Oct-6 transcriptional factor a possible biomarker for leprosy diagnosis.

Leprosy is an infectious disease caused by Mycobacterium leprae that affects the skin and nerves. The nerve damage in leprosy may be related to alterations in transcriptional factors, such as Krox-20, Oct-6, Sox-10. Thirty skin biopsies in leprosy patients and 15 non-leprosy skin biopsies were evaluated using RT-qPCR to assess Krox-20, Oct-6, and Sox-10 and these data was related with S-100 immunohistochemistry. Changes in gene expression were observed in the skin and dermal nerves of leprosy patients in Oct-6 and Sox-10. When comparing Oct-6 with S-100 IHC as diagnostic tests for leprosy, Oct-6 showed a sensitivity of 73.3%, and specificity of 100%, while S-100 IHC showed a sensitivity of 96.6% and specificity of 100%. Our data suggest Oct-6 could be an auxiliary biomarker specific to detecting changes in dermal nerves in leprosy and thus useful to health workers and pathologists with no expertise to observe nerve injuries in leprosy.

Two Signal Recognition Particle Sequences Are Present in the Amino-Terminal Domain of the C-Tailed Protein SciP.

During their synthesis, the C-tailed membrane proteins expose the membrane-spanning segment late from the ribosome and consequently can insert into the membrane only posttranslationally. However, the C-tailed type 6 secretion system (T6SS) component SciP uses the bacterial signal recognition particle (SRP) system for membrane targeting, which operates cotranslationally. Analysis of possible sequence regions in the amino-terminal part of the protein revealed two candidates that were then tested for whether they function as SRP signal peptides. Both sequences were tested positive as synthetic peptides for binding to SRP. In addition, purified ribosomes with stalled nascent chains exposing either sequence were capable of binding to SRP and SRP-FtsY complexes with high affinity. Together, the data suggest that both peptides can serve as an SRP signal sequence promoting an early membrane targeting of SciP during its synthesis. Like observed for multispanning membrane proteins, the two cytoplasmic SRP signal sequences of SciP may also facilitate a retargeting event, making the targeting more efficient.IMPORTANCE C-tail proteins are anchored in the inner membrane with a transmembrane segment at the C terminus in an N-in/C-out topology. Due to this topology, membrane insertion occurs only posttranslationally. Nevertheless, the C-tail-anchored protein SciP is targeted cotranslationally by SRP. We report here that two amino-terminal hydrophobic stretches in SciP are individually recognized by SRP and target the nascent protein to FtsY. The presence of two signal sequences may enable a retargeting mechanism, as already observed for multispanning membrane proteins, to make the posttranslational insertion of SciP by YidC more efficient.

Mir-29b Mediates the Neural Tube versus Neural Crest Fate Decision during Embryonic Stem Cell Neural Differentiation.

During gastrulation, the neuroectoderm cells form the neural tube and neural crest. The nervous system contains significantly more microRNAs than other tissues, but the role of microRNAs in controlling the differentiation of neuroectodermal cells into neural tube epithelial (NTE) cells and neural crest cells (NCCs) remains unknown. Using embryonic stem cell (ESC) neural differentiation systems, we found that miR-29b was upregulated in NTE cells and downregulated in NCCs. MiR-29b promoted the differentiation of ESCs into NTE cells and inhibited their differentiation into NCCs. Accordingly, the inhibition of miR-29b significantly inhibited the differentiation of NTE cells. A mechanistic study revealed that miR-29b targets DNA methyltransferase 3a (Dnmt3a) to regulate neural differentiation. Moreover, miR-29b mediated the function of Pou3f1, a critical neural transcription factor. Therefore, our study showed that the Pou3f1-miR-29b-Dnmt3a regulatory axis was active at the initial stage of neural differentiation and regulated the determination of cell fate.

Dysregulation of the SIRT1/OCT6 Axis Contributes to Environmental Stress-Induced Neural Induction Defects.

