Role of the hindbrain in dorsoventral but not anteroposterior axial specification of the inner ear

An early and crucial event in vertebrate inner ear development is the acquisition of axial identities that in turn dictate the positions of all subsequent inner ear components. Here, we focus on the role of the hindbrain in establishment of inner ear axes and show that axial specification occurs well after otic placode formation in chicken. Anteroposterior (AP) rotation of the hindbrain prior to specification of this axis does not affect the normal AP orientation and morphogenesis of the inner ear. By contrast, reversing the dorsoventral (DV) axis of the hindbrain results in changing the DV axial identity of the inner ear. Expression patterns of several ventrally expressed otic genes such as NeuroD, Lunatic fringe (Lfng) and Six1 are shifted dorsally, whereas the expression pattern of a normally dorsal-specific gene, Gbx2, is abolished. Removing the source of Sonic Hedgehog (SHH) by ablating the floor plate and/or notochord, or inhibiting SHH function using an antibody that blocks SHH bioactivity results in loss of ventral inner ear structures. Our results indicate that SHH, together with other signals from the hindbrain, are important for patterning the ventral axis of the inner ear. Taken together, our studies suggest that tissue(s) other than the hindbrain confer AP axial information whereas signals from the hindbrain are necessary and sufficient for the DV axial patterning of the inner ear

Violation of cell lineage restriction compartments in the chick hindbrain

Previous cell lineage studies indicate that the repeated neuromeres of the chick hindbrain, the rhombomeres, are cell lineage restriction compartments. We have extended these results and tested if the restrictions are absolute. Two different cell marking techniques were used to label cells shortly after rhombomeres form (stage 9+ to 13) so that the resultant clones could be followed up to stage 25. Either small groups of cells were labelled with the lipophilic dye DiI or single cells were injected intracellularly with fluorescent dextran. The majority of the descendants labelled by either technique were restricted to within a single rhombomere. However, in a small but reproducible proportion of the cases (greater than 5%), the clones expanded across a rhombomere boundary. Neither the stage of injection, the stage of analysis, the dorsoventral position, nor the rhombomere identity correlated with the boundary crossing. Judging from the morphology of the cells, both neurons and non-neuronal cells were able to expand over a boundary. These results demonstrate that the rhombomere boundaries represent cell lineage restriction barriers which are not impenetrable in normal development

Ancient Pbx-Hox signatures define hundreds of vertebrate developmental enhancers

Background: Gene regulation through cis-regulatory elements plays a crucial role in development and disease. A major aim of the post-genomic era is to be able to read the function of cis-regulatory elements through scrutiny of their DNA sequence. Whilst comparative genomics approaches have identified thousands of putative regulatory elements, our knowledge of their mechanism of action is poor and very little progress has been made in systematically de-coding them. Results: Here, we identify ancient functional signatures within vertebrate conserved non-coding elements (CNEs) through a combination of phylogenetic footprinting and functional assay, using genomic sequence from the sea lamprey as a reference. We uncover a striking enrichment within vertebrate CNEs for conserved binding-site motifs of the Pbx-Hox hetero-dimer. We further show that these predict reporter gene expression in a segment specific manner in the hindbrain and pharyngeal arches during zebrafish development. Conclusions: These findings evoke an evolutionary scenario in which many CNEs evolved early in the vertebrate lineage to co-ordinate Hox-dependent gene-regulatory interactions that pattern the vertebrate head. In a broader context, our evolutionary analyses reveal that CNEs are composed of tightly linked transcription-factor binding-sites (TFBSs), which can be systematically identified through phylogenetic footprinting approaches. By placing a large number of ancient vertebrate CNEs into a developmental context, our findings promise to have a significant impact on efforts toward de-coding gene-regulatory elements that underlie vertebrate development, and will facilitate building general models of regulatory element evolution

Rhombomere of origin determines autonomous versus environmentally regulated expression of Hoxa3 in the avian embryo

