Julien Ghislain

Control of myelination in Schwann cells: a Krox20 cis-regulatory element integrates Oct6, Brn2 and Sox10 activities

Myelination in Schwann cells is governed by several transcription factors, including the POU proteins Oct6 and Brn2, the high mobility group protein Sox10 and the zinc‐finger protein Krox20. How the function of these factors is integrated in the control of myelination has not been established. Previously, we identified an enhancer element controlling Krox20 expression throughout myelination in Schwann cells. In this paper, cell culture experiments were combined with transgenesis to identify transcription factors acting directly upstream of Krox20. The results show that during the promyelin–myelin transition, Krox20 expression is directly activated by Oct6 and Brn2 acting on this enhancer. In addition, the enhancer‐dependent synergism between these POU proteins and Sox10 suggests that Krox20 expression requires this combination of factors. These results resolve previous controversy concerning the mechanism of action of Oct6 and Brn2 during myelination and provide an explanation for myelin deficiencies in Waardenberg–Hirschsprung disease patients whereby Sox10 mutations could lead to a loss of Krox20 expression.

Peripheral Myelin Maintenance Is a Dynamic Process Requiring Constant Krox20 Expression

Onset of myelination in Schwann cells is governed by several transcription factors, including Krox20/Egr2, and mutations affecting Krox20 result in various human hereditary peripheral neuropathies, including congenital hypomyelinating neuropathy (CHN) and Charcot-Marie-Tooth disease (CMT). Similar molecular information is not available on the process of myelin maintenance. We have generated conditional Krox20 mutations in the mouse that allowed us to develop models for CHN and CMT. In the latter case, specific inactivation of Krox20 in adult Schwann cells results in severe demyelination, involving rapid Schwann cell dedifferentiation and increased proliferation, followed by an attempt to remyelinate and a block at the promyelinating stage. These data establish that Krox20 is not only required for the onset of myelination but that it is also crucial for the maintenance of the myelinating state. Furthermore, myelin maintenance appears as a very dynamic process in which Krox20 may constitute a molecular switch between Schwann cell myelination and demyelination programs.

Neural crest patterning: autoregulatory and crest-specific elements co-operate for Krox20 transcriptional control.

Neural crest patterning constitutes an important element in the control of the morphogenesis of craniofacial structures. Krox20, a transcription factor gene that plays a critical role in the development of the segmented hindbrain, is expressed in rhombomeres (r) 3 and 5 and in a stream of neural crest cells migrating from r5 toward the third branchial arch. We have investigated the basis of the specific neural crest expression of Krox20 and identified a cis-acting enhancer element (NCE) located 26 kb upstream of the gene that is conserved between mouse, man and chick and can recapitulate the Krox20 neural crest pattern in transgenic mice. Functional dissection of the enhancer revealed the presence of two conserved Krox20 binding sites mediating direct Krox20 autoregulation in the neural crest. In addition, the enhancer included another essential element containing conserved binding sites for high mobility group (HMG) box proteins and which responded to factors expressed throughout the neural crest. Consistent with this the NCE was strongly activated in vitro by Sox10, a crest-specific HMG box protein, in synergism with Krox20, and the inactivation of Sox10 prevented the maintenance of Krox20 expression in the migrating neural crest. These results suggest that the dependency of the enhancer on both crest- (Sox10) and r5- (Krox20) specific factors limits its activity to the r5-derived neural crest. This organisation also suggests a mechanism for the transfer and maintenance of rhombomere-specific gene expression from the hindbrain neuroepithelium to the emerging neural crest and may be of more general significance for neural crest patterning.

Live analysis of endodermal layer formation identifies random walk as a novel gastrulation movement.

During gastrulation, dramatic movements rearrange cells into three germ layers expanded over the entire embryo [1-3]. In fish, both endoderm and mesoderm are specified as a belt at the embryo margin. Mesodermal layer expansion is achieved through the combination of two directed migrations. The outer ring of precursors moves toward the vegetal pole and continuously seeds mesodermal cells inside the embryo, which then reverse their movement in the direction of the animal pole [3-6]. Unlike mesoderm, endodermal cells internalize at once and must therefore adopt a different strategy to expand over the embryo [7, 8]. With live imaging of YFP-expressing zebrafish endodermal cells, we demonstrate that in contrast to mesoderm, internalized endodermal cells display a nonoriented/noncoordinated movement fit by a random walk that rapidly disperses them over the yolk surface. Transplantation experiments reveal that this behaviour is largely cell autonomous, induced by TGF-beta/Nodal, and dependent on the downstream effector Casanova. At midgastrulation, endodermal cells switch to a convergence movement. We demonstrate that this switch is triggered by environmental cues. These results uncover random walk as a novel Nodal-induced gastrulation movement and as an efficient strategy to transform a localized cell group into a layer expanded over the embryo.