Environmental stresses are increasingly acknowledged as core causes of abnormal neural induction leading to neural tube defects (NTDs). However, the mechanism responsible for environmental stress-triggered neural induction defects remains unknown. Here, we report that a spectrum of environmental stresses, including oxidative stress, starvation, and DNA damage, profoundly activate SIRT1, an NAD+-dependent lysine deacetylase. Both mouse embryos and in vitro differentiated embryonic stem cells (ESCs) demonstrated a negative correlation between the expression of SIRT1 and that of OCT6, a key neural fate inducer. Activated SIRT1 radically deacetylates OCT6, triggers an OCT6 ubiquitination/degradation cascade, and consequently increases the incidence of NTD-like phenotypes in mice or hinders neural induction in both human and mouse ESCs. Together, our results suggest that early exposure to environmental stresses results in the dysregulation of the SIRT1/OCT6 axis and increases the risk of NTDs.

The sodium channel isoform transition at developing nodes of Ranvier in the peripheral nervous system: dependence on a Genetic program and myelination-induced cluster formation.

Among sodium channel isoforms, Nav 1.6 is selectively expressed at nodes of Ranvier in both the CNS and the PNS. However, non-Nav 1.6 isoforms such as Nav 1.2 are also present at the CNS nodes in early development but gradually diminish later. It has been proposed that myelination is part of a glia-neuron signaling mechanism that produces this change in nodal isoform expression. The present study used isoform-specific antibodies to demonstrate that, in the PNS, four other neuronal sodium channel isoforms were also clustered at nodes in early development but eventually disappeared during maturation. To study possible roles of myelination in such transitions, we investigated the nodal expression of selected isoforms in the sciatic nerve of the transgenic mouse Oct6(ΔSCE/ßgeo) , whose PNS myelination is delayed in the first postnatal week but eventually resumes. We found that delayed myelination retarded the formation of nodal channel clusters and altered the expression-elimination patterns of sodium channel isoforms, resulting in significantly reduced expression levels of non-Nav 1.6 isoforms in such delayed nodes. However, delayed myelination did not significantly affect the gene expression, protein synthesis, or axonal trafficking of any isoform studied. Rather, we found evidence for a developmentally programmed increase in neuronal Nav 1.6 expression with constant or decreasing neuronal expression of other isoforms that were unaffected by delayed myelination. Thus our results suggest that, in the developmental isoform switch of the PNS, myelination does not play a signaling role as that proposed for the CNS but rather serves only to form nodal clusters from existing isoform pools.

Transcription initiation factor IIB involves in Schwann cell differentiation after rat sciatic nerve crush.

Transcription Initiation Factor IIB (TFIIB), as a general transcription factor, plays an essential role in preinitiation complex assembly and transcription initiation by recruiting RNA polymerase II to the promoter. However, its distribution and function in peripheral system lesion and repair were still unknown. Here, we investigated the spatiotemporal expression of TFIIB in an acute sciatic nerve crush model in adult rats. Western blot analysis revealed that TFIIB was expressed in normal sciatic nerve. It gradually increased, reached a peak at the seventh day after crush, and then returned to the normal level at 4 weeks. We observed that TFIIB expressed mainly increased in Schwann cells and co-localized with Oct-6. In vitro, we induced Schwann cell differentiation with cyclic adenosine monophosphate (cAMP) and found that TFIIB expression was increased in the differentiated process. TFIIB-specific siRNA inhibited cAMP-induced Schwann cell morphological change and the expression of P0. Collectively, we hypothesized peripheral nerve crush-induced upregulation of TFIIB in the sciatic nerve was associated with Schwann cell differentiation.

Changes in the BAG1 expression of Schwann cells after sciatic nerve crush.

Bcl-2-associated athanogene-1 (BAG1), a co-chaperone for Hsp70/Hsc70, is a multifunctional protein, which has been shown to suppress apoptosis and enhance neuronal differentiation. However, the expression and roles of BAG1 in peripheral system lesions and repair are still unknown. In this study, we investigated the dynamic changes in BAG1 expression in an acute sciatic nerve crush model in adult rats. Western blot analysis revealed that BAG1 was expressed in normal sciatic nerves. BAG1 expression increased progressively after sciatic nerve crush, reached a peak 2 weeks post-injury, and then returned to the normal level 4 weeks post-injury. Spatially, we observed that BAG1 was mainly expressed in Schwann cells and that BAG1 expression increased in Schwann cells after injury. In vitro, we found that BAG1 expression increased during the cyclic adenosine monophosphate (cAMP)-induced Schwann cell differentiation process. BAG1-specific siRNA inhibited cAMP-induced Schwann cell differentiation. In conclusion, we speculated that BAG1 was upregulated in the sciatic nerve after crush, which was associated with Schwann cell differentiation.

