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Cell Growth & Differentiation Vol. 10, 525-536, July 1999
© 1999 American Association for Cancer Research

Expression of the Microphthalmia-associated Basic Helix-Loop-Helix Leucine Zipper Transcription Factor Miin Avian Neuroretina Cells Induces aPigmented Phenotype1

Nathalie Planque, Nathalie Turque2, Karin Opdecamp3, Manuella Bailly, Patrick Martin and Simon Saule4

Centre National de la Recherche Scientifique EP 560, Institut Pasteur de Lille, 59019 Lille cedex, France


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The microphthalmia gene (mi) appears to be required for pigment cell development, based on its mutation in mi mice. The mi gene encodes a basic helix-loop-helix leucine zipper transcription factor (Mi) with tissue-restricted expression. To investigate the role of mi in cell proliferation and pigmentation, we transfected neuroretina (NR) cells with a recombinant virus expressing the murine mi cDNA. The virus induced the proliferation of chicken NR cells in response to fibroblast growth factor 2, which enabled them to form colonies in soft agar. In contrast to control cultures, transfected chicken NR cells or quail NR cells became rapidly pigmented and strongly expressed the QNR-71 mRNA encoding a melanosomal protein. These results demonstrate that Mi not only acts as pigmentation inducer but is also able to modulate the response of cells to growth factors.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The vertebrate embryonic eye offers exceptional advantages for the investigation of the molecular basis of differentiation. The retina is formed by two distinct cellular layers that originate from the neuroectoderm. The outer layer, known as the RPE,5 contains a single cell type that can be related to melanocytes on the basis of its melanin content. The inner layer that forms the sensory NR is more complex and contains several strata of neurons. The embryonic cells of the RPE and the NR share the characteristic of remaining plastic after they have reached their terminal differentiation stage. Indeed, cells from the RPE have the potential to transdifferentiate into lens or neuronal cells (1, 2, 3) , and conversely, cells from the NR have the potential to transdifferentiate into pigmented cells (4 , 5) . Cell differentiation is the product of differential gene expression, and transcription factors do regulate such events.

We have previously observed that the avian retrovirus MC29 expressing the v-Myc transcription factor is able to stimulate QNR (Coturnix coturnix japonica) cells to become pigmented (6) . Conversely, this virus is able to induce neuronal traits in infected quail RPE (7) . This suggests that Myc is able to modulate gene expression, leading to these transdifferentiated phenotypes. myc encodes a b-HLH-ZIP transcription factor. b-HLH-ZIP proteins bind to DNA by the basic domain, dimerize through the helix-loop-helix domain, and are stabilized by the leucine zipper domain. The transcription activation domain is located NH2-terminal to the DNA-binding domain. Recently, specific genes containing the core hexanucleotide CACGTG or CATGTG sequences have been found to be activated by the c-Myc protein, including the p53 gene (8) , the embryonically expressed gene ECA39 (9) , the ornithine decarboxylase gene (10) , the CDC25 cell cycle phosphatase gene (11) , and the melanocyte-specific gene QNR-71 (12) .

Among the b-HLH-ZIP family members, the microphthalmia-associated transcription factor (Mi; Ref. 13) is of special interest in relation to the NR to RPE transdifferentiation phenomenon, because it has been shown to play a critical role in melanocyte development. Indeed, mice that bear severe mutations in the mi gene are completely unpigmented and deaf because they lack the neural crest-derived melanocytes that normally populate the skin and inner ear (14) . These mice are also microphthalmic because of an abnormal development of the RPE that appears unpigmented and often pluristratified (15 , 16) , suggesting a role for Mi in both cellular differentiation and control of cell division. In fact, in mi severe mutants, neural crest-derived melanoblasts cannot be identified, showing that the Mi function is required very early in the development of the melanocyte lineage, suggesting a possible role for this factor in the determination of these cells (17) . Sequence relationships within the b-HLH-ZIP domain show that Mi is most closely related to the transcription factors TFEB, TFEC, and TFE3, all of which can form in vitro stable heterodimers (18) . Mi regulates the expression of genes involved in the melanogenesis, including QNR-71, which encodes a melanosomal protein (12) and several enzyme-encoding genes controlling the melanin synthesis. These genes include the tyrosinase (19 , 20) and the tyrosinase-related peptide 1 (21) . These genes are regulated by Mi through a direct binding at the CATGTG hexameric motif (E-box) present in the promoter region in a M-box (12 , 18 , 20 , 21) . The molecular features of Mi as well as its fundamental role in melanocyte development rises the possibility for this factor to play a role similar to that played by v-Myc in the NR-to-RPE transdifferentiation phenomenon.

