Pascale Gilardi-Hebenstreit

Several receptor tyrosine kinase genes of the Eph family are segmentally expressed in the developing hindbrain

Abstract Pattern formation in the hindbrain involves a segmentation process leading to the formation of metameric units, manifested as successive swellings known as rhombomeres (r). In search for genes involved in cell-cell interactions during hindbrain segmentation, we have screened for protein kinase genes with restricted expression patterns in this region of the CNS. We present the cloning of three novel mouse genes, Sek-2, Sek-3 and Sek-4 (members of the Eph subfamily of putative transmembrane receptor protein tyrosine kinases (RTKs)), the identification of their chromosomal locations, and the analysis of their expression between 7.5 and 10.5 days of development. Before morphological segmentation, Sek-2 is transcribed in a transverse stripe corresponding to prospective r4 and the adjacent mesoderm, suggesting possible roles both in hindbrain segmentation and signalling between neuro-epithelium and mesoderm. Sek-3 and Sek-4 have common domains of expression, including r3, r5 and part of the midbrain, as well as specific domains in the diencephalon, telencephalon, spinal cord and in mesodermal and neural crest derivatives. Together with our previous finding that Sek ( Sek-1 ) is expressed in r3 and r5 (Gilardi-Hebenstreit et al., 1992; Nieto et al., 1992), these data indicate that members of the Eph family of RTKs may co-operate in the segmental patterning of the hindbrain.

EGR1 and EGR2 Involvement in Vertebrate Tendon Differentiation

The molecules involved in vertebrate tendon formation during development remain largely unknown. To date, only two DNA-binding proteins have been identified as being involved in vertebrate tendon formation, the basic helix-loop-helix transcription factor Scleraxis and, recently, the Mohawk homeobox gene. We investigated the involvement of the early growth response transcription factors Egr1 and Egr2 in vertebrate tendon formation. We established that Egr1 and Egr2 expression in tendon cells was correlated with the increase of collagen expression during tendon cell differentiation in embryonic limbs. Vertebrate tendon differentiation relies on a muscle-derived FGF (fibroblast growth factor) signal. FGF4 was able to activate the expression of Egr genes and that of the tendon-associated collagens in chick limbs. Egr gene misexpression experiments using the chick model allowed us to establish that either Egr gene has the ability to induce de novo expression of the reference tendon marker scleraxis, the main tendon collagen Col1a1, and other tendon-associated collagens Col3a1, Col5a1, Col12a1, and Col14a1. Mouse mutants for Egr1 or Egr2 displayed reduced amounts of Col1a1 transcripts and a decrease in the number of collagen fibrils in embryonic tendons. Moreover, EGR1 and EGR2 trans-activated the mouse Col1a1 proximal promoter and were recruited to the tendon regulatory regions of this promoter. These results identify EGRs as novel DNA-binding proteins involved in vertebrate tendon differentiation by regulating type I collagen production.

Krox20 and kreisler co-operate in the transcriptional control of segmental expression of Hoxb3 in the developing hindbrain

In the segmented vertebrate hindbrain, the Hoxa3 and Hoxb3 genes are expressed at high relative levels in the rhombomeres (r) 5 and 6, and 5, respectively. The single enhancer elements responsible for these activities have been identified previously and shown to constitute direct targets of the transcription factor kreisler, which is expressed in r5 and r6. Here, we have analysed the contribution of the transcription factor Krox20, present in r3 and r5. Genetic analyses demonstrated that Krox20 is required for activity of the Hoxb3 r5 enhancer, but not of the Hoxa3 r5/6 enhancer. Mutational analysis of the Hoxb3 r5 enhancer, together with ectopic expression experiments, revealed that Krox20 binds to the enhancer and synergizes with kreisler to promote Hoxb3 transcription, restricting enhancer activity to their domain of overlap, r5. These analyses also suggested contributions from an Ets‐related factor and from putative factors likely to heterodimerize with kreisler. The integration of multiple independent inputs present in overlapping domains by a single enhancer is likely to constitute a general mechanism for the patterning of subterritories during vertebrate development.

How to build a vertebrate hindlbrain. lessons from genetics

Abstract During vertebrate embryogenesis, the hindbrain is the site of a segmentation process which leads to the formation, along the anterior-posterior axis, of 7–8 metameres called rhombomeres. This phenomenon plays an essential role in early hindbrain regionalisation and in the specification of the pattern of developing structures in this region of the brain. Data accumulated during the last 10 years have also shown that rhombomeres are units of gene expression and of cell lineage. Hence, a number of regulatory genes are expressed according to segment-specific patterns in the hindbrain and have been implicated in the pattern formation process. In this review, we focus on the analysis of the function and regulation of these genes along the different steps of hindbrain segmentation, from segment delimitation to acquisition of positional identity. On this basis, we propose a model for the control of early hindbrain development.

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.

