T. M. (1996). Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity. Cell 87, 661-673.[Medline]
Ericson, J., Rashbass, P., Schedl, A., Brenner-Morton, S., Kawakami, A., van Heyningen, V., Jessell, T. M. and Briscoe, J. (1997). Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell 90, 169-180.[Medline]
Estivill-Torrús, G., Vitalis, T., Fernández-Llebrez, P. and Price, D. J. (2001). The transcription factor Pax6 is required for development of the diencephalic dorsal midline secretory radial glia that form the subcommissural organ. Mech. Dev. 109, 215-224.[Medline]
Erskine, L., Patel, K. and Clarke, J. D. (1998). Progenitor dispersal and the origin of early neuronal phenotypes in the chick embryo spinal cord. Dev. Biol. 199, 26-41.[Medline]
Gehring W. J. and Ikeo, K. (1999). Pax6: mastering eye morphogenesis and eye evolution. Trends Genet. 15, 371-377.[Medline]
Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. A. and Scott, M. P. (1996). Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by Hedgehog. Genes Dev. 10, 301-312.[Abstract]
Götz, M., Stoykova, A. and Gruss, P. (1998). Pax6 controls radial glia differentiation in the cerebral cortex. Neuron, 21, 1031-1044.[Medline]
Goulding, M. D., Lumsden, A. and Gruss, P. (1993). Signals from the notochord and floor plate regulate the region-specific expression of two Pax genes in the developing spinal cord. Development 117, 1001-1016.
Gruss, P. and Walther, C. (1992). Pax in development. Cell 69, 719-722.[Medline]
Heisenberg, C. P., Tada, M., Rauch, G. J., Saude, L., Concha, M. L., Geisler, R., Stemple, D. L., Smith, J. C. and Wilson, S. W. (2000). Sliberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation. Nature 405, 76-81.[Medline]
Hill, R. E. and Hanson, I. M. (1992). Molecular genetics of the Pax gene family. Curr. Opin. Cell Biol. 4, 967-972.[Medline]
Hynes, M., Stone, D. M., Dowd, M., Pitts-Meek, S., Goddard, A., Gurney, A. and Rosenthal, A. (1997). Control of cell pattern in the neural tube by the zinc finger transcription factor and oncogene Gli-1. Neuron 19, 15-26.[Medline]
Inoue, T., Nakamura, S. and Osumi, N. (2000). Fate mapping of the mouse prosencephalic neural plate. Dev. Biol. 219, 373-383.[Medline]
Inoue, T., Tanaka, T., Takeichi, M., Chisaka, O., Nakamura, S. and Osumi, N. (2001). Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development. Development 128, 561-569.
Ishii, Y., Nakamura, S. and Osumi, N. (2000). Demarcation of early mammalian cortical development by differential expression of fringe genes. Dev. Brain Res. 119, 307-320.[Medline]
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1, 20-29.[Medline]
Jönsson, M. and Andersson, T. (2001). Repression of Wnt-5a impairs DDR1 phosphorylation and modifies adhesion and migration of mammary cells. J. Cell Sci. 114, 2043-2053.
Lee, J., Platt, K. A., Censullo, P. and Ruiz i Altaba, A. (1997). Gli1 is a target of Sonic hedgehog that induces ventral neural tube development. Development 124, 2537-2552.
Lee, K. J. and Jessell, T. M. (1999). The specification of dorsal cell fates in the vertebrate central nervous system. Annual Review of Neuroscience (ed. W. M. Cowan, E. M. Shooter, C. F. Stenens and R. F. Thompson), pp. 261-294. California: Annual Reviews.