To study whether Mi, which shares some target genes with Myc (12) , is able to play a similar role in NR cells, we studied the effect of Mi accumulation on NR cell proliferation, differentiation, and transformation. CNR cells provide an attractive model because proliferation (growth as monolayer) and transformation (growth under anchorage-independent conditions) can be studied separately (22 , 23) . We have previously shown that FGF2, a fundamental regulatory molecule, stimulates the growth of CNR-overexpressing various transcription factors and allows them to form colonies in soft agar (24 , 25) . FGF family members are necessary for NR and RPE development. Addition of FGF2 to the optic vesicles during the culture period in vitro causes the presumptive pigmented epithelium to differentiate as neural retina and conversely, addition of FGF2 neutralizing antibodies to these optic vesicles inhibited neural retina development but did not interfere with normal development of the pigmented epithelium (26) .

In this study, we analyze in CNR and QNR cells the biological properties of an avian retrovirus encoding the murine mi cDNA. This virus expresses a p66/68 (Mr 66,000/68,000) protein reacting with a polyclonal antibody made against the COOH terminus of Mi. We found that mi-expressing CNR cells are responsive to FGF2 both in liquid and semisolid medium. Furthermore, CNR or QNR cells transfected with this viral molecular clone become rapidly pigmented in vitro, in contrast to the control cells transfected with the empty DNA vector. To test the possibility that expression of other b-HLH-ZIP proteins induce such pigmented phenotype in NR cells, we transfected viral molecular clones containing either the mi-related TFEB (16) or the very distantly related USF (27) cDNAs. Pigmentation was found only with the TFEB-containing vector, but the mi-containing vector was found to be 8-fold more efficient in pigmentation induction than the TFEB containing vector. v-myc-expressing MC29-QNR cells that became pigmented also expressed the endogenous Mi, and this expression increased with the passages of the culture. These results show that, in addition to its previously documented role as a transcription factor acting on the pigmentation genes (28) , Mi is able to modulate the growth factor responsiveness of expressing cells, and this function may be critical for the genesis of the abnormalities observed in vivo either in the mouse mi mutants or in the human Waardenburg syndrome type 2, an hereditary disorder associated with melanocyte abnormalities (29 , 30) .


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Effects of the LE-mi Virus on the Proliferation and FGF2 Sensitivity of CNR Cells.
Primary CNR cells cotransfected with the LE-mi (the SFCV-LE-mi virus was created by the insertion of the 1.8-kbp HindIII-XbaI fragment of the mi cDNA into the avian retroviral vector SFCV-LE; Ref. 12 ) and the helper virus RAV1 DNA were morphologically untransformed after G418 selection, suggesting that, as reported for fibroblastic cell lines (28) , Mi was not able alone to transform primary cells (Fig. 1)Citation . Similar results were obtained without helper virus, suggesting that the RAV1 virus did not influence the Mi functions. We have previously shown that CNR cells expressing nuclear transcription factors were stimulated to proliferate by FGF2 (24 , 25) . To determine whether Mi could also induce this positive response, we tested the effect of increasing amount of FGF2 on the proliferation in low-serum containing medium of LE-mi-CNR cells. In contrast to the control CNR cells transfected with the empty LE vector, which died without FGF2, CNR cells transfected with LE-mi responded positively to FGF2, and the stimulating effect of this growth factor was dose dependent (Fig. 2)Citation .



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Fig. 1. Morphology of normal (a) and mi-expressing (b) CNR cells. Dissociated CNR cells dissected from 8-day-old chicken embryos were plated in DMEM-F-12 medium supplemented with 10% FCS, 1% MEM/100x vitamins, and 10 µg/ml conalbumin. Dishes (60-mm diameter) containing 107 dissociated cells were transfected at 37°C with pSFCV-LE mi (LE-mi) and RAV-1 proviral DNA or with the empty retroviral vector pSFCV-LE (LE) and RAV-1. The transfected cells were selected in G418 containing complete medium for 10 days. Actin filaments were labeled with nitrobenzoxadiazole-labeled phallacidin, as described previously (22) .

 


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Fig. 2. Ability of FGF2 to stimulate the growth of LE-mi-CNR cells. Low-density cultures of G418-selected CNR cells transfected with LE-mi or LE were grown in 22-mm diameter gelatinized wells (4 x 104 cells) with 1 ml of medium containing 1% FCS, 1% MEM/100x vitamins, 10 µg/ml conalbumin, and 5 µg/ml insulin. FGF2 was added 2 days after the day of plating at the concentration indicated in the figure. After 4 days in culture, duplicate wells were trypsinized, and cells were counted. •, LE-mi CNR cells; {blacksquare}, LE CNR cells.

 
Effects of FGF2 on Anchorage-independent Cell Growth.
The ability of cells to grow under anchorage-independent conditions has been shown to correlate with transformation (31) . We, therefore, tested the ability of LE-mi-CNR cells to respond to FGF2 when they were maintained in soft agar in the presence of 10% serum-supplemented medium. As shown in Table 1Citation , cells cotransfected with LE and the proviral RAV-1 DNA did not form any colony in absence or in presence of FGF2. LE-mi-CNR cells did grow in soft agar, but only in presence of FGF2. We concluded that the presence of Mi allows the CNR cells to become transformed in the presence of FGF2.