The role of C-terminal binding protein 2 in Schwann cell differentiation after sciatic nerve crush.

C-terminal binding protein 2 (CtBP2), as a transcriptional repressor, plays an essential role in development and tumorigenesis. However, its distribution and function in peripheral system lesion and repair are still unknown. Here, we investigated the spatiotemporal expression of CtBP2 in rat sciatic nerve crush model. Western blot analysis revealed that CtBP2 was expressed in normal sciatic nerve. It gradually decreased, reached minimal levels at 7 days after crush, and then returned to the normal level at 4 weeks. We observed that CtBP2 is mainly expressed in Schwann cells (SCs). In vitro, we induced SC differentiation via cyclic adenosine monophosphate (cAMP) and found that CtBP2 expression was downregulated during the process of differentiation. CtBP2-specific siRNA inhibited the cAMP-induced expression of the immature SC marker P75(NTR), and exogenous CtBP2 expression upregulated the expression of P75(NTR). Taken together, we hypothesized that peripheral nerve crush-induced downregulation of CtBP2 in the sciatic nerve was associated with SC differentiation, and CtBP2 likely played an important role in peripheral nerve injury and regeneration.

Peripheral axons of the adult zebrafish maxillary barbel extensively remyelinate during sensory appendage regeneration.

Myelination is a cellular adaptation allowing rapid conduction along axons. We have investigated peripheral axons of the zebrafish maxillary barbel (ZMB), an optically clear sensory appendage. Each barbel carries taste buds, solitary chemosensory cells, and epithelial nerve endings, all of which regenerate after amputation (LeClair and Topczewski [2010] PLoS One 5:e8737). The ZMB contains axons from the facial nerve; however, myelination within the barbel itself has not been established. Transcripts of myelin basic protein (mbp) are expressed in normal and regenerating adult barbels, indicating activity in both maintenance and repair. Myelin was confirmed in situ by using toluidine blue, an anti-MBP antibody, and transmission electron microscopy (TEM). The adult ZMB contains ∼180 small-diameter axons (<2 µm), approximately 60% of which are myelinated. Developmental myelination was observed via whole-mount immunohistochemistry 4-6 weeks postfertilization, showing myelin sheaths lagging behind growing axons. Early-regenerating axons (10 days postsurgery), having no or few myelin layers, were disorganized within a fibroblast-rich collagenous scar. Twenty-eight days postsurgery, barbel axons had grown out several millimeters and were organized with compact myelin sheaths. Fiber types and axon areas were similar between normal and regenerated tissue; within 4 weeks, regenerating axons restored ∼85% of normal myelin thickness. Regenerating barbels express multiple promyelinating transcription factors (sox10, oct6 = pou3f1; krox20a/b = egr2a/b) typical of Schwann cells. These observations extend our understanding of the zebrafish peripheral nervous system within a little-studied sensory appendage. The accessible ZMB provides a novel context for studying axon regeneration, Schwann cell migration, and remyelination in a model vertebrate.

Gbx2 directly restricts Otx2 expression to forebrain and midbrain, competing with class III POU factors.

Otx2 plays essential roles in rostral brain development, and its counteraction with Gbx2 has been suggested to determine the midbrain-hindbrain boundary (MHB) in vertebrates. We previously identified the FM enhancer that is conserved among vertebrates and drives Otx2 transcription in forebrain/midbrain from the early somite stage. In this study, we found that the POU homeodomain of class III POU factors (Brn1, Brn2, Brn4, and Oct6) associates with noncanonical target sequence TAATTA in the FM enhancer. MicroRNA-mediated knockdown of Brn2 and Oct6 diminished the FM enhancer activity in anterior neural progenitor cells (NPCs) differentiated from P19 cells. The class III POU factors associate with the FM enhancer in forebrain and midbrain but not in hindbrain. We also demonstrated that the Gbx2 homeodomain recognizes the same target TAATTA in the FM enhancer, and Gbx2 associates with the FM enhancer in hindbrain. Gbx2 misexpression in the anterior NPCs repressed the FM enhancer activity and inhibited Brn2 association with the enhancer, whereas Gbx2 knockdown caused ectopic Brn2 association in the posterior NPCs. These results suggest that class III POU factors and Gbx2 share the same target site, TAATTA, in the FM enhancer and that their region-specific binding restricts Otx2 expression at the MHB.