Liem, K. F., Tremml, G. and Jessell, T. M. (1997). A role for the roof plate and its resident TGF-ß related proteins in neuronal patterning in the dorsal spinal cord. Cell 91, 127-138.[Medline]
Liu, I. S., Chen, J. D., Ploder, L., Vidgen, D., van der Kooy, D., Kalnins, V. I. and McInnes, R. R. (1994). Developmental expression of a novel murine homeobox gene (Chx10): evidence for roles in determination of the neuroretina and inner nuclear layer. Neuron 13, 377-393.[Medline]
Lu, S., Bogarad, L. D., Murtha, M. T. and Ruddle, F. H. (1992). Expression pattern of a murine homeobox gene, Dbx, displays extreme spatial restriction in embryonic forebrain and spinal cord. Proc. Natl. Acad. Sci. USA 89, 8053-8057.[Abstract]
Lu, S., Wise, T. L. and Ruddle, F. H. (1994). Mouse homeobox gene Dbx: sequence, gene structure and expression pattern during mid-gestation. Mech. Dev. 47, 187-195.[Medline]
Marquardt, T., Ashery-Padan, R., Andrejewski, N., Scardigli, R., Guillemot, F. and Gruss, P. (2001). Pax6 is required for the multipotent state of retinal progenitor cells. Cell 105, 43-55.[Medline]
Marusich, M. F., Furnraux, H. M., Henion, P. D. and Weston, J. A. (1994). Hu neuronal proteins are expressed in proliferating neurogenic cells. J. Neurobiol. 25, 143-155.[Medline]
Matsunaga, E., Araki, I. and Nakamura, H. (2000). Pax6 defines the di-mesencephalic boundary by repressing En1 and Pax2. Development 127, 2357-2365.
Matsuo, T., Osumi-Yamashita, N., Noji, S., Ohuchi, H., Koyama, E., Myokai, F., Matsuo, N., Taniguchi, S., Doi, H., Iseki, S. et al. (1993). A mutation in the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells. Nat. Genet. 3, 299-304.[Medline]
Muhr, J., Andersson, E., Persson, M., Jessell, T. M. and Ericson, J. (2001). Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube. Cell 104, 861-873.[Medline]
Osumi, N. (2001). The role of Pax6 in brain patterning. Tohoku J. Exp. Med. 193, 163-174.[Medline]
Osumi, N. and Nakafuku, M. (1998). Pax-6 is involved in specification of ventral cell types in the hindbrain. In Neural Development: Keio Univ. Symposia for Life Science and Medicine Vol. 2 (ed. K. Uyemura, K. Kawamura and T. Yazaki), pp. 1117-1124. Tokyo: Springer-Verlag.
Osumi, N. and Inoue, T. (2001). Gene transfer into cultured mammalian embryos by electroporation. Methods 24, 35-42.[Medline]
Osumi, N., Hirota, A., Ohuchi, H., Nakafuku, M., Iimura, T., Kuratani, S., Fujiwara, M., Noji, S. and Eto, K. (1997). Pax-6 is involved in the specification of hindbrain motor neuron subtype. Development 124, 2961-2972.
Pierani, A., Brenner-Morton, S., Chiang, C. and Jessell, T. M. (1999). A sonic hedgehog-independent, retinoid-activated pathway of neurogenesis in the ventral spinal cord. Cell 97, 903-915.[Medline]
Pierani, A., Moran-Rivard, L., Sunshine, M. J., Littman, D. R., Goulding, M. and Jessell, T. M. (2001). Control of interneuron fate in the developing spinal cord by the progenitor homeodomain protein Dbx1. Neuron 29, 367-384.[Medline]
Pratt, T., Vitalis, T., Warren, N., Edgar, J. M., Mason, J. O. and Price, D. J. (2000). A role for Pax6 in the normal development of dorsal thalamus and its cortical connections. Development 127, 5167-5178.
Qiu, M., Shimamura, K., Sussel, L., Chen, S. and Rubenstein, J. L. (1998). Control of anteroposterior and dorsoventral domains of Nkx-6.1 gene expression relative to other Nkx genes during vertebrate CNS development. Mech. Dev. 72, 77-88.[Medline]
Roelink, H., Augsburger, A., Heemskerk, J., Korzh, V., Norlin, S., Ruiz i Altaba, A., Tanabe, Y., Placzek, M., Edlund, T., Jessell, T. M. and Dodd, J. (1994). Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord. Cell 76, 761-775.[Medline]
Sander, M., Paydar, S., Ericson, J., Briscoe, J., Berber, E., German, M., Jessell, T. M. and Rubenstein, J. L. (2000). Ventral neural patterning by Nkx homeobox genes: Nkx6.1 controls somatic motor neuron and ventral interneuron fates. Genes Dev. 14, 2134-2139.