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Table 1 Effect of FGF2 on CNR cell transformation

 
Protein Products of LE-mi in CNR Cells.
To obtain rabbit antiserum that was able to specifically recognize the Mi, we expressed in bacteria the 222 amino acids from the Mi COOH terminus, fused in-frame with the first 98 amino acids of the polymerase of the phage MS2 and injected rabbits with gel-purified peptides. The anti-Mi serum (serum MiC) was tested in immunoprecipitation using [35S]methionine-labeled reticulocyte lysate with a vector encoding the Mi protein. SDS-PAGE analysis of anti-Mi immunoprecipitates revealed a predominant protein with a relative molecular mass of 66 kDa, which was not recognized by the cognate preimmune serum (Fig. 3A)Citation . Mi protein was precipitated even when the serum was incubated with an excess of MS2 peptide, suggesting that the protein was recognized through specific determinants to the Mi segment of the immunogen (data not shown).



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Fig. 3. Expression and characterization of Mi proteins. A, characterization of the serum directed against Mi protein. The murine mi cDNA inserted in the pSG5 expression vector was translated in [35S]methionine-containing reticulocyte lysate. Translated proteins were immunoprecipitated with rabbit serum MiC prepared against bacterially expressed Mi COOH-terminal peptide (Lane 1) and with cognate preimmune serum (Lane 2). Lane 3, translated crude Mi protein. B, detection of Mi by immunoprecipitation. Selected LE-mi-transfected CNR cells or LE-transfected CNR cells were labeled with [35S]methionine/cysteine for 30 min. Labeled cells were solubilized in RIPA buffer. Cell lysates were then immunoprecipitated with the serum MiC (Lanes 2 and 4) and with cognate preimmune serum (Lanes 3 and 5). Lane 1, LE-mi-CNR lysate were first immunoprecipitated with the serum MiC, and proteins recovered from the immunocomplexes bound to protein A-Sepharose were immunoprecipitated again with the same serum. C: a, phase contrast of selected LE-mi-CNR. b, subcellular localization of Mi assayed by indirect immunofluorescence on fixed cells with serum MiC. Anti-Mi-immunoreactive proteins were detected with tetramethylrhodamine isomer R-labeled swine antirabbit immunoglobulins secondary reagent. c, phase contrast of selected LE-CNR used as control. d, subcellular localization of Mi assayed by indirect immunofluorescence. Nuclei are labeled in LE-mi-CNR cells but not in control cells.

 
To confirm that the expected proteins were found in LE-mi-transfected CNR cells, cells were labeled with [35S]methionine/cysteine, and lysates were immunoprecipitated with the serum MiC. Results are shown in Fig. 3BCitation . Because we observed a doublet protein, to determine whether these proteins were Mi-associated proteins, we performed an immunoprecipitation experiment with serum MiC, and then the proteins recovered from the immunocomplexes bound to protein A-Sepharose were immunoprecipitated again with the same serum. If the Mr 66,000 proteins recovered with serum MiC were Mi-associated proteins and not Mi isoforms, we did not expect to find them in the second immunoprecipitation. Fig. 3BCitation (Lane 1) demonstrates that this is not the case because these proteins were recovered with the second immunoprecipitation. This result suggests that some of the numerous in-frame AUGs present in the mi sequence are used to direct the synthesis of the low molecular weight Mi products in the virally transfected cells. Alternatively, because a mitogen-activated protein kinase-dependent Mi phosphorylation has been reported (32) , the high molecular weight Mi protein may represent a phosphorylated form of the transcription factor. In addition, Fig. 3CCitation shows, by immunodetection with serum MiC, that Mi is localized in the nuclei of the G418-selected cells.

Effects of b-HLH-ZIP Proteins on the Transdifferentiation of CNR Cells.
Transfection of the culture, followed by G418 selection, resulted in the appearance of pigmented foci in the LE-mi-transfected cells, 8 days after the selection but not in the LE control-transfected CNR (Table 2)Citation . Because we observed the induction of a pigmented phenotype in the NR cells expressing either Myc or Mi, we asked whether this pigmentation-inducing ability is a general property of the b-HLH-ZIP transcription factors. Therefore, we tested whether Mi-related transcription factor TFEB or the distantly related USF would also be efficient in pigmentation induction. We transfected the CNR cells with the LE-TFEB and the LE-USF vectors. Transfection of the cultures followed by G418-selection resulted in the appearance of pigmented foci only in the LE-TFEB (with a very low efficiency) but not in the LE-USF transfected cells (Table 2)Citation . To verify that the LE-TFEB molecular clone expressed an active protein, we tested TFEB functionally. We cotransfected equal amounts of plasmid pMT2.2 (the 2.2-kb fragment 5' of the transcriptional start site of the mouse tyrosinase gene cloned upstream the luciferase coding sequence; Ref. 33 ), together with increasing amounts of expression vectors LE-mi or LE-TFEB into RPE cells. Cell lysates were collected 2 days after transfection, and the levels of luciferase activity present in the lysates determined. Cotransfection of pMT2.2 with 5 µg of the vector expressing Mi or TFEB protein resulted in a similar increase of luciferase activity relative to the vector control (Fig. 4B)Citation , suggesting that Mi and TFEB are similarly efficient in tyrosinase promoter transactivation. In addition, TFEB mRNA was identified by a digoxigenin-labeled TFEB riboprobe in the G418-selected LE-TFEB cells but not in the LE control cells (Fig. 4A)Citation . In addition, no pigmented transdifferentiation could be observed in CNR cells overexpressing various transcription factors that are unrelated to the b-HLH-ZIP transcription factors but able to induce a FGF2 response of cells, such as Fos, Jun, Ski, E1A, myb, myb-ets, and erbA (data not shown).