Chimeric EWSR1-FLI1 regulates the Ewing sarcoma susceptibility gene EGR2 via a GGAA microsatellite

Deciphering the ways in which somatic mutations and germline susceptibility variants cooperate to promote cancer is challenging. Ewing sarcoma is characterized by fusions between EWSR1 and members of the ETS gene family, usually EWSR1-FLI1, leading to the generation of oncogenic transcription factors that bind DNA at GGAA motifs. A recent genome-wide association study identified susceptibility variants near EGR2. Here we found that EGR2 knockdown inhibited proliferation, clonogenicity and spheroidal growth in vitro and induced regression of Ewing sarcoma xenografts. Targeted germline deep sequencing of the EGR2 locus in affected subjects and controls identified 291 Ewing-associated SNPs. At rs79965208, the A risk allele connected adjacent GGAA repeats by converting an interspaced GGAT motif into a GGAA motif, thereby increasing the number of consecutive GGAA motifs and thus the EWSR1-FLI1–dependent enhancer activity of this sequence, with epigenetic characteristics of an active regulatory element. EWSR1-FLI1 preferentially bound to the A risk allele, which increased global and allele-specific EGR2 expression. Collectively, our findings establish cooperation between a dominant oncogene and a susceptibility variant that regulates a major driver of Ewing sarcomagenesis.

Hindbrain patterning requires fine-tuning of early krox20 transcription by Sprouty 4

Vertebrate hindbrain segmentation is an evolutionarily conserved process that involves a complex interplay of transcription factors and signalling pathways. Fibroblast growth factor (FGF) signalling plays a major role, notably by controlling the expression of the transcription factor Krox20 (Egr2), which is required for the formation and specification of two segmental units: rhombomeres (r) 3 and 5. Here, we explore the molecular mechanisms downstream of FGF signalling and the function of Sprouty 4 (Spry4), a negative-feedback regulator of this pathway, in zebrafish. We show that precise modulation of FGF signalling by Spry4 is required to determine the appropriate onset of krox20 transcription in r3 and r5 and, ultimately, rhombomere size in the r3-r5 region. FGF signalling acts by modulating the activity of krox20 initiator enhancer elements B and C; in r5, we show that this regulation is mediated by direct binding of the transcription factor MafB to element B. By contrast, FGF signalling does not control the krox20 autoregulatory element A, which is responsible for amplification and maintenance of krox20 expression. Therefore, early krox20 transcription sets the blueprint for r3-r5 patterning. This work illustrates the necessity for fine-tuning in a common and fundamental patterning process, based on a bistable cell-fate choice involving the coupling of an extracellular gradient with a positive-feedback loop. In this mode of patterning, precision and robustness can be achieved by the introduction of a negative-feedback loop, which, in the hindbrain, is mediated by Spry4.

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.

A functional interaction between Irx and Meis patterns the anterior hindbrain and activates krox20 expression in rhombomere 3

Patterning of the vertebrate hindbrain involves a segmentation process leading to the formation of seven rhombomeres along the antero-posterior axis. While recent studies have shed light on the mechanisms underlying progressive subdivision of the posterior hindbrain into individual rhombomeres, the early events involved in anterior hindbrain patterning are still largely unknown. In this paper we demonstrate that two zebrafish Iroquois transcription factors, Irx7 and Irx1b, are required for the proper formation and specification of rhombomeres 1 to 4 and, in particular, for krox20 activation in r3. We also show that Irx7 functionally interacts with Meis factors to activate the expression of anterior hindbrain markers, such as hoxb1a, hoxa2 and krox20, ectopically in the anterior neural plate. Then, focusing on krox20 expression, we show that the effect of Irx7 and Meis1.1 is mediated by element C, a conserved cis-regulatory element involved in krox20 activation in the hindbrain. Together, our data point to an essential function of Iroquois transcription factors in krox20 activation and, more generally, in anterior hindbrain specification.

Disruption of Krox20–Nab Interaction in the Mouse Leads to Peripheral Neuropathy with Biphasic Evolution

Krox20/Egr2 is a zinc finger transcription factor that plays essential roles in several developmental processes, including peripheral nervous system myelination by Schwann cells, where it acts as a master gene regulator. Krox20 is known to interact with cofactors of the Nab family and a mutation affecting isoleucine 268, which prevents this interaction, has been shown to result in congenital hypomyelinating neuropathy in humans. To further investigate the role of this interaction, we have introduced such a mutation, Krox20I268F , in the mouse germ line. Clinical, immunohistochemical, and ultrastructural analyses of the homozygous mutants reveal that they develop a severe hypomyelination phenotype that mimics the human syndrome. Furthermore, a time-course analysis of the disease indicates that it follows a biphasic evolution, the hypomyelination phase being followed by a dramatic demyelination. Although for the regulation of most analyzed Krox20 target genes the mutation behaves as a loss of function, this is not the case for a few of them. This differential effect indicates that the molecular function of the Krox20–Nab interaction is target dependent and might explain the degradation of the residual myelin, because of imbalances in its composition. In conclusion, this work provides a novel and useful model for severe human peripheral neuropathies.