Sasaki, H., Nishizaki, Y., Hui, C., Nakafuku, M. and Kondoh, H. (1999). Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: implication of Gli2 and Gli3 as primary mediators of Shh signaling. Development 126, 3915-3924.
Schedl, A., Ross, A., Lee, M., Engelkam, D., Rashbass, P., van Heyningen, V. and Hastie, N. D. (1996). Influence of PAX6 gene dosage on development: overexpression causes severe eye abnormalities. Cell 86, 71-82.[Medline]
Sharma, K., Sheng, H. Z., Lettieri, K., Li, H., Karavanov, A., Potter, S., Westphal, H. and Pfaff, S. L. (1998). LIM homeodomain factors Lhx3 and Lhx4 assign subtype identities for motor neurons. Cell 95, 817-828.[Medline]
Shoji, H., Ito, T., Wakamatsu, Y., Hayasaka, N., Ohsaki. K., Oyanagi, M., Kominami, R., Kondoh, H. and Takahashi, N. (1996). Regionalized expression of the Dbx family homeobox genes in the embryonic CNS of the mouse. Mech. Dev. 56, 25-39.[Medline]
Stone, D. M., Hynes, M., Armanini, M., Swanson, T. A., Gu, Q., Johnson, R. L., Scott, M. P., Pennica, D., Goddard, A., Phillips, H. et al. (1996). The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature 384, 129-134.[Medline]
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.
Stoykova, A., Treichel, D., Hallonet, M. and Gruss, P. (2000). Pax6 modulates the dorsoventral patterning of the mammalian telencephalon. J. Neurosci. 20, 8042-8050.
Sun, T., Pringle, N. P., Hardy, A. P., Richardson, W. D. and Smith, H. K. (1998). Pax6 influences the time and site of origin of glial precursors in the ventral neural tube. Mol. Cell. Neurosci. 12, 228-239.[Medline]
Tanabe, Y., William, C. and Jessell, T. M. (1998). Specification of motor neuron identity by the MNR2 homeodomain protein. Cell 95, 67-80.[Medline]
Teleman, A. A., Strigini, M. and Cohen, S. M. (2001). Shaping morphogen gradients. Cell 105, 559-562.[Medline]
Thaler, J., Harrison, K., Sharma, K., Lettieri, K., Kehrl, J. and Pfaff, S. L. (1999). Active suppression of interneuron programs within developing motor neurons revealed by analysis of homeodomain factor HB9. Neuron 4, 675-687.
Toresson, H., Potter, S. S. and Campbell, K. (2000). Genetic control of dorsal-ventral identity in the telencephalon: opposing roles for Pax6 and Gsh2. Development 127, 4361-4371.
Tsuchida, T., Ensini, M., Morton, S. B., Baldassare, M., Edlund, T., Jessell, T. M. and Pfaff, S. L. (1994). Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Cell 79, 957-970.[Medline]
van Heyningen, V. (1998). Developmental eye disease a genome era paradigm. Clin. Genet. 54, 272-282.[Medline]
Varela-Echavarría, A., Pfaff, S. L. and Guthrie, S. (1996). Differential expression of LIM homeobox genes among motor neuron subpopulations in the developing chick brain stem. Mol. Cell. Neurosci. 8, 242-257.[Medline]
Wakamatsu, Y., Mochii, M., Vogel, K. S. and Weston, J. A. (1998). Avian neural crest-derived neurogenic precursors undergo apoptosis on the lateral migration pathway. Development 125, 4205-4213.
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.
Yamasaki, T., Kawaji, K., Ono, K., Bito, H., Hirano, T., Osumi, N. and Kengaku, M. (2001). Pax6 regulates granule cell polarization during parallel fiber formation in the developing cerebellum. Development 128, 3133-3144.
Yun, K., Potter, S. and Rubenstein, J. L. (2001). Gsh2 and Pax6 play complementary roles in dorsoventral patterning of the mammalian telencephalon. Development 128, 193-205.