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Table 2 Effect of viruses on avian NR cell pigmentation

 


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Fig. 4. Transfection of CNR cells with the LE-TFEB expression vector. A, TFEB expression in the G418-resistant CNR cells, as revealed by nonradioactive in situ hybridization (a). Note that the G418-resistant CNR cells obtained after LE transfection were not labeled (b). B, activity of Mi and TFEB proteins encoded by the LE vectors. Quail RPE cells were transfected with 1 µg of pMT2.2, a plasmid that contains a 2.2-kb fragment 5' of the transcriptional start site of the mouse tyrosinase gene cloned upstream the luciferase coding sequence, and 5 µg of either the LE-TFEB or LE-mi expression plasmid. The vector control used was the empty LE DNA. Cells were solubilized, and luciferase activities were assayed.

 
Effects of the LE-mi Virus on the Differentiation of QNR Cells.
Primary QNR cells cotransfected with LE-mi and RAV-1 DNA were also morphologically untransformed and, like the CNR cells, they became rapidly pigmented (Fig. 5ACitation and Table 2Citation ). However, several G418 foci were not pigmented in the LE-mi-transfected cells. To study the correlation between Mi expression and pigmentation, we performed an immunodetection of Mi into the G418-selected foci directly on the culture plates. All pigmented foci showed labeled nuclei with serum MiC (Fig. 5B)Citation . In contrast, none of the foci with unlabeled nuclei were pigmented, consistent with the fact that no pigment cells were detected in the control plate, therefore suggesting that Mi was critical for pigment accumulation in the cells. No obvious differences in the number of apoptotic bodies in the Mi-expressing or nonexpressing cells could be found after the Hoechst 33258 staining (Fig. 5Ba)Citation , suggesting that the particular compaction of the apoptotic nuclei is not present in the G418-selected cells. This was further demonstrated by using TUNEL, a method that reveals apoptotic cells by detecting DNA stand breaks (34) . Similar number of positive nuclei could be observed in the LE-mi pigmented foci (Fig. 5C, a–c)Citation or in the nonpigmented control QNR LE cells (Fig. 5C, d–f)Citation .



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Fig. 5. Expression of Mi products by immunofluorescence in LE-mi-transfected QNR cells. A, after G418-selection, some of the LE-mi-QNR foci were pigmented (b and d), in contrast of the LE-QNR foci (a and c). a and b, cells were fixed in 4% PFA in PBS. Pigmented foci were clearly visible. c and d, cells were stained with Coomassie Blue to visualize pigmented and nonpigmented foci. B: a, nuclei were stained with Hoescht 33258 in PBS. b, subcellular localization of Mi was assayed by indirect immunofluorescence on fixed QNR cells with serum MiC. Anti-Mi-immunoreactive proteins were detected with FITC-labeled swine antirabbit immunoglobulins secondary reagent. c, pigment cells were observed by direct light microscopy. Nuclei were labeled in pigmented LE-mi-QNR cells (arrows) but not in nonpigmented cells (arrowheads). C: a, LE-mi-QNR nuclei were stained with Hoescht 33258 in PBS. b, TUNEL assay. DNA of fixed cells is labeled by the addition of fluorescein dUTP at strand breaks by terminal transferase (arrow). c, pigment cells were observed by direct light microscopy. d, LE-QNR nuclei were stained with Hoescht 33258 in PBS. e, TUNEL assay. f, cells were observed by direct light microscopy (no pigment found).

 
Expression of the Quail Mi in the MC29-QNR.
We previously showed that Mi was able to increase the amount of QNR-71 proteins in RPE. Because we found that QNR-71 was not expressed in QNR but was induced after several subcultures in vitro (12) , we asked whether Mi would be able to modulate the amount of QNR-71 mRNA in the LE-mi-transfected QNR cells. As shown in Fig. 6ACitation , the amount of QNR-71 mRNA detected in LE-mi-transfected cells shows a 10-fold increase, compared to the nontransfected or LE-transfected control cells (using the actin gene as internal control; data not shown). As expected, the QNR-71 proteins were expressed in mi-expressing QNR cells but not in the control cells, as shown by immunoprecipitation of lysates labeled with [35S]methionine/cysteine (Fig. 6B)Citation .