Mitogen-activated protein kinase p38 regulates Krox-20 to direct Schwann cell differentiation and peripheral myelination.

We previously reported that addition of extracellular matrix (ECM) extracts to rat Schwann cell-dorsal root ganglion neuron (DRGN) co-cultures activated mitogen-activated protein kinase (MAPK) p38, whereas inhibition blocked myelination. Here, we used p38 pharmacological inhibitors and gene silencing to assess their effects on downstream kinases and key transcription factors. We show that p38α regulates expression of the master transcription factor, Krox-20, required for the onset of myelination in Schwann cell-DRGNs, as assessed by immunocytochemistry and qRT-PCR. p38 activity is also required for the expression of the cell cycle inhibitor p27(kip1) , associated with Schwann cell differentiation. Three potential effectors of p38 were explored: MAPK-activated protein kinase-2 (MK2), mitogen and stress-activated protein kinase-1 (MSK-1), and the transcription factor cAMP response element-binding protein (CREB). Inhibition of MK2 with CMPD1 or gene knockdown with siRNAs reduced numbers of Krox-20-positive Schwann cells and expression of myelin proteins MBP and MAG. ECM activated CREB and increased Krox-20 expression, whereas CREB1 gene silencing reduced Krox-20. Furthermore, two nonselective inhibitors of MSK-1 (H89 and R0-318820) decreased ECM-induced CREB phosphorylation and, similar to anti-MSK-1 siRNAs, reduced Krox-20-positive cells. In addition, p38 modulated the expression of two transcription factors involved in the regulation of Krox-20 [suppressed cAMP-inducible protein (SCIP) and Sox10], but not Sox2, an antagonist of Krox-20. Collectively, our results show that p38 primarily directs Schwann cell differentiation and peripheral myelination by regulating Krox-20 expression through its downstream effectors, MK2 and MSK-1/CREB, and transcription factors SCIP and Sox10.

Regulation of Schwann cell differentiation and proliferation by the Pax-3 transcription factor.

Pax-3 is a paired domain transcription factor that plays many roles during vertebrate development. In the Schwann cell lineage, Pax-3 is expressed at an early stage in Schwann cells precursors of the embryonic nerve, is maintained in the nonmyelinating cells of the adult nerve, and is upregulated in Schwann cells after peripheral nerve injury. Consistent with this expression pattern, Pax-3 has previously been shown to play a role in repressing the expression of the myelin basic protein gene in Schwann cells. We have studied the role of Pax-3 in Schwann cells and have found that it controls not only the regulation of cell differentiation but also the survival and proliferation of Schwann cells. Pax-3 expression blocks both the induction of Oct-6 and Krox-20 (K20) by cyclic AMP and completely inhibits the ability of K20, the physiological regulator of myelination in the peripheral nervous system, to induce myelin gene expression in Schwann cells. In contrast to other inhibitors of myelination, we find that Pax-3 represses myelin gene expression in a c-Jun-independent manner. In addition to this, we find that Pax-3 expression alone is sufficient to inhibit the induction of apoptosis by TGFß1 in Schwann cells. Expression of Pax-3 is also sufficient to induce the proliferation of Schwann cells in the absence of added growth factors and to reverse K20-induced exit from the cell cycle. These findings indicate new roles for the Pax-3 transcription factor in controlling the differentiation and proliferation of Schwann cells during development and after peripheral nerve injury.

Gpr126 is essential for peripheral nerve development and myelination in mammals.

In peripheral nerves, Schwann cells form the myelin sheath that insulates axons and allows rapid propagation of action potentials. Although a number of regulators of Schwann cell development are known, the signaling pathways that control myelination are incompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nerve development in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, and expression of differentiated Schwann cell markers, including Pou3f1, Egr2, myelin protein zero and myelin basic protein, is reduced. Ultrastructural studies of Gpr126-/- mice showed that axonal sorting by Schwann cells is delayed, Remak bundles (non-myelinating Schwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinating stage. Additionally, ectopic perineurial fibroblasts form aberrant fascicles throughout the endoneurium of the mutant sciatic nerve. This analysis shows that Gpr126 is required for Schwann cell myelination in mammals, and defines new roles for Gpr126 in axonal sorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.

