Gross Joshua B, Gottlieb Aaron A, Barlow Linda A
Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford St, MCZ 115, Cambridge, MA 02138, USA.
Dev Biol. 2003 Dec 15;264(2):467-81. doi: 10.1016/j.ydbio.2003.08.024.
Taste buds are multicellular receptor organs innervated by the VIIth, IXth, and Xth cranial nerves. In most vertebrates, taste buds differentiate after nerve fibers have reached the lingual epithelium, suggesting that nerves induce taste buds. However, under experimental conditions, taste buds of amphibians develop independently of innervation. Thus, rather than being induced by nerves, the developing taste periphery likely regulates ingrowing nerve fibers. To test this idea, we devised a culture approach using axolotl embryos. Gustatory neurons were generated from cultured epibranchial placodes, and when cultured alone, axon outgrowth was random over 4 days, a time period coincident with axon growth to the periphery in vivo. In contrast, cocultures of placodal neurons with oropharyngeal endoderm (OPE), the normal taste bud-containing target for these neurons, resulted in neurite growth toward the target tissue. Unexpectedly, placodal neurons also grew toward flank ectoderm (FE), which these neurons do not encounter in vivo. To compare further the impact of OPE and FE explants on gustatory neurons, cocultures were extended and examined at 6, 8, and 10 days, when, in vivo, placodal fibers have innervated the epithelium but prior to taste bud formation, when taste buds have differentiated and are innervated, and when the mouth has opened and larvae have begun to feed, respectively. The behavior of placodal axons with respect to target type did not differ between OPE and FE cocultures at 6 days. However, by 8 days, differences in axonal outgrowth were observed with respect to target type, and these differences were enhanced by 10 days in vitro. Most clearly, exuberant placodal fibers grew in 10-day OPE cocultures, and numerous neurites had invaded OPE explants by this time, whereas gustatory neurites were sparse in FE cocultures, and rarely approached and almost never contacted FE explants. Thus, embryonic endoderm destined to give rise to taste buds specifically attracts its innervation early in development, as placodal neurons send out axons. Later, when gustatory axons synapse with differentiated taste buds in vivo, the OPE provides trophic support for cultured gustatory neurons.
味蕾是由第七、第九和第十对脑神经支配的多细胞感受器器官。在大多数脊椎动物中,味蕾在神经纤维到达舌上皮后才分化,这表明神经诱导了味蕾的形成。然而,在实验条件下,两栖动物的味蕾在没有神经支配的情况下也能发育。因此,发育中的味觉外周组织可能不是由神经诱导的,而是对向内生长的神经纤维起到调节作用。为了验证这一观点,我们设计了一种使用蝾螈胚胎的培养方法。味觉神经元由培养的鳃上板产生,单独培养时,轴突在4天内随机生长,这段时间与体内轴突向外周生长的时间一致。相比之下,将鳃上板神经元与口咽内胚层(OPE)(这些神经元正常的含味蕾靶组织)共培养时,神经突会朝着靶组织生长。出乎意料的是,鳃上板神经元也会朝着体侧外胚层(FE)生长,而这些神经元在体内不会接触到体侧外胚层。为了进一步比较OPE和FE外植体对味觉神经元的影响,共培养持续进行,并在第6、8和10天进行检查,这三个时间点分别对应于体内鳃上板纤维支配上皮但在味蕾形成之前、味蕾分化并被神经支配时以及口张开且幼虫开始进食时。在第6天,OPE和FE共培养中,鳃上板轴突相对于靶组织类型的行为没有差异。然而,到第8天,观察到轴突生长在靶组织类型方面存在差异,并且在体外培养10天时这些差异更加明显。最明显的是,在10天的OPE共培养中,大量的鳃上板纤维生长,此时许多神经突侵入了OPE外植体,而在FE共培养中味觉神经突稀疏,很少接近且几乎从不接触FE外植体。因此,注定要产生味蕾的胚胎内胚层在发育早期特异性地吸引其神经支配,因为鳃上板神经元发出轴突。后来,当味觉轴突在体内与分化的味蕾形成突触时,OPE为培养的味觉神经元提供营养支持。