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Fig. 6. Expression of QNR-71 in mi-expressing cells. A, Northern blot analysis of QNR-71 transcription in mi-expressing cells. Total RNA was extracted from normal QNR, QNR transfected with LE-mi (QNR-mi) or QNR transfected with the control empty vector LE (QNR-LE), as indicated at the top. QNR cells dissected from 8-day-old embryos were passaged four times (QNR P4) in vitro. A 20-µg aliquot of total RNA was hybridized with 32P-labeled QNR-71 probe encompassing the complete QNR-71 open reading frame. B, detection of QNR-71 proteins in mi-expressing cells. Normal QNR or LE-mi-transfected QNR cells were labeled with [35S]methionine/cysteine for 45 min, solubilized in 1% SDS-5% 2-mercaptoethanol-containing solution, boiled 5 min., and diluted in RIPA buffer. Cell lysates were then immunoprecipitated with a rabbit serum QNR-71 (Lanes 1 and 4) and with cognate preimmune serum (Lanes 2 and 3).

 
QNR cells infected with the avian retrovirus MC29 expressing the v-Myc nuclear transcription factor were induced to transdifferentiate along the pigmented pathway (6 , 12) . Because this phenotype is only marginally induced by TFEB, a factor more closely related to Mi than Myc, we studied the possibility that the pigmented phenotype induced by v-Myc involved endogenous mi expression. We performed Northern blot experiments with a quail mi cDNA probe as well as an immunodetection of nuclear proteins with serum MiC. As shown in Fig. 7ACitation , numerous nuclei were labeled in the MC29-infected QNR cells, whereas none could be detected either in the noninfected control cells at P5 (Fig. 7Ad)Citation or with the preimmune serum (data not shown), and the amount of labeled nuclei increased from 0 to 42% of the cells with the number of passages in vitro (Fig. 7B)Citation .



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Fig. 7. Expression of mi by immunofluorescence in MC29-infected QNR cells. A, subcellular localization of Mi protein was assayed by indirect immunofluorescence with serum MiC on fixed MC29-QNR cells grown for 24 h onto 16 mm glass coverslips. a, MC29-QNR after three passages (P3). b, MC29-QNR after five passages (P5). c, MC29-QNR after seven passages (P7). d, control QNR cells infected by the helper virus after only five passages (P5). Anti-Mi-immunoreactive proteins were detected with FITC-labeled swine antirabbit immunoglobulin secondary reagent. Note that, in control QNR P5, the nuclei were not labeled. B, percentage of MiC-labeled nuclei in the different MC29-QNR. At least 500 cells were scored, except for P1 and P3, in which all of the positive cells on the coverslip were counted. Except for the P3 culture, the coverslips were covered by an equivalent number of cells. Note that the culture appeared fully transformed as early as P1.

 
To identify a mRNA transcript for the mi gene, we performed PCR amplifications using two oligonucleotides located at the junction of exons 4 and 5, upstream of the basic domain, and at the end of the zipper domain in exon 9. As shown in Fig. 8Citation , we amplified a 400-bp fragment from the quail RPE E8 and E14.5 cells and reverse-transcribed the RNA using a random priming oligonucleotide. The protein sequence deduced from this quail cDNA was 90.6% identical to the human MI product (including the basic helix-loop-helix domain and part of the leucine zipper domain; amino acids 142–284) and 77% identical to the human TFE3, which is the best homology score with the related TFE members. Comparison with the chicken Mi sequence (35) shows 99.7% identity, suggesting that we have isolated a fragment of the quail mi cDNA and not a quail TFE member. We observed a deletion of 6 amino acids (amino acids 187–192) at the same place than in the TFE members upstream of the basic region also absent in the Mi sequence deduced from some mouse cDNA clones (13) . No quail mi cDNAs containing these 6 amino acids have been thus far isolated. Using this cDNA fragment as probe, we performed Northern blot experiments on QNR cells infected with MC29 after several passages in vitro. As shown in Fig. 9Citation , the amount of mi mRNA increased in the MC29-QNR cells after several passages of the culture, whereas a similar experiment performed with MC29-RPE cells showed that the amount of mi RNA remained constant in the culture.



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Fig. 8. Protein sequence comparison of the quail Mi protein with those of the chicken, human, and murine Mi and with the other TFE members. Numbers, amino acid positions in Mi. Boxes, conserved amino acids; dark gray boxes, shared amino acids; light gray boxes, amino acids with similar physicochemical character. Gaps (-) are introduced to allow maximal alignment. Accession numbers of the sequences used were as follows: chicken Mi, GenBank D88363 (35) ; murine Mi, GenBank Z23066; human MI, GenBank Z29678; human TFE3, EMBL 51330; human TFEB, GenBank M33782; rat TFEC, GenBank, L08812.