Peripheral nerve pathology, including aberrant Schwann cell differentiation, is ameliorated by doxycycline in a laminin-α2-deficient mouse model of congenital muscular dystrophy.

The most common form of childhood congenital muscular dystrophy, Type 1A (MDC1A), is caused by mutations in the human LAMA2 gene that encodes the laminin-α2 subunit. In addition to skeletal muscle deficits, MDC1A patients typically show a loss of peripheral nerve function. To identify the mechanisms underlying this loss of nerve function, we have examined pathology and cell differentiation in sciatic nerves and ventral roots of the laminin-α2-deficient (Lama2(-/-)) mice, which are models for MDC1A. We found that, compared with wild-type, sciatic nerves of Lama2(-/-) mice had a significant increase in both proliferating (Ki67+) cells and premyelinating (Oct6+) Schwann cells, but also had a significant decrease in both immature/non-myelinating [glial fibrillary acidic protein (GFAP)(+)] and myelinating (Krox20+) Schwann cells. To extend our previous work in which we found that doxycycline, which has multiple effects on mammalian cells, improves motor behavior and more than doubles the median life-span of Lama2(-/-) mice, we also determined how nerve pathology was affected by doxycycline treatment. We found that myelinating (Krox20+) Schwann cells were significantly increased in doxycycline-treated compared with untreated sciatic nerves. In addition, doxycycline-treated peripheral nerves had significantly less pathology as measured by assays such as amount of unmyelinated or disorganized axons. This study thus identified aberrant proliferation and differentiation of Schwann cells as key components of pathogenesis in peripheral nerves and provided proof-of-concept that pharmaceutical therapy can be of potential benefit for peripheral nerve dysfunction in MDC1A.

Aberrant CpG island hypermethylation and down-regulation of Oct-6 mRNA expression in human hepatocellular carcinoma.

BACKGROUND: Aberrant CpG island hypermethylation is a major epigenetic mechanism that can inactivate the transcription of cancer-related genes. PURPOSE: This study aimed to investigate whether Oct-6 transcription was regulated by CpG island methylation in hepatocellular carcinoma (HCC). METHODS: Quantitative real-time PCR and the MassARRAY platform (Sequenom) were employed in 38 HCC tissues samples and four cell lines. RESULTS: The levels of Oct-6 mRNA were decreased by more than twofold in 31 of 38 tumor tissues compared to that of adjacent non-cancerous tissues. Among the 31 tumor tissues with lower levels of Oct-6 mRNA, 17 tumor tissues also had higher methylation levels in Oct-6 CpG island. Based on these results, we hypothesized that CpG island hypermethylation may down-regulate Oct-6 mRNA expression in HCC. To confirm this hypothesis, we also analyzed the changes in Oct-6 mRNA expression and CpG island methylation in four HCC cell lines (Huh7, Bel-7402, HepG2 and SMMC-7721) after treatment with 0.1, 0.5 and 2.5 µM 5-Aza-2-deoxycytidine (5-Aza-CdR), a demethylating agent. The results demonstrated that the CpG island methylation levels decreased and Oct-6 mRNA levels increased in a dose-dependent manner in both Huh7 and Bel7402 cells, but there were only slight changes in HepG2 cell. Interestingly, there were no significant alterations of Oct-6 mRNA levels observed in SMMC7721 cell; although lower levels of CpG island methylation were detected after treatment with 5-Aza-CdR. CONCLUSIONS: Our study shows that CpG island hypermethylation contributes to down-regulation of Oct-6 mRNA expression in HCC.

Octamer-binding factor 6 (Oct-6/Pou3f1) is induced by interferon and contributes to dsRNA-mediated transcriptional responses.

