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

Birth of ophthalmic trigeminal neurons initiates early in the placodal ectoderm

The largest of the cranial ganglia, the trigeminal ganglion, relays cutaneous sensations of the head to the central nervous system. Its sensory neurons have a dual origin from both ectodermal placodes and neural crest. Here, we show that the birth of neurons derived from the chick ophthalmic trigeminal placode begins prior to their ingression (HH11), as early as HH8, and considerably earlier than previously suspected (HH16). Furthermore, cells exiting the cell cycle shortly thereafter express the ophthalmic trigeminal placode marker Pax3 (HH9). At HH11, these postmitotic Pax3+ placode cells begin to express the pan-neuronal marker neurofilament while still in the ectoderm. Analysis of the ectodermal origin and distribution of these early postmitotic neurons reveals that the ophthalmic placode extends further rostrally than anticipated, contributing to neurons that reside in and make a significant contribution to the ophthalmic trigeminal nerve. These data redefine the timing and extent of neuron formation from the ophthalmic trigeminal placode

Early regulative ability of the neuroepithelium to form cardiac neural crest

The cardiac neural crest (arising from the level of hindbrain rhombomeres 6–8) contributes to the septation of the cardiac outflow tract and the formation of aortic arches. Removal of this population after neural tube closure results in severe septation defects in the chick, reminiscent of human birth defects. Because neural crest cells from other axial levels have regenerative capacity, we asked whether the cardiac neural crest might also regenerate at early stages in a manner that declines with time. Accordingly, we find that ablation of presumptive cardiac crest at stage 7, as the neural folds elevate, results in reformation of migrating cardiac neural crest by stage 13. Fate mapping reveals that the new population derives largely from the neuroepithelium ventral and rostral to the ablation. The stage of ablation dictates the competence of residual tissue to regulate and regenerate, as this capacity is lost by stage 9, consistent with previous reports. These findings suggest that there is a temporal window during which the presumptive cardiac neural crest has the capacity to regulate and regenerate, but this regenerative ability is lost earlier than in other neural crest populations

Age-Dependent Neurotransmitter Plasticity of Ciliary Ganglion Neurons

We have examined neurotransmitter plasticity in postmitotic cholinergic neurons isolated from 6.5- to 11-day-old embryonic quail ciliary ganglia. Purified neurons were labeled with DiI, transplanted into the trunk of young chick embryos, and assayed for catecholamine content and [^3H]thymidine uptake 4 to 5 days later. For ciliary neurons derived from 6.5- to 8-day-old embryos, as many as 25% (average of 9% overall) expressed catecholamines in the host sympathetic ganglia, migratory stream, aortic plexuses, and adrenal medulla. In contrast, neurons from >8-day-old ganglia did not acquire or produce detectable catecholamines, indicating a limited time period over which phenotypic conversion can occurin vivo.As a control, ciliary neurons were also injected into the head mesenchyme of young embryos; no catecholamine expression was observed. Interestingly, after transplantation some Dil-labeled postmitotic ciliary neurons took up [^3H]thymidine with or without phenotypic change. These results suggest that phenotypic plasticity in ciliary neurons is age-dependent, is location-dependent, and may involve resumption of DNA replication, a characteristic feature of some differentiating adrenergic sympathetic neurons. Apoptosis of a few proliferating transplanted cells may be induced independently or in association with transmitter change

Both neural crest and placode contribute to the ciliary ganglion and oculomotor nerve

The chick ciliary ganglion is a neural crest-derived parasympathetic ganglion that innervates the eye. Here, we examine its axial level of origin and developmental relationship to other ganglia and nerves of the head. Using small, focal injections of DiI, we show that neural crest cells arising from both the caudal half of the midbrain and the rostral hindbrain contribute to the ciliary as well as the trigeminal ganglion. Precursors to both ganglia have overlapping migration patterns, moving first ventrolaterally and then rostrally toward the optic vesicle. At the level of the midbrain/forebrain junction, precursors to the ciliary ganglion separate from the main migratory stream, turn ventromedially, and condense in the vicinity of the rostral aorta and Rathke's pouch. Ciliary neuroblasts first exit the cell cycle at early E2, prior to and during ganglionic condensation, and neurogenesis continues through E5.5. By E3, markers of neuronal differentiation begin to appear in this population. By labeling the ectoderm with DiI, we discovered a new placode, caudal to the eye and possibly contiguous to the trigeminal placode, that contributes a few early differentiating neurons to the ciliary ganglion, oculomotor nerve, and connecting branches to the ophthalmic nerve. These results suggest for the first time a dual neural crest and placodal contribution to the ciliary ganglion and associated nerves

Neuronal Differentiation from Postmitotic Precursors in the Ciliary Ganglion

In the chick ciliary ganglion, neuronal number is kept constant between St. 29 and St. 34 (E6–E8) despite a large amount of cell death. Here, we characterize the source of neurogenic cells in the ganglion as undifferentiated neural crest-derived cells. At St. 29, neurons and nonneuronal cells in the ciliary ganglion expressed the neural crest markers HNK-1 and p75^(NTR). Over 50% of the cells were neurons at St. 29; of the nonneuronal cells, a small population expressed glial markers, whereas the majority was undifferentiated. When placed in culture, nonneuronal cells acquired immunoreactivity for HuD, suggesting that they had commenced neuronal differentiation. The newly differentiated neurons arose from precursors that did not incorporate bromodeoxyuridine. To test whether these precursors could undergo neural differentiation in vivo, purified nonneuronal cells from St. 29 quail ganglia were transplanted into chick embryos at St. 9–14. Subsequently, quail cells expressing neuronal markers were found in the chick ciliary ganglion. The existence of this precursor pool was transient because nonneuronal cells isolated from St. 38 ganglia failed to form neurons. Since all ciliary ganglion neurons are born prior to St. 29, these results demonstrate that there are postmitotic neural crest-derived precursors in the developing ciliary ganglion that can differentiate into neurons in the appropriate environment