 


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Fig. 9. Northern blot analysis of mi expression in MC29-transformed cells. Total RNA was extracted from normal QNR, QNR-MC29, RPE, or RPE-MC29 at several passages as indicated at the top. A 20-µg aliquot of total RNA was hybridized with 32P-labeled miQ probe.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Several studies performed both in vivo and in vitro suggest that the differentiation of neural retina and pigmented epithelium from the optic vesicle epithelium is not due strictly to intrinsic differences between these two tissues but rather that specific inducing factors are produced by tissues surrounding the optic vesicle to pattern the differentiation of the eye (26) . Among these factors, members of the FGF family are necessary for neural retina and pigmented epithelium development (26) . A fundamental question concerns the transcriptional mechanisms by which multipotent cells become committed to particular cell fates. Clues about these underlying mechanisms may be inferred from observations of various experimentally induced transdifferentiation events, in which embryonic cells occur from one phenotype to another (1 , 3 , 5 , 6 , 36) . Such transdifferentiation events may reveal aspects of the molecular mechanisms responsible for the determination of the relationship between the two cell types. For example, FGF2 induces RPE to generate neural retina in vitro (3) . However, FGF2 was also found responsible for the transdifferentiation of avian neural crest-derived Schwann cell precursor into melanocytes (36) .

In previous work, we found that the b-HLH-ZIP v-myc oncogene induced the appearance of pigmented cells in cultured embryonic QNR (12) . We report here that another b-HLH-ZIP transcription factor Mi also induced this phenotype when expressed in these cells. However, in contrast to Myc, Mi was unable to induce a morphological transformation, but in the presence of FGF2, mi-expressing CNR cells were able to grow in soft agar, suggesting that FGF2 and Mi cooperate to induce a transformed phenotype in these cells. Interestingly, genetic evidence obtained from the mouse indicate that Mi is required for cells derived from the neural crest to respond to growth factors to differentiate as melanocytes (17) . Therefore, overexpression of Mi may induce the cells to proliferate in response to a growth factor, allowing them to grow in soft agar. Because this cell growth-promoting effect is observed in complete medium (which, in contrast to the low-serum containing medium, allows the control CNR cells to survive without FGF2) and because no obvious difference in the number of apoptotic nuclei could be observed in the G418-selected QNR cells expressing or not Mi, we do not favor the hypothesis that Mi promotes growth by suppressing apoptosis of NR cells. In addition to this effect on cell proliferation, Mi induced the differentiation of a pigmented phenotype in NR cells. Thus, these results may explain several observations made in the mouse mi mutants. In these mice, the lack of Mi in the neural crest-derived melanocytes is responsible for their disappearance, due to the lack of growth factor responsiveness (17) . In contrast, in the QNR, the permanent expression of mi results in terminal differentiation of poorly dividing cells. Indeed, after passages in vitro, the mi-expressing cells were rapidly lost, and only a few Mi-positive nuclei (1% of the starting mi-expressing QNR) could be recovered after three passages in vitro. This indicates that mi-expressing pigmented QNR cells are not able to proliferate. Interestingly, in several mi mutants, the RPE is stratified (15 , 16) , suggesting that the lack of functional Mi allows the RPE to overproliferate.

Another point we examined was whether the endogenous Mi is required for the v-Myc-induced pigmentation in QNR cell transdifferentiation. We observed mi expression both at the RNA and protein level in MC29-infected QNR cells, and the amount of mi increased after the passages of the cells at both the RNA and protein levels. In contrast, in the MC29-infected RPE, the mi RNA remained at the same level as in the noninfected culture, suggesting that the mi increase observed in MC29-QNR cells is linked with the transdifferentiation process occurring in these cells and may be the key event leading to the pigmentation. An interesting observation is that, in contrast to LE-mi-QNR, in the MC29-QNR cells, the pigmented differentiation occurred in highly proliferating cells. This is not the first observation that v-Myc induced simultaneously proliferation and differentiation (37) . However, it remains possible that, in the mi-expressing cells, Mi counteracts the v-Myc proliferative effect. Because Mi and Myc are not expected to form heterodimers (18) , Mi could inhibit the expression of Myc target genes involved in the cellular proliferation by a direct binding on the E-box in the promoter of these genes [even if the promoter of some genes, like QNR-71 (12) and Tyrosinase (data not shown), are independently activated by both Myc and Mi]. Thus, in a culture of vigorously proliferating cells, cells expressing Mi may be withdrawn from the cell cycle and differentiate as pigmented cells.