We have investigated the pattern and regulation of Hoxa3 expression in the hindbrain and associated neural crest cells in the chick embryo, using whole mount in situ hybridization in conjunction with DiI labeling of neural crest cells and microsurgical manipulations. Hoxa3 is expressed in the neural plate and later in the neural tube with a rostral border of expression corresponding to the boundary between rhombomeres (r) 4 and 5. Initial expression is diffuse and becomes sharp after boundary formation. Hoxa3 exhibits uniform expression within r5 after formation of rhombomeric borders. Cell marking experiments reveal that neural crest cells migrating caudally, but not rostrally, from r5 and caudally from r6 express Hoxa3 in normal embryo. Results from transposition experiments demonstrate that expression of Hoxa3 in r5 neural crest cells is not strictly cell-autonomous. When r5 is transposed with r4 by rostrocaudal rotation of the rhomobomeres, Hoxa3 is expressed in cells migrating lateral to transposed r5 and for a short time, in condensing ganglia, but not by neural crest within the second branchial arch. Since DiI-labeled cells from transposed r5 are present in the second arch, Hoxa3-expressing neural crest cells from r5 appear to down-regulate their Hoxa3 expression in their new environment. In contrast, when r6 is transposed to the position of r4 after boundary formation, Hoxa3 is maintained in both migrating neural crest cells and those positioned within the second branchial arch and associated ganglia. These results suggest that Hoxa3 expression is cell-autonomous in r6 and its associated neural crest. Our results suggest that neural crest cells expressing the same Hox gene are not eqivalent; they respond differently to environmental signals and exhibit distinct degrees of cell autonomy depending upon their rhombomere of origin

Dorsal hindbrain ablation results in rerouting of neural crest migration and changes in gene expression, but normal hyoid development

Our previous studies have shown that hindbrain neural tube cells can regulate to form neural crest cells for a limited time after neural fold removal (Scherson, T., Serbedzija, G., Fraser, S. E. and Bronner-Fraser, M. (1993). Development 188, 1049-1061; Sechrist, J., Nieto, M. A., Zamanian, R. T. and Bronner-Fraser, M. (1995). Development 121, 4103-4115). In the present study, we ablated the dorsal hindbrain at later stages to examine possible alterations in migratory behavior and/or gene expression in neural crest populations rostral and caudal to the operated region. The results were compared with those obtained by misdirecting neural crest cells via rhombomere rotation. Following surgical ablation of dorsal r5 and r6 prior to the 10 somite stage, r4 neural crest cells migrate along normal pathways toward the second branchial arch. Similarly, r7 neural crest cells migrate primarily to the fourth branchial arch. When analogous ablations are performed at the 10- 12 somite stage, however, a marked increase in the numbers of DiI/Hoxa-3-positive cells from r7 are observed within the third branchial arch. In addition, some DiI-labeled r4 cells migrate into the depleted hindbrain region and the third branchial arch. During their migration, a subset of these r4 cells up-regulate Hoxa-3, a transcript they do not normally express. Krox20 transcript levels were augmented after ablation in a population of neural crest cells migrating from r4, caudal r3 and rostral r3. Long-term survivors of bilateral ablations possess normal neural crest-derived cartilage of the hyoid complex, suggesting that misrouted r4 and r7 cells contribute to cranial derivatives appropriate for their new location. In contrast, misdirecting of the neural crest by rostrocaudal rotation of r4 through r6 results in a reduction of Hoxa-3 expression in the third branchial arch and corresponding deficits in third arch-derived structures of the hyoid apparatus. These results demonstrate that neural crest/tube progenitors in the hindbrain can compensate by altering migratory trajectories and patterns of gene expression when the adjacent neural crest is removed, but fail to compensate appropriately when the existing neural crest is misrouted by neural tube rotation

Swimming Rhythm Generation in The Caudal Hindbrain of The Lamprey

The spinal cord has been well established as the site of generation of the locomotor rhythm in vertebrates, but studies have suggested that the caudal hindbrain in larval fish and amphibians can also generate locomotor rhythms. Here, we investigated whether the caudal hindbrain of the adult lamprey (Petromyzon marinus and Ichthyomyzon unicuspis) has the ability to generate the swimming rhythm. The hindbrain-spinal cord transition zone of the lamprey contains a bilateral column of somatic motoneurons that project via the spino-occipital (S-O) nerves to several muscles of the head. In the brainstem-spinal cord-muscle preparation, these muscles were found to burst and contract rhythmically with a left-right alternation when swimming activity was evoked with a brief electrical stimulation of the spinal cord. In the absence of muscles, the isolated brainstem-spinal cord preparation also produced alternating left-right bursts in S-O nerves (i.e., fictive swimming), and the S-O nerve bursts preceded the bursts occurring in the first ipsilateral spinal ventral root. After physical isolation of the S-O region using transverse cuts of the nervous system, the S-O nerves still exhibited rhythmic bursting with left-right alternation when glutamate was added to the bathing solution. We conclude that the S-O region of the lamprey contains a swimming rhythm generator that produces the leading motor nerve bursts of each swimming cycle, which then propagate down the spinal cord to produce forward swimming. The S-O region of the hindbrain-spinal cord transition zone may play a role in regulating speed, turning, and head orientation during swimming in lamprey

