Identification of Phox2b-regulated genes by expression profiling of cranial motoneuron precursors

<p>Abstract</p> <p>Background</p> <p>Branchiomotor neurons comprise an important class of cranial motor neurons that innervate the branchial-arch-derived muscles of the face, jaw and neck. They arise in the ventralmost progenitor domain of the rhombencephalon characterized by expression of the homeodomain transcription factors Nkx2.2 and Phox2b. Phox2b in particular plays a key role in the specification of branchiomotor neurons. In its absence, generic neuronal differentiation is defective in the progenitor domain and no branchiomotor neurons are produced. Conversely, ectopic expression of Phox2b in spinal regions of the neural tube promotes cell cycle exit and neuronal differentiation and, at the same time, induces genes and an axonal phenotype characteristic for branchiomotor neurons. How Phox2b exerts its pleiotropic functions, both as a proneural gene and a neuronal subtype determinant, has remained unknown.</p> <p>Results</p> <p>To gain further insights into the genetic program downstream of Phox2b, we searched for novel Phox2b-regulated genes by cDNA microarray analysis of facial branchiomotor neuron precursors from heterozygous and homozygous <it>Phox2b </it>mutant embryos. We selected for functional studies the genes encoding the axonal growth promoter Gap43, the Wnt antagonist Sfrp1 and the transcriptional regulator Sox13, which were not previously suspected to play roles downstream of <it>Phox2b </it>and whose expression was affected by <it>Phox2b </it>misexpression in the spinal cord. While <it>Gap43 </it>did not produce an obvious phenotype when overexpressed in the neural tube, <it>Sfrp1 </it>induced the interneuron marker Lhx1,5 and <it>Sox13 </it>inhibited neuronal differentiation. We then tested whether <it>Sfrp1 </it>and <it>Sox13</it>, which are down-regulated by Phox2b in the facial neuron precursors, would antagonize some aspects of <it>Phox2b </it>activity. Co-expression of <it>Sfrp1 </it>prevented <it>Phox2b </it>from repressing Lhx1,5 and alleviated the commissural axonal phenotype. When expressed together with <it>Sox13</it>, <it>Phox2b </it>was still able to promote cell cycle exit and neuronal differentiation, but the cells failed to relocate to the mantle layer and to extinguish the neural stem cell marker Sox2.</p> <p>Conclusion</p> <p>Our results suggest novel roles for <it>Sfrp1 </it>and <it>Sox13 </it>in neuronal subtype specification and generic neuronal differentiation, respectively, and indicate that down-regulation of <it>Sfrp1 </it>and <it>Sox13 </it>are essential aspects of the genetic program controlled by Phox2b in cranial motoneurons.</p

Die Bedeutung von SoxE-Transkriptionsfaktoren für die Entwicklung von Neuralleistenderivaten