An interesting question is whether other b-HLH-ZIP factor related to Mi would be sufficient to induce a pigmented phenotype in the NR. USF, an ubiquitously expressed b-HLH-ZIP factor, is not able to induce a pigmented phenotype, even if this factor, like Mi or Myc, is able to bind to an E-box. The ability of Mi to discriminate between CATGTG E-box elements is dictated by the nature of the base at position -4 relative to the center of the E-box (38) , and it is not known whether USF is also able to recognize and transactivate the promoters bearing this subset of E-box. In contrast to USF, we observed that expression of TFEB mRNA in NR cells (a tissue not expected to express this gene; Ref. 16 ) resulted in the appearance of few pigmented foci, with a 8-fold less efficiency than Mi, whereas the two molecular clones exhibited a similar activity as transcription factors. The nuclei of the TFEB pigmented cells were not labeled with the MIC antiserum (data not shown), suggesting that, in contrast to Myc, TFEB was not inducing the endogenous microphthalmia gene.

Eventually, because several data have shown that FGF2 is an inducer of neural retina (26) , it will be of primary interest to determine the effect of FGF2 on mi expression and activity in the early embryo. From our results, it will be tempting to postulate that FGF2 antagonizes the Mi activity with respect to the pigmentation process. Indeed, Halaban et al. (39) reported that Mi expression in melanocytes was down-regulated by FGF2, and recently, Mochii et al. (35) reported a similar Mi down-regulation in dedifferentiated RPE cultured with this factor. In addition, FGF2 was found dispensable in Mi-/- quail RPE in the induction of the RPE transdifferentiation (40) .


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture, Growth Assay, and Soft Agar Cloning.
Dissociated NR cells dissected from 8-day-old avian embryos were plated in DMEM-F-12 medium containing 10% FCS, 1% MEM vitamins x100, and 10 µg/ml conalbumin (complete medium). Dishes (60-mm diameter) containing 107 dissociated cells in complete medium were transfected with various viruses cDNA at 37°C. The transfected cells were passaged one time on gelatin-coated dishes (100-mm diameter) and selected for 10 days in complete medium containing G418 (400 µg/ml for CNR and 200 µg/ml for QNR) before testing. The LE-mi vector was described previously (12) . The LE-TFEB vector was constructed by inserting the human EcoRI-HindIII fragment (16) into the LE vector digested by the same restriction enzymes, and the LE-USF was constructed by inserting the EcoRI Human USF1 (27) fragment into the LE vector.

To study the sensitivity of the cells to growth factors, we seeded 4 x 104 cells in 22-mm wells, after the selection process, in DMEM-F12 containing 1% FCS, 1% MEM/100x vitamins, 10 µg/ml conalbumin, and 5 µg/ml of insulin. FGF2 was added 2 days after the day of plating. After 4 days, the cultures were trypsinized, and the number of cells was determined with a Coulter counter.

To assess the anchorage-independent growth of transfected CNR, we suspended 4 x 104 cells in 2 ml of soft agar containing medium (complete medium plus 0.4% agar noble: cloning medium). This medium was layered on a hardened base layer of medium containing 1.2% agarose in 35-mm tissue culture dishes. Twenty ng of FGF2 were added every 2 days on the top of the soft agar until the end of the experiment. Colonies were counted 14 days later, using a phase-contrast microscope (SK2; Olympus).

Expression of Proteins in Bacteria.
To produce the Mi COOH terminus, the appropriate fragment was obtained from pSG5mi plasmid as template in PCR experiments using oligonucleotides 5'-TAGG GAT CCC ATG TTG GCT AAA GAG AGG CAG-3' (position +640) and 5'-ATC AAG CTT ACT AAC ACG CAT GCT CCGT-3' (position +1296). The expected band was sequenced, subjected to BamHI-HindIII restriction enzyme digestion, and inserted between the BamHI-HindIII sites of a pLC24 vector. The subcloning results in the in-frame fusion of the desired sequence to the first 98 amino acids of the polymerase of phage MS2. For expression, plasmids grown in LE392({lambda}) were transferred into an Escherichia coli host (SG4044) containing a temperature-sensitive repressor of the PL promoter. Cultures of exponentially growing bacteria carrying the desired vector were induced at 42°C for 3 h. Bacterial pellets were resuspended in TE buffer and incubated with 1 mg/ml lysozyme and 0.5% NP40. Bacterial DNA was digested by 100 µg/ml DNaseI. After centrifugation, pellets were washed twice in TE buffer added with 0.5% NP40 and once in TE buffer, boiled in sample buffer, and electrophoresed in a 15% SDS-PAGE. Proteins were visualized by staining with Coomassie Brilliant Blue and recovered from the gel.

Cell Labeling and Immunoprecipitation.
NR cells starved in methionine-, cysteine-, and glutamine-free medium for 15 min were incubated for 30 min in the presence of 30 µCi·ml-1 of L-[35S]methionine/cysteine (Amersham; specific activity, of 1000 Ci·mmol-1), lysed in RIPA buffer, and immunoprecipitated with rabbit serum MiC and rabbit serum QNR-71. Immunoprecipitated proteins were analyzed by SDS-PAGE followed by autoradiography.