Krox20 hindbrain cis-regulatory landscape: interplay between multiple long-range initiation and autoregulatory elements.

The vertebrate hindbrain is subject to a transient segmentation process leading to the formation of seven or eight metameric territories termed rhombomeres (r). This segmentation provides the basis for the subsequent establishment of hindbrain neuronal organization and participates in the patterning of the neural crest involved in craniofacial development. The zinc-finger gene Krox20 is expressed in r3 and r5, and encodes a transcription factor that plays a key role in hindbrain segmentation, coordinating segment formation, specification of odd- and even-numbered rhombomeres, and cell segregation between adjacent segments, through the regulation of numerous downstream genes. In order to further elucidate the genetic network underlying hindbrain segmentation, we have undertaken the analysis of the cis-regulatory sequences governing Krox20 expression. We have found that the control of Krox20 transcription relies on three very long-range (200 kb) enhancer elements (A, B and C) that are conserved between chick, mouse and human genomes. Elements B and C are activated at the earliest stage of Krox20 expression in r5 and r3-r5, respectively, and do not require the Krox20 protein. These elements are likely to function as initiators of Krox20 expression. Element B contains a binding site for the transcription factor vHNF1, the mutation of which abolishes its activity, suggesting that vHNF1 is a direct initiator of Krox20 expression in r5. Element A contains Krox20-binding sites, which are required, together with the Krox20 protein, for its activity. This element therefore allows the establishment of a direct positive autoregulatory loop, which takes the relay of the initiator elements and maintains Krox20 expression. Together, our studies provide a basis for a model of the molecular mechanisms controlling Krox20 expression in the developing hindbrain and neural crest.

Rostral hindbrain patterning involves the direct activation of a Krox20 transcriptional enhancer by Hox/Pbx and Meis factors

The morphogenesis of the vertebrate hindbrain involves the generation of metameric units called rhombomeres (r), and Krox20 encodes a transcription factor that is expressed in r3 and r5 and plays a major role in this segmentation process. Our knowledge of the basis of Krox20 regulation in r3 is rather confusing, especially concerning the involvement of Hox factors. To investigate this issue, we studied one of the Krox20 hindbrain cis-regulatory sequences, element C, which is active in r3-r5 and which is the only initiator element in r3. We show that element C contains multiple binding sites for Meis and Hox/Pbx factors and that these proteins synergize to activate the enhancer. Mutation of these binding sites allowed us to establish that Krox20 is under the direct transcriptional control of both Meis (presumably Meis2) and Hox/Pbx factors in r3. Furthermore, our data indicate that element C functions according to multiple modes, in Meis-independent or -dependent manners and with different Hox proteins, in r3 and r5. Finally, we show that the Hoxb1 and Krox20 expression domains transiently overlap in prospective r3, and that Hoxb1 binds to element C in vivo, supporting a cell-autonomous involvement of Hox paralogous group 1 proteins in Krox20 regulation. Altogether, our data clarify the molecular mechanisms of an essential step in hindbrain patterning. We propose a model for the complex regulation of Krox20, involving a novel mode of initiation, positive and negative controls by Hox proteins, and multiple direct and indirect autoregulatory loops.

FGF-receptor signalling controls neural cell diversity in the zebrafish hindbrain by regulating olig2 and sox9.

The mechanisms underlying the generation of neural cell diversity are the subject of intense investigation, which has highlighted the involvement of different signalling molecules including Shh, BMP and Wnt. By contrast, relatively little is known about FGF in this process. In this report we identify an FGF-receptor-dependent pathway in zebrafish hindbrain neural progenitors that give rise to somatic motoneurons, oligodendrocyte progenitors and differentiating astroglia. Using a combination of chemical and genetic approaches to conditionally inactivate FGF-receptor signalling, we investigate the role of this pathway. We show that FGF-receptor signalling is not essential for the survival or maintenance of hindbrain neural progenitors but controls their fate by coordinately regulating key transcription factors. First, by cooperating with Shh, FGF-receptor signalling controls the expression of olig2, a patterning gene essential for the specification of somatic motoneurons and oligodendrocytes. Second, FGF-receptor signalling controls the development of both oligodendrocyte progenitors and astroglia through the regulation of sox9, a gliogenic transcription factor the function of which we show to be conserved in the zebrafish hindbrain. Overall, for the first time in vivo, our results reveal a mechanism of FGF in the control of neural cell diversity.