Order and coherence in the fate map of the zebrafish nervous system

The zebrafish is an excellent vertebrate model for the study of the cellular interactions underlying the patterning and the morphogenesis of the nervous system. Here, we report regional fate maps of the zebrafish anterior nervous system at two key stages of neural development: the beginning (6 hours) and the end (10 hours) of gastrulation. Early in gastrulation, we find that the presumptive neurectoderm displays a predictable organization that reflects the future anteroposterior and dorsoventral order of the central nervous system. The precursors of the major brain subdivisions (forebrain, midbrain, hindbrain, neural retina) occupy discernible, though overlapping, domains within the dorsal blastoderm at 6 hours. As gastrulation proceeds, these domains are rearranged such that the basic order of the neural tube is evident at 10 hours. Furthermore, the anteroposterior and dorsoventral order of the progenitors is refined and becomes aligned with the primary axes of the embryo. Time-lapse video microscopy shows that the rearrangement of blastoderm cells during gastrulation is highly ordered. Cells near the dorsal midline at 6 hours, primarily forebrain progenitors, display anterior-directed migration. Cells more laterally positioned, corresponding to midbrain and hindbrain progenitors, converge at the midline prior to anteriorward migration. These results demonstrate a predictable order in the presumptive neurectoderm, suggesting that patterning interactions may be well underway by early gastrulation. The fate maps provide the basis for further analyses of the specification, induction and patterning of the anterior nervous system, as well as for the interpretation of mutant phenotypes and gene-expression patterns

Rhombomere rotation reveals that multiple mechanisms contribute to the segmental pattern of hindbrain neural crest migration

Hindbrain neural crest cells adjacent to rhombomeres 2 (r2), r4 and r6 migrate in a segmental pattern, toward the first, second and third branchial arches, respectively. Although all rhombomeres generate neural crest cells, those arising from r3 and r5 deviate rostrally and caudally (J. Sechrist, G. Serbedzija, T. Scherson, S. Fraser and M. Bronner-Fraser (1993) Development 118, 691–703). We have altered the rostrocaudal positions of the cranial neural tube, adjacent ectoderm/mesoderm or presumptive otic vesicle to examine tissue influences on this segmental migratory pattern. After neural tube rotation, labeled neural crest cells follow pathways generally appropriate for their new position after grafting. For example, when r3 and r4 were transposed, labeled r3 cells migrated laterally to the second branchial arch whereas labeled r4 cells primarily deviated caudally toward the second arch, with some cells moving rostrally toward the first. In contrast to r4 neural crest cells, transposed r3 cells leave the neural tube surface in a polarized manner, near the r3/4 border. Surprisingly, some labeled neural crest cells moved directionally toward small ectopic otic vesicles that often formed in the ectoderm adjacent to grafted r4. Similarly, they moved toward grafted or displaced otic vesicles. In contrast, surgical manipulation of the mesoderm adjacent to r3 and r4 had no apparent effects. Our results offer evidence that neural crest cells migrate directionally toward the otic vesicle, either by selective attraction or pathway-derived cues

In ovo time-lapse analysis after dorsal neural tube ablation shows rerouting of chick hindbrain neural crest

Previous analyses of single neural crest cell trajectories have suggested important roles for interactions between neural crest cells and the environment, and amongst neural crest cells. To test the relative contribution of intrinsic versus extrinsic information in guiding cells to their appropriate sites, we ablated subpopulations of premigratory chick hindbrain neural crest and followed the remaining neural crest cells over time using a new in ovo imaging technique. Neural crest cell migratory behaviors are dramatically different in ablated compared with unoperated embryos. Deviations from normal migration appear either shortly after cells emerge from the neural tube or en route to the branchial arches, areas where cell-cell interactions typically occur between neural crest cells in normal embryos. Unlike the persistent, directed trajectories in normal embryos, neural crest cells frequently change direction and move somewhat chaotically after ablation. In addition, the migration of neural crest cells in collective chains, commonly observed in normal embryos, was severely disrupted. Hindbrain neural crest cells have the capacity to reroute their migratory pathways and thus compensate for missing neural crest cells after ablation of neighboring populations. Because the alterations in neural crest cell migration are most dramatic in regions that would normally foster cell-cell interactions, the trajectories reported here argue that cell-cell interactions have a key role in the shaping of the neural crest migration