In recent years, Sox transcription factors have come under increasing scrutiny as important regulators of diverse developmental processes. Members of the subgroup E of Sox proteins emerged as indispensable modulators through all stages of neural crest cell development. This study adressed the impact of the two SoxE proteins Sox10 and Sox8 on the development of adrenal chromaffin cells and myelinating Schwann cells as two prominent neural crest derivatives. During development of the adrenal gland, Sox10 and Sox8 were co-expressed in neural crest cells giving rise to the adrenal medulla. Whereas Sox8 expression declined with ongoing development, Sox10 persisted at least until birth. Sox10 deficiency in mice resulted in a complete loss of the chromaffin cell population due to increased apoptosis of neural crest cells prior to immigration into the adrenal anlagen. Sox10-deficient cells already lacked Sox8 expression before dying apoptotically, arguing that Sox10 regulates Sox8 in this cell type. In contrast, loss of Sox8 did not affect Sox10 expression. Because of functional redundancy and compensation by Sox10, loss of Sox8 was without phenotypic consequence in the corresponding mouse mutant. When Sox8 was expressed instead of Sox10, it rescued half of the chromaffin cell population indicating that Sox8 is partly capable of replacing Sox10 if its expression can be maintained in the absence of Sox10. The analysis of hypomorphic Sox10 mouse mutants additionally revealed the importance of an intact dimerization domain and the conserved K2 domain for the generation of chromaffin cells in appropriate numbers. The early loss of chromaffin cells in all mutants demonstrated the essential importance of Sox10 for survival at this developmental stage. In Schwann cells, Sox10 was already identified as a key factor for specification and was additionally implicated in terminal differentiation and myelination. Sox10 induced expression of Krox20 as the master regulator of myelination through activation of the MSE, an enhancer downstream of the Krox20 gene. Seven high-affinity binding sites existed for Sox10 in the MSE and contributed to Sox10-dependent transactivation. Sox10 functioned synergistically with Oct6. For synergism, the DNA-binding domain of Sox10 was necessary as were the transactivation domain and the conserved K2 domain. Oct6 on the other hand required the DNA-binding POU domain but not the transactivation domain. The importance of the K2 domain for induction of Krox20 was also demonstrated in vivo in a hypomorphic mouse mutant where Schwann cells expressed a Sox10 protein without K2 domain. In these mice, Schwann cells failed to initiate expression of Krox20 despite Oct6 expression. This proved that Sox10 is indispensable in Schwann cells for terminal differentiation and myelination.Transkriptionsfaktoren der Sox-Protein-Familie erlangen immer mehr Aufmerksamkeit als wichtige Regulatoren zahlreicher entwicklungsbiologischer Vorgänge. Mitglieder der Untergruppe E haben sich als unverzichtbar in allen Stadien der Neuralleistenentwicklung erwiesen. Das Augenmerk dieser Studie liegt auf der Bedeutung der beiden SoxEProteine Sox10 und Sox8 für die Entwicklung chromaffiner Zellen des Nebennierenmarks und myelinisierender Schwann Zellen des peripheren Nervensystems, deren Ursprung in der Neuralleiste liegt. In Vorläuferzellen des Nebennierenmarks waren Sox8 und Sox10 anfangs koexprimiert, bevor Sox8 im Gegensatz zu Sox10 nach und nach verschwand. Im Mausmodell hatte der Verlust von Sox10 ein vollständiges Fehlen chromaffiner Zellen zur Folge, die aufgrund erhöhter Apoptose schon vor der Einwanderung in die Nebennierenanlagen zugrunde gingen. Auch vor dem apoptotischen Absterben exprimierten Sox10-defiziente Zellen kein Sox8, was auf eine Regulation von Sox8 durch Sox10 hinweist. Im Gegensatz dazu hatte der Verlust von Sox8 weder Auswirkungen auf die Expression von Sox10 noch auf die sonstige Entwicklung chromaffiner Zellen. Daher konnte Sox10 durch seine funktionelle Redundanz den Verlust von Sox8 ausgleichen. Wurde Sox8 anstelle von Sox10 exprimiert, konnte es einen signifikanten Teil der chromaffinen Population aufrecht erhalten. Hier zeigte sich, dass Sox8 bei ausreichender Expression Sox10 teilweise ersetzen konnte. Bei der zusätzlichen Analyse hypomorpher Mausmutanten wurde die Bedeutung der Dimerisierungsdomäne von Sox10 und der konservierten K2-Domäne für die Entwicklung chromaffiner Zellen deutlich. Die Beobachtung, dass die Anzahl chromaffiner Zellen in allen untersuchten Mausmutanten schon sehr früh erniedrigt war, ließ auf die besondere Bedeutung von Sox10 für das Überleben dieser Zellen in frühen Entwicklungsstadien schließen. In Schwann Zellen ist Sox10 entscheidend an der Spezifizierung beteiligt, vermutlich aber auch an der terminalen Differenzierung und Myelinisierung. Sox10 induziert Krox20, den Haupt-Regulator der Myelinisierung im peripheren Nervensystem, indem es in Synergie mit Oct6 ein Enhancer-Element transaktivert, das die Schwann Zell-Expression von Krox20 steuert. Dieses Enhancer-Element enthält sieben hoch-affine Bindestellen für Sox10, die nachweislich an der Aktivierung beteiligt sind. Für die synergistische Aktivierung war die DNA-bindende HMG-Domäne von Sox10 ebenso von Nöten wie die Transaktivierungsdomäne und die konservierte K2-Domäne. Im Oct6-Protein war die DNA-bindende POU-Domäne von vergleichbarer Wichtigkeit, während die Transaktivierungsdomäne nicht vorhanden sein musste. Die Bedeutung der K2-Domäne zeigte sich auch in vivo anhand einer hypomorphen Mausmutante mit deletierter K2-Domäne. Infolge dieser Mutation wiesen Schwann Zellen trotz intakter Expression von Oct6 kein Krox20 auf. Somit konnte die unverzichtbare Funktion von Sox10 für die terminale Differenzierung myelinisierender Schwann Zellen bestätigt werden