Immunofluorescence Study.
Transfected cells cultured on 16-mm microscope coverslips or on the G418-selected cells directly in the Petri dishes, fixed for 20 min with 4% paraformaldehyde in PBS, and then treated with serum MiC. Anti-Mi-reactive proteins were detected with FITC-labeled goat antirabbit immunoglobulins secondary reagent.

TUNEL.
Apoptotic cells detection was performed by enzymatic (terminal deoxynucleotidyl transferase) in situ labeling (incorporation of FITC-labeled dUTP) in apoptosis-induced DNA strands using the In Situ Cell Death Detection Kit (Boehringer Mannheim).

RT-PCR Experiment.
Total RNA (1 µg) from RPE cells dissected from 8- and 14.5-day-old quail embryos was converted to cDNA using the oligonucleotide random hexamers (Perkin-Elmer), 39 units of rRNasin (Promega), 1 µl of 25 mM dNTP mix, 1 µl of 100 mM dithiothreitol, and 1 µl of 1 mg/ml BSA; and 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) were added in the appropriate buffer; and the mixture was incubated for 45 min at 37°C. A 1-µl aliquot was prepared for PCR (Taq polymerase, Taq buffer; Eurogentech) with 0.25 µg of the oligonucleotide primers 5'-TTG CAA ATG GCA AAT ACG TT-3' (position +442) and 5'-TGG CTG CAG TTC TCA AGA AC-3' (position +1022). Amplifications were carried out in a Cetus apparatus: 1.0 min of denaturation at 94°C, 90 s of annealing at 50°C, and 90 s of elongation at 72°C. Reaction products were cloned in the PCRII vector (PCRII-miQ). Individual clones were isolated and sequenced.

LUC Reporter Plasmid Transfection and Luciferase Assays.
The tyrosinase promoter fragment (-2236/+42) was obtained from R. Ballotti (33) and contains the E-box responsible for the mi activation of this promoter. Quail pigmented epithelium cells were seeded at 5 x 105 cells per 60-mm dish in DMEM-10% FCS, 24 h prior to transfection, performed by the calcium phosphate method. Cells were cotransfected with 1 µg of tyrosinase LUC construct. For the experiments with the vector expressing the TFEB or the Mi proteins, cells were cotransfected with 1 µg of the LUC construct and 5 µg of expression vectors.

RNA Extraction and Northern Analysis.
RNA was extracted using the guanidinium-isothiocyanate-cesium chloride method. Twenty µg of total cellular RNA were denatured at 68°C in a formamide-formaldehyde mixture, separated by electrophoresis in a 1% agarose-2.2 M formaldehyde gel, transferred to nitrocellulose filter in 20x SSC, and hybridized to DNA probes labeled by random priming. Hybridization was performed by the standard procedure, and the filters were exposed to Kodak XAR-5 films.

The miQ probe was a 0.4-kbp EcoRI fragment of the PCRII-miQ. QNR-71 probe was the 1.7-kbp EcoRI-BglII fragment containing the full open reading frame. The {alpha}-actin probe (41) was a 513-bp PvuII-XbaI fragment from pHMaA-1 and was provided by F. Radvanyi. The purified inserts were labeled to high specific activity (2–4 x 109 dpm/µg) with a random primer labeling system (Boehringer Mannheim).

In Situ Hybridization.
Riboprobes were made by in vitro transcription of appropriate plasmids with either T3 or T7 polymerase and incorporation of digoxigenin-labeled UTP. The template for TFEB was described previously (16) and exclude the conserved b-HLH-ZIP region. Nonradioactive in situ hybridization was performed as described previously (16 , 17) .


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut Pasteur de Lille, the Association Francaise Retinitis Pigmentosa, the Association pour la Recherche contre le Cancer, the Ligue Nationale Contre le Cancer, and the Groupements des Entreprises Françaises dans la Lutte contre le Cancer. Back

2 Present address: Laboratory of Developmental Neurogenetics, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892. Back

3 Present address: Université Libre de Bruxelles, Laboratoire d’Embryologie Moléculaire, Rue des Chevaux, 67, B-1640 Rhode-St-Genèse, Brussels, Belgium. Back

4 To whom requests for reprints should be addressed, at Centre National de la Recherche Scientifique EP 560, Institut Pasteur de Lille, 1 Rue Calmette, 59019 Lille cedex, France. Phone: (33) 3 20 87 11 21; Fax: (33) 3 20 87 11 11; E-mail: saule{at}infobiogen.fr Back

5 The abbreviations used are: RPE, retinal pigment epithelium; NR, neuroretina; QNR, quail NR; b-HLH-ZIP, basic helix-loop-helix leucine zipper; CNR, chicken NR; FGF, fibroblast growth factor; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; TE, 50 mM Tris-1 mM EDTA; RIPA, radioimmunoprecipitation assay. Back

Received for publication 1/27/99. Revision received 4/12/99. Accepted for publication 4/23/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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