Adult neural stem cell cycling in vivo requires thyroid hormone and its alpha receptor.

Thyroid hormones (TH) are essential for brain development. However, information on if and how this key endocrine factor affects adult neurogenesis is fragmentary. We thus investigated the effects of TH on proliferation and apoptosis of stem cells in the subventricular zone (SVZ), as well as on migration of transgene-tagged neuroblasts out of the stem cell niche. Hypothyroidism significantly reduced all three of these processes, inhibiting generation of new cells. To determine the mechanisms relaying TH action in the SVZ, we analyzed which receptor was implicated and whether the effects were played out directly at the level of the stem cell population. The alpha TH receptor (TRalpha), but not TRbeta, was found to be expressed in nestin positive progenitor cells of the SVZ. Further, use of TRalpha mutant mice showed TRalpha to be required to maintain full proliferative activity. Finally, a direct TH transcriptional effect, not mediated through other cell populations, was revealed by targeted gene transfer to stem cells in vivo. Indeed, TH directly modulated transcription from the c-myc promoter reporter construct containing a functional TH response element containing TRE but not from a mutated TRE sequence. We conclude that liganded-TRalpha is critical for neurogenesis in the adult mammalian brain.

Early activation of transcription factor expression in Schwann cells by progesterone.

Progesterone (PROG) promotes the myelination of sciatic nerves during regeneration after cryolesion. But, little is known about the molecular mechanisms by which the hormone exerts its effects. This could be initiated by the regulation of transcription factor expression in Schwann cells, which produce the myelin sheaths in the peripheral nervous system. We investigated by RT-PCR whether PROG activated expression of transcription factors: Egr-1 (Krox-24) Egr-2 (Krox-20), Egr-3, c-jun, jun B, jun D, c-Fos, Fos B, Fra-1, Fra-2, CREB, ATF 4, SCIP and Sox-10 in cultured Schwann cells. PROG triggered a quick (visible as soon as 15 min), strong (6 to 18-fold) and transient (1-2 h) stimulation of Egr-1, Egr-2, Egr-3 and Fos B genes expression. Expression of other genes remained unaffected by PROG treatment. The same expression pattern was obtained in the MSC 80 line (mouse Schwann cells), but not in the NIH-3T3 and CHO lines. Estradiol and testosterone induced different patterns of transcription factor gene activation in Schwann cells. Serum stimulated all genes activated by PROG in addition c-fos, fra-1 and fra-2. The PROG effects were blocked by Actinomycin D and by RU 486. This suggests that the activation of these genes occurs at the transcriptional level via the interaction of the hormone with its cognate receptor. Thus, PROG can regulate Schwann cell functions and differentiation by transiently activating specific transcription factors.

Rapid effects of triiodothyronine on immediate-early gene expression in Schwann cells.

In the peripheral nervous system, triiodothyronine (T3) plays an important role in the development and regeneration of nerve fibers and in myelin formation. However, the target genes of T3 in peripheral nerves remain to be identified. We investigated whether T3 activated genes of transcription factors in Schwann cells. Expression of egr-1 (krox-24), egr-2 (krox-20), egr-3, c-jun, junB, c-fos, fosB, fra-1, fra-2, and CREB genes was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in Schwann cells isolated from neonatal rat sciatic nerves and in the cell lines MSC-80 (mouse Schwann cells), NIH-3T3 (mouse fibroblasts), and CHO (Chinese hamster ovary cells). Some of these transcription factors have been shown to be involved in Schwann cell differentiation. T3 triggered a rapid (15-30 min), transient (1-2-h) and strong (6- to 15-fold) stimulation of Egr-1, Egr-2, Egr-3, Jun B, c-Fos, and Fos B mRNA expression in Schwann cells. In contrast, expression of c-Jun, Fra-1, Fra-2, and CREB mRNA was not affected by T3. The stimulatory effects of T3 could be abolished by adding actinomycin D. T3 triggered the same pattern of gene stimulation in the mouse Schwann cell line MSC80, but not in the NIH-3T3 and CHO cell lines. Serum activated all the genes that responded to T3 and in addition fra-1 and fra-2, but not c-jun and CREB. Immunoblotting showed that the increase in Egr-1 and c-Fos mRNA levels was accompanied by an increase in the corresponding proteins. In addition, shifts of the protein bands indicated a posttranslational modification of the two proteins. These effects of T3 are likely to be mediated by the intracellular T3 receptor, as the D-isomer RT3 and T0, which do not bind to T3 receptors, proved ineffective. The present data suggested that T3 may regulate Schwann cell functions and differentiation by transiently activating the expression of specific transcription factors.

Expression of the microphthalmia-associated basic helix-loop-helix leucine zipper transcription factor Mi in avian neuroretina cells induces a pigmented phenotype.

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.

Overexpression of A-myb induces basic fibroblast growth factor-dependent proliferation of chicken neuroretina cells.