Crazy Little Thing Called Sox—New Insights in Oligodendroglial Sox Protein Function

In the central nervous system, oligodendrocytes wrap axons with myelin sheaths, which is essential for rapid transfer of electric signals and their trophic support. In oligodendroglia, transcription factors of the Sox protein family are pivotal regulators of a variety of developmental processes. These include specification, proliferation, and migration of oligodendrocyte precursor cells as well as terminal differentiation to mature myelinating oligodendrocytes. Sox proteins are further affected in demyelinating diseases and are involved in remyelination following damage of the central nervous system. Here we summarize and discuss latest findings on transcriptional regulation of Sox proteins, their function, target genes, and interaction with other transcription factors and chromatin remodelers in oligodendroglia with physiological and pathophysiological relevance

MicroRNA miR‐204 regulates proliferation and differentiation of oligodendroglia in culture

Oligodendrocytes wrap and physically shield axons of the central nervous system with myelin sheaths, resulting in rapid signal transduction and accurate neuronal function. The complex oligodendroglial development from immature oligodendrocyte precursor cells (OPCs) to myelinating oligodendrocytes (OLs) is profoundly dependent on the activity of transcription factors of the Sox protein family. Target genes of the crucial regulator Sox10 have recently been expanded to microRNAs. Here, we report miR‐204 as a novel transcriptional target of Sox10. Regulatory regions of miR‐204 show responsiveness to and binding of Sox10 in reporter gene assays and electromobility shift assays. Once expressed, miR‐204 inhibits OPC proliferation and facilitates differentiation into OLs in the presence of Sox10 as evident from overexpression in primary rat and mouse oligodendroglial cultures. Phenotypes are at least in part caused by miR‐204‐dependent repression of the pro‐proliferative Ccnd2 and the differentiation inhibiting Sox4. These findings argue that the transcriptional activator Sox10 forces oligodendroglial cells to exit the cell cycle and start differentiation by gene inhibition via miR‐204 induction

Targeted Deletion of Sox10 by Wnt1-cre Defects Neuronal Migration and Projection in the Mouse Inner Ear

Sensory nerves of the brainstem are mostly composed of placode-derived neurons, neural crest-derived neurons and neural crest-derived Schwann cells. This mixed origin of cells has made it difficult to dissect interdependence for fiber guidance. Inner ear-derived neurons are known to connect to the brain after delayed loss of Schwann cells in ErbB2 mutants. However, the ErbB2 mutant related alterations in the ear and the brain compound interpretation of the data. We present here a new model to evaluate exclusively the effect of Schwann cell loss on inner ear innervation. Conditional deletion of the neural crest specific transcription factor, Sox10, using the rhombic lip/neural crest specific Wnt1-cre driver spares Sox10 expression in the ear. We confirm that neural crest-derived cells provide a stop signal for migrating spiral ganglion neurons. In the absence of Schwann cells, spiral ganglion neurons migrate into the center of the cochlea and even out of the ear toward the brain. Spiral ganglion neuron afferent processes reach the organ of Corti, but many afferent fibers bypass the organ of Corti to enter the lateral wall of the cochlea. In contrast to this peripheral disorganization, the central projection to cochlear nuclei is normal. Compared to ErbB2 mutants, conditional Sox10 mutants have limited cell death in spiral ganglion neurons, indicating that the absence of Schwann cells alone contributes little to the embryonic survival of neurons. These data suggest that neural crest-derived cells are dispensable for all central and some peripheral targeting of inner ear neurons. However, Schwann cells provide a stop signal for migratory spiral ganglion neurons and facilitate proper targeting of the organ of Corti by spiral ganglion afferents

Sox10 conditional mutants have no Schwann cells and show different distribution of neurons.

