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The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex three-dimensional structure. To achieve this complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tissues surrounding the otic primordium, and more particularly the adjacent segmented hindbrain, have been implicated in specifying structures along the anteroposterior and dorsoventral axes of the inner ear. In this work we have first characterized the generation and axial specification of the otic neurogenic domain, and second, we have investigated the effects of the mutation of kreisler/MafB - a gene transiently expressed in rhombomeres 5 and 6 of the developing hindbrain - in early otic patterning and cell specification. We show that kr/kr embryos display an expansion of the otic neurogenic domain, due to defects in otic patterning. Although many reports have pointed to the role of FGF3 in otic regionalisation, we provide evidence that FGF3 is not sufficient to govern this process. Neither Krox20 nor Fgf3 mutant embryos, characterized by a downregulation or absence of Fgf3 in r5 and r6, display ectopic neuroblasts in the otic primordium. However, Fgf3-/-Fgf10-/- double mutants show a phenotype very similar to kr/kr embryos: they present ectopic neuroblasts along the AP and DV otic axes. Finally, partial rescue of the kr/kr phenotype is obtained when Fgf3 or Fgf10 are ectopically expressed in the hindbrain of kr/kr embryos. These results highlight the importance of hindbrain-derived signals in the regulation of otic neurogenesis. © 2008 Elsevier Inc. All rights reserved., This work was supported by the grant BFU2006-05604 from the Spanish Ministry of Education and Science to C.P. and BFU2007-61030, Tercel, Ciberned and Junta de Castilla y León to T.S. C.V.E. was supported by a FI fellowship from AGAUR (Generalitat de Catalunya), and by CONACYT (Mexico); E.D.F. was supported by a FPI fellowship from the MEC, Spain.

The homeodomain transcription factor vHNF1 plays an essential role in the patterning of the caudal segmented hindbrain, where it participates in the definition of the boundary between rhombomeres (r) 4 and 5 and in the specification of the identity of r5 and r6. Understanding the molecular basis of vHnf1 own expression therefore constitutes an important issue to decipher the regulatory network governing hindbrain patterning. We have identified a highly conserved 800-bp enhancer element located in the fourth intron of vHnf1 and whose activity recapitulates vHnf1 neural expression in transgenic mice. Functional analysis of this enhancer revealed that it contains two types of essential motifs, a retinoic acid response element and two half T-MARE sites, indicating that it integrates direct inputs from the retinoic acid signaling cascade and MAF-related factors. Our data suggest that MAFB, which is itself regulated by vHNF1, acts as a positive modulator of vHnf1 in r5 and r6, whereas another MAF-related factor is absolutely required for the expression of vHnf1 in both the hindbrain and the spinal cord. We propose a model accounting for the initiation and maintenance phases of vHnf1 expression and for the establishment of the r4/r5 boundary, based on cooperative contributions of Maf factors and retinoic acid signaling.

The bZip transcription factor Mafb is expressed in two segments of the developing vertebrate hindbrain: the rhombomeres 5 and 6. Loss of Mafb expression in the mouse mutant kreisler leads to elimination of r5 and to alterations of r6 regional identity. Here, we further investigated the role of Mafb in hindbrain patterning using gain-of-function experiments in the chick embryo. Our work has revealed novel functions for Mafb, including a positive autoregulatory activity, the capacity to repress Hoxb1 expression, and the capacity to synergise with or antagonise Krox20 activity. These different activities appear to be spatially restricted in the hindbrain, presumably due to interactions with other factors. Reinvestigation of the kreisler mutation indicated that it also results in an ectopic activation of Mafb in rhombomere 3, accounting for the previously described molecular alterations of this rhombomere in the mutant. Together, these data allow us to refine our view of the dual function of Mafb in both segmentation and specification of anteroposterior identity in the hindbrain.

Krox-20 and mafB/kr encode transcription factors involved in the control of hindbrain development and are expressed in rhombomeres (r) 3 and 5 and 5 and 6, respectively. To analyse the regulation of the expression of these genes by positional cues, focusing on the stages just preceding the formation of rhombomeres, we have performed ectopic grafts involving single prospective rhombomeres (pr) or couples of pr on 4–6 somite avian embryos. Transplantation of pr6 in the pr5 position leads to Krox-20 activation and grafting of pr7 in the pr5 position results in mafB/kr activation. Furthermore, pr6 grafted in the pr5 position develops an r5-like cytoarchitecture. These data establish that rostral transplantation can lead to anteriorization within the hindbrain. However, additional experiments indicate that the competence of the transplanted tissue for such anteriorization appears limited and that transformations corresponding to shifts of a single rhombomere are favoured. We also show that caudal transplantation of pr5 into the pr6 position can lead to a down-regulation of Krox-20 expression consistent with posteriorization, suggesting that caudalizing influences are present within the nonsomitic hindbrain after the 4- to 6-somite stage. Finally, combinations of extirpation and grafting experiments suggest that the regulation of mafB/kr expression in the r6–r7 region may involve anteriorizing influences in addition to previously identified posteriorizing signals from the somitic region.

13 páginas, 7 figuras., After segmentation of the vertebrate hindbrain, expression of the zinc-finger geneKrox-20and the receptor tyrosine kinase geneSek-1is precisely restricted to rhombomeres (r) 3 and 5. This precise segmental expression is likely to reflect a critical requirement for these rhombomeres to acquire a distinct and homogeneous identity and raises the question as to how this relates to the intermingling and restriction of cell movement during segmentation. We have analysedKrox-20andSek-1expression in the mouse and chick hindbrain at single-cell resolution using whole-mountin situhybridisation and immunocytochemistry. We find that, in the mouse, the presumptive r3 and r5 expression domains each arise as narrow stripes that then broaden, suggestive of a recruitment of cells to an r3/r5 identity and/or a segmental regulation of cell proliferation. In addition, we find that expression of these genes initially occurs in fuzzy domains, and that these are progressively restricted to segmental domains, although occasional “violating” cells are observed even after segmentation. We propose that the establishment and maintenance of these segmental domains may involve both a dynamic regulation of r3/r5 identity and the restriction of cell movement across rhombomere boundaries., This work was supported by the Medical Research Council, the European Union Biotechnology program (BIO2CT-930060), and by the Spanish Ministry of Education (DGICYT-PB92-0045).