A-Myb behaves similarly to c-Myb in chicken neuroretina cells in its ability to induce fibroblast-like differentiation, to promote growth in the presence of basic fibroblast growth factor (bFGF), and to induce Pax-6 and mim-1 expression. The one difference between c-Myb and A-Myb in these cells is that the former but not the latter protein causes colony formation in soft agar in the presence of bFGF.

The homeobox-containing Engrailed (En-1) product down-regulates the expression of Pax-6 through a DNA binding-independent mechanism.

By in situ hybridization of quail neuroretinas, we observed that Engrailed (En-1) is expressed both in the ganglionic and the amacrine cell layers, similarly to Pax-6. Because we observed a decrease of Pax-6 expression in the neuroretina of hatched animals, we studied the effect of the chicken En-1 and En-2 proteins on Pax-6 expression. En-1 and to some extent En-2 were able to repress the basal and the p46Pax-6-activated transcription from the two Pax-6 promoters. Infection of retinal pigmented epithelium by a virus encoding the En-1 protein repressed the endogenous Pax-6, and a similar effect was observed with a homeodomain-deleted En-1. In vitro interaction indicates that En proteins are able to interact with the p46Pax-6 through the paired domain. This interaction negatively regulates the DNA-binding properties of the p46Pax-6. These results suggest an interplay between En-1 and Pax-6 during the central nervous system development and indicate that En-1 may be a negative regulator of Pax-6.

Hydrocortisone modulates the expression of c-ets-1 and 72 kDa type IV collagenase in chicken dermis during early feather morphogenesis.

At the onset of chicken feather morphogenesis, dermal cells migrate along bundles of collagen fibers to colonize areas where bud outgrowth takes place. Chicken embryos treated with hydrocortisone during the critical phase of dermal rearrangement show featherless skin areas in which the dermis exhibits an increase of interstitial collagen. We had previously demonstrated that c-ets-1 is a nuclear transcription factor expressed in the dermis at the beginning of feather morphogenesis. Here we study, by in situ mRNA hybridization, the expression of c-ets-1 in the dermis of chicken embryos treated with hydrocortisone. We found that, among the two distinct products (p54 and p68) encoded by the chicken c-ets-1, the expression of the p68 product increased while expression of p54 decreased after hydrocortisone treatment. Since Ets-1 regulates matrix-metalloproteinases genes, we analyzed the expression of the 72 kDa type IV collagenase in both normal and hydrocortisone-treated embryos. We demonstrated that 72 kDa type IV collagenase mRNA expression decreased in the dermis after hydrocortisone treatment and that its expression correlated with that of p54c-ets-1. Taken together, these results indicate that hydrocortisone modulates c-ets-1 expression. In addition, they raise the interesting possibility that c-ets-1 might be involved in an altered pattern of feather development mediated by the accumulation of collagen due to a decrease in collagenase activities.

Characterization of a new melanocyte-specific gene (QNR-71) expressed in v-myc-transformed quail neuroretina.

Quail neuroretina cells (QNR) infected with the v-myc-expressing retrovirus MC29 become pigmented after several passages in vitro. After differential screening of a cDNA library constructed from these cells, we have isolated a cDNA clone (QNR-71) which identifies an RNA expressed only in the pigmented layer of the retina and in the epidermis. This gene can also be induced in other cell types transformed by MC29, suggesting that QNR-71 may be regulated by the v-myc protein. Sequence analysis showed that the QNR-71 cDNA exhibits stretches of homologies with melanosomal proteins encoding genes. From bacterially expressed QNR-71 peptides we obtained rabbit antisera able to specifically recognize two proteins of 95 and 100 kDa in pigmented retinal cells, but not in the neuroretina. To study the regulation of QNR-71, we used promoter fragments linked to the CAT reporter gene, in transient co-expression assay. We observed an increase in CAT expression with a c-MYC and microphtalmia (mi) expression vectors. Both MYC and mi activate the QNR-71 promoter through direct binding to a CATGTG site present in the promoter fragment.

Differential expression between PAX-6 and EN proteins in medulloblastoma.

A collection of 28 medulloblastomas was analyzed for expression of the developmental control genes PAX-6 and EN by immunohistochemical staining. Sixteen medulloblastomas expressed both EN and PAX-6 but, when differentiation could be assessed in the positive areas, PAX-6 is expressed in the less differentiated cells. Since Drosophila en encodes a negative regulator, we overexpressed the chicken en-1 in retinal pigmented epithelium cells. This resulted in Pax-6 down regulation. These results suggest a regulatory loop between PAX-6 and EN, two molecular markers of medulloblastoma.

Quail Pax-6 (Pax-QNR) mRNAs are expressed from two promoters used differentially during retina development and neuronal differentiation.