<p>These sections are cut parallel to the facial nerve (FN) to obtain cross sections through the vestibular (Scarpa’s) ganglion and cochlear nerve between the ear and the brain. Vestibular ganglion (VG) neurons are anterior and adjacent to the cochlear nerve (CN) in control mice (A, A’). In Sox10 mutants, vestibular and spiral ganglion neurons co-mingle next to the facial nerve (B). Schwann cells with small, elongated and heterochromatin rich nuclei are readily visible between nerve fibers (arrows in C) and the developing spiral ganglion (E) in control mice. No such cells exist in the Sox10 mutants (D, F). Abbreviations: CN, cochlear nerve; FN, facial nerve; SPG; spiral ganglion; VG, vestibular ganglion. Bar indicates 100 um in A–F.</p

Sox8 and Sox10 jointly maintain myelin gene expression in oligodendrocytes

In Schwann cells of the vertebrate peripheral nervous system, induction of myelination and myelin maintenance both depend on the HMG-domain-containing transcription factor Sox10. In oligodendrocytes of the central nervous system, Sox10 is also essential for the induction of myelination. Its role in late phases of myelination and myelin maintenance has not been studied so far. Here, we show that these processes are largely unaffected in mice that lack Sox10 in mature oligodendrocytes. As Sox10 is co-expressed with the related Sox8, we also analyzed oligodendrocytes and myelination in Sox8-deficient mice. Again, we could not detect any major abnormalities. Expression of many myelin genes was only modestly reduced in both mouse mutants. Dramatic reductions in expression levels and phenotypic disturbances became only apparent once Sox8 and Sox10 were both absent. This argues that Sox8 and Sox10 are jointly required for myelin maintenance and impact myelin gene expression. One direct target gene of both Sox proteins is the late myelin gene Mog. Our results point to at least partial functional redundancy between both related Sox proteins in mature oligodendrocytes and are the first report of a substantial function of Sox8 in the oligodendroglial lineage

ErbB2 mutants with Sox10 conditional mutants have similar unusual spiral ganglion position but differ in fiber projections and apoptotic neuron ratio.

<p>Injection of lipophilic dyes into cochlear nuclei label spiral ganglion cells in the center of cochlea (A, B) and elongated radial fibers (RF). Only Sox10 mutants show a massive projection of spiral ganglion afferents to the lateral wall (insert in B). ErbB2 mutants have a severe reduction in radial fibers with very few extending into the organ of Corti (shown by PLP-eGFP) and the lateral wall (LW). In contrast, Sox10 mutants have a denser organ of Corti innervation (shown with Myo7a immunoreactivity in D) and many fibers to the lateral wall. One in 5 spiral ganglion neurons of ErbB2 mutants is apoptotic (E) and rare apoptotic profiles are also found in Sox10 mutants (F). Markers for apoptosis such as anti-activated caspase3 immunocytochemistry (G’) and PSVue (G”) confirm the occasional dying cell (G, G’”) in Sox10 conditional mutant mice. Abbreviations: LW, lateral wall; OC, organ of Corti; PLP-GFP, phospholipoprotein-green fluorescent protein; RF, radial fiber. Bar indicates 100 μm in A, B, D, 50 μm in C, E, F and 10 μm in G.</p

Afferents to vestibular epithelia are reduced and disorganized.

<p>Neural crest-derived Sox10 positive cells play a guidance function in the vestibular innervation of the ear. Compared with control mice of E18.5 (A), Sox10 conditional mutants show a reduced innervation to the canal cristae(B, D) and disorganized fibers routing from one canal crista to another and occasionally to nowhere (B, D). Note the smaller size of epithelia and fiber projections in the younger Sox10 mutant (B). Abbreviations: PC, posterior crista; AC, anterior crista; HC, horizontal crista; U, utricle. Bar indicates 100 μm.</p