BACKGROUND: Octamer-binding factor 6 (Oct-6, Pou3f1, SCIP, Tst-1) is a transcription factor of the Pit-Oct-Unc (POU) family. POU proteins regulate key developmental processes and have been identified from a diverse range of species. Oct-6 expression is described to be confined to the developing brain, Schwann cells, oligodendrocyte precursors, testes, and skin. Its function is primarily characterised in Schwann cells, where it is required for correctly timed transition to the myelinating state. In the present study, we report that Oct-6 is an interferon (IFN)-inducible protein and show for the first time expression in murine fibroblasts and macrophages. RESULTS: Oct-6 was induced by type I and type II IFN, but not by interleukin-6. Induction of Oct-6 after IFNbeta treatment was mainly dependent on signal transducer and activator of transcription 1 (Stat1) and partially on tyrosine kinase 2 (Tyk2). Chromatin immunopreciptitation experiments revealed binding of Stat1 to the Oct-6 promoter in a region around 500 bp upstream of the transcription start site, a region different from the downstream regulatory element involved in Schwann cell-specific Oct-6 expression. Oct-6 was also induced by dsRNA treatment and during viral infections, in both cases via autocrine/paracrine actions of IFNalpha/beta. Using microarray and RT-qPCR, we furthermore show that Oct-6 is involved in the regulation of transcriptional responses to dsRNA, in particular in the gene regulation of serine/threonine protein kinase 40 (Stk40) and U7 snRNA-associated Sm-like protein Lsm10 (Lsm10). CONCLUSION: Our data show that Oct-6 expression is not as restricted as previously assumed. Induction of Oct-6 by IFNs and viruses in at least two different cell types, and involvement of Oct-6 in gene regulation after dsRNA treatment, suggest novel functions of Oct-6 in innate immune responses.

NFIA controls telencephalic progenitor cell differentiation through repression of the Notch effector Hes1.

The balance between self-renewal and differentiation of neural progenitor cells is an absolute requirement for the correct formation of the nervous system. Much is known about both the pathways involved in progenitor cell self-renewal, such as Notch signaling, and the expression of genes that initiate progenitor differentiation. However, whether these fundamental processes are mechanistically linked, and specifically how repression of progenitor self-renewal pathways occurs, is poorly understood. Nuclear factor I A (Nfia), a gene known to regulate spinal cord and neocortical development, has recently been implicated as acting downstream of Notch to initiate the expression of astrocyte-specific genes within the cortex. Here we demonstrate that, in addition to activating the expression of astrocyte-specific genes, Nfia also downregulates the activity of the Notch signaling pathway via repression of the key Notch effector Hes1. These data provide a significant conceptual advance in our understanding of neural progenitor differentiation, revealing that a single transcription factor can control both the activation of differentiation genes and the repression of the self-renewal genes, thereby acting as a pivotal regulator of the balance between progenitor and differentiated cell states.

A G protein-coupled receptor is essential for Schwann cells to initiate myelination.

The myelin sheath allows axons to conduct action potentials rapidly in the vertebrate nervous system. Axonal signals activate expression of specific transcription factors, including Oct6 and Krox20, that initiate myelination in Schwann cells. Elevation of cyclic adenosine monophosphate (cAMP) can mimic axonal contact in vitro, but the mechanisms that regulate cAMP levels in vivo are unknown. Using mutational analysis in zebrafish, we found that the G protein-coupled receptor Gpr126 is required autonomously in Schwann cells for myelination. In gpr126 mutants, Schwann cells failed to express oct6 and krox20 and were arrested at the promyelinating stage. Elevation of cAMP in gpr126 mutants, but not krox20 mutants, could restore myelination. We propose that Gpr126 drives the differentiation of promyelinating Schwann cells by elevating cAMP levels, thereby triggering Oct6 expression and myelination.

Replacement of related POU transcription factors leads to severe defects in mouse forebrain development.

Related transcription factors of the POU protein family show extensive overlap of expression in vivo and exhibit very similar biochemical properties in vitro. To study functional equivalence of class III POU proteins in vivo, we exchanged the Oct-6 gene by Brn-1 in the mouse. Brn-1 can fully replace Oct-6 in Schwann cells and rescue peripheral nervous system development in these mice. The same mice, however, exhibit severe defects in forebrain development arguing that Oct-6 and Brn-1 are not functionally equivalent in the central nervous system. The cause of the observed forebrain phenotype is complex, but anteriorly expanded Wnt1 expression contributes. Oct-6 normally represses Wnt1 expression in the early diencephalon and replacement by Brn-1 as a weaker inhibitor is no longer sufficient to maintain the necessary level of repression in the mouse mutant. The extent of functional equivalence between related transcription factors is thus strongly dependent on the analyzed tissue.