During investigations on the regulation of the Pax-6 gene, we characterized a cDNA from quail neuroretina showing a 5' untranslated region distinct from that previously described and initiated from an internal promoter. Using RNase protection and primer extension mapping, we localized this second quail Pax-6 promoter, termed P1. As reported for the already described P0 promoter, P1 was also transactivated in vitro by the p46Pax-QNR protein. RNase protection assays performed with quail neuroretina RNA showed that P1-initiated mRNAs were detected before the P0-initiated mRNAs, remained constant up to embryonic day 8, and decreased slowly thereafter whereas, P0-initiated mRNAs accumulated up to embryonic day 8. In contrast, quail retinal pigmented epithelium expressed only the P1-initiated mRNAs. Transformation of these cells by the v-myc oncogene induced neuronal traits in the culture, which thereafter, in addition to the P1-initiated mRNAs, expressed Pax-QNR from the P0 promoter. These results suggest that expression of the quail Pax-6 gene is under the control of different regulators through alternate promoters, P0 being activated at the onset of neuronal differentiation.

C-Myb acts as transcriptional activator of the quail PAX6 (PAX-QNR) promoter through two different mechanisms.

To understand the regulation of the Pax-6 gene, which plays an important role in eye development, we have characterized the promoter region of the quail Pax-6(Pax-QNR) gene. In addition to TATA and CAAT boxes, sequence analysis revealed several putative cis-regulatory elements among which three myb-responsive elements (MRE). C-myb encodes a nuclear, DNA-binding phosphoprotein that functions as transcriptional regulator. Co-transfection in quail embryo cells of the Pax-QNR/pax-6 promoter with a vector expressing the 75 kDa c-myb protein resulted in an increase in Pax-QNR promoter activity. By footprinting experiments we identified multiple binding sites for the myb protein within the promoter region. Protein containing the myb DNA-binding domain fused to the VP16-transactivation domain was fully efficient in Pax-QNR promoter transactivation, demonstrating that myb can transactivate through a direct binding on DNA. However, a myb truncated protein devoid of DNA-binding domain was also able to transactivate the Pax-QNR promoter. These results show that this promoter can be transactivated by the myb protein directly as well as indirectly. Finally we show by in situ hybridization that c-myb is strongly expressed in the developing neuroretina, simultaneously with Pax-QNR. These observations suggest that the c-myb protein may be a regulator of Pax-QNR/pax-6.

Pax-QNR/Pax-6, a paired box- and homeobox-containing gene expressed in neurons, is also expressed in pancreatic endocrine cells.

After differential screening of a cDNA library constructed from quail neuroretina cells infected with the v-myc containing avian retrovirus MC29, we have isolated a cDNA clone Pax-QNR, homologous to the murine Pax6 which is mutated in the autosomal dominant mutation small eye (Sey) of the mouse and aniridia in man. Here we report the characterization of Pax-QNR/Pax-6 expression in the chicken, quail, and mouse pancreas. In situ hybridization performed with E3 chick embryos demonstrated that, in addition to the documented expression of Pax-QNR/Pax-6 in the neural tube, this gene is also expressed in the pancreatic bud. This expression is later restricted to discrete parts of the organ. From bacterially expressed Pax-QNR peptides we obtained rabbit antisera (paired domain, serum 11; domain between paired and homeo, serum 12; homeodomain, serum 13; and carboxyl-terminal part, serum 14) capable of specifically recognizing Pax-QNR/Pax-6 proteins (48, 46 kilodaltons) in cell lines derived from alpha- and beta-pancreatic cells, but not from exocrine derived cell lines. We conclude that Pax-QNR/Pax-6 represents another gene expressed both in the endocrine pancreas and neuro-ectodermic tissues.

Hydrocortisone perturbs the cell proliferation pattern during feather morphogenesis: evidence for disturbance of cephalocaudal orientation.

In this study, we have monitored the spatial distribution of S-phase cells during successive stages of normal feather morphogenesis using the specific marker BrdU. We also disturbed the development program by administration of hydrocortisone on the chorioallantoic membrane of 6.5-day chick embryos and examined the resulting pattern of BrdU incorporation. Our results show that a specific spatio-temporal pattern of cell proliferation occurs during successive stages of feather development and that this pattern accounts for the growth of feather buds according to the cephalocaudal orientation. Our experimental analysis showed that the stage-dependent alteration of feather morphogenesis (as shown by Züst, Ann. Embryol. Morphogen. 4, 1971 and confirmed by Démarchez et al., Dev. Biol. 106, 1984), is based on a stage-dependent alteration of the proliferation pattern in the epidermis. Forty-eight hours after treatment, non-induced epidermis ceases DNA synthesis and is unable to form placodes. Induced epidermis at the placodal and dermal condensation stages fails to produce the cohorts of S-phase cells responsible for the caudal outgrowth and the slanting shape of the buds. These young buds display anarchic proliferation in the whole epidermis possibly resulting in the appearance of "curly" feathers. Together, these results show the importance of the spatial pattern of ectodermal and mesodermal cell proliferation during the normal feather morphogenesis. Moreover, they corroborate the particular role of epidermis both in the establishment of feather rudiments and in the cephalocaudal orientation of the feathers.