The Independent Evolution of Dorsal Pallia in Multiple Vertebrate Lineages.

Comparative neurobiologists have long wondered when and how the dorsal pallium (e.g., mammalian neocortex) evolved. For the last 50 years, the most widely accepted answer has been that this structure was already present in the earliest vertebrates and, therefore, homologous between the major vertebrate lineages. One challenge for this hypothesis is that the olfactory bulbs project throughout most of the pallium in the most basal vertebrate lineages (notably lampreys, hagfishes, and lungfishes) but do not project to the putative dorsal pallia in teleosts, cartilaginous fishes, and amniotes (i.e., reptiles, birds, and mammals). To make sense of these data, one may hypothesize that a dorsal pallium existed in the earliest vertebrates and received extensive olfactory input, which was subsequently lost in several lineages. However, the dorsal pallium is notoriously difficult to delineate in many vertebrates, and its homology between the various lineages is often based on little more than its topology. Therefore, we suspect that dorsal pallia evolved independently in teleosts, cartilaginous fishes, and amniotes. We further hypothesize that the emergence of these dorsal pallia was accompanied by the phylogenetic restriction of olfactory projections to the pallium and the expansion of inputs from other sensory modalities. We do not deny that the earliest vertebrates may have possessed nonolfactory sensory inputs to some parts of the pallium, but such projections alone do not define a dorsal pallium.

Electrosensory ampullary organs are derived from lateral line placodes in cartilaginous fishes.

Ampullary organ electroreceptors excited by weak cathodal electric fields are used for hunting by both cartilaginous and non-teleost bony fishes. Despite similarities of neurophysiology and innervation, their embryonic origins remain controversial: bony fish ampullary organs are derived from lateral line placodes, whereas a neural crest origin has been proposed for cartilaginous fish electroreceptors. This calls into question the homology of electroreceptors and ampullary organs in the two lineages of jawed vertebrates. Here, we test the hypothesis that lateral line placodes form electroreceptors in cartilaginous fishes by undertaking the first long-term in vivo fate-mapping study in any cartilaginous fish. Using DiI tracing for up to 70 days in the little skate, Leucoraja erinacea, we show that lateral line placodes form both ampullary electroreceptors and mechanosensory neuromasts. These data confirm the homology of electroreceptors and ampullary organs in cartilaginous and non-teleost bony fishes, and indicate that jawed vertebrates primitively possessed a lateral line placode-derived system of electrosensory ampullary organs and mechanosensory neuromasts.

Evolution of centralized nervous systems: two schools of evolutionary thought.

Understanding the evolution of centralized nervous systems requires an understanding of metazoan phylogenetic interrelationships, their fossil record, the variation in their cephalic neural characters, and the development of these characters. Each of these topics involves comparative approaches, and both cladistic and phenetic methodologies have been applied. Our understanding of metazoan phylogeny has increased greatly with the cladistic analysis of molecular data, and relaxed molecular clocks generally date the origin of bilaterians at 600-700 Mya (during the Ediacaran). Although the taxonomic affinities of the Ediacaran biota remain uncertain, a conservative interpretation suggests that a number of these taxa form clades that are closely related, if not stem clades of bilaterian crown clades. Analysis of brain-body complexity among extant bilaterians indicates that diffuse nerve nets and possibly, ganglionated cephalic neural systems existed in Ediacaran organisms. An outgroup analysis of cephalic neural characters among extant metazoans also indicates that the last common bilaterian ancestor possessed a diffuse nerve plexus and that brains evolved independently at least four times. In contrast, the hypothesis of a tripartite brain, based primarily on phenetic analysis of developmental genetic data, indicates that the brain arose in the last common bilaterian ancestor. Hopefully, this debate will be resolved by cladistic analysis of the genomes of additional taxa and an increased understanding of character identity genetic networks.

Variation in reptilian brains and cognition.

The class Reptilia is monophyletic, if all synapsid tetrapods are excluded and birds are included. The phylogenetic position of turtles within the reptilian clade is still problematic, but recent microRNA data suggest that turtles are the sister group to lepidosaurians. Brain-body data for approximately 60 reptilian taxa indicate that the relative brain size for a given body weight varies some six-fold among reptiles, with some turtles and lizards having relatively large brains and other turtles and lizards having relatively small brains. Snakes appear to be characterized by relatively small brains, and crocodilians appear to possess the largest brains among living reptiles, with the exception of birds. Data on the relative size of major brain divisions among tetrapods are limited, but the telencephalic and cerebellar hemispheres account for much of the variation. Telencephalic hemispheres in reptiles are approximately twice as large as those in amphibians, and the relative size of the telencephalic hemispheres in monitor lizards and crocodilians approaches that in basal birds and mammals. New data on the relative volumes of telencephalic pallial divisions in tetrapods reveal that the dorsal ventricular ridge, a ventral pallial derivative, accounts for much of the increase in pallial size that characterizes reptiles. Studies of spatial and visual cognition in nonavian reptiles reveal that they learn mazes and make visual discriminations as rapidly as most birds and mammals. Studies of social cognition and novel behavior, including play, reveal levels of complexity not previously believed to exist among nonavian reptiles. Given this level of neural and cognitive complexity, it is possible that consciousness has evolved numerous times, independently, among reptiles.

Comparative analysis of the organization of the cholinergic system in the brains of two holostean fishes, the Florida gar Lepisosteus platyrhincus and the bowfin Amia calva.

The cholinergic system in the brain has been widely studied in most vertebrate groups, but there is no information available about this neurotransmission system in the brains of holostean fishes, a primitive and poorly understood group of actinopterygian fishes. The present study provides the first detailed information on the distribution of cholinergic cell bodies and fibers in the central nervous system in two holostean species, the Florida gar, Lepisosteus platyrhincus, and the bowfin, Amia calva. Immmunohistochemistry against the enzyme choline acetyltransferase (ChAT) revealed distinct groups of ChAT-immunoreactive (ChAT-ir) cells in the habenula, isthmic nucleus, laterodorsal tegmental nucleus, octavolateral area, reticular formation, cranial nerve motor nuclei and the motor column of the spinal cord, all of which seem to be highly conserved among vertebrates. Some ChAT-ir cells were detected in the basal telencephalon that appear in actinopterygians for the first time in the evolution of this neurotransmission system, whereas the remarkable cholinergic population in the optic tectum is a peculiar characteristic, the presence of which varies throughout evolution, although it is present in all teleosts studied. Abundant cholinergic fibers were found in the pretectal region and optic tectum, where they probably modulate vision, and in the hypothalamus and the interpeduncular neuropil. Some interspecific differences were also observed, such as the presence of ChAT-ir cells in the supraoptoparaventricular band only in Lepisosteus and in in the nucleus subglomerulosus only in Amia. In addition, ChAT-ir fibers in the olfactory bulb were detected only in Amia. Comparison of these results with those from other classes of vertebrates, and a segmental analysis to correlate cell populations, reveal that the pattern of the cholinergic system in holosteans is very close to that in ancestral actinopterygian fishes, as recently described in the bichir (Cladistia), although an important evolutionary novelty in holosteans is the presence of cholinergic cells in the basal telencephalon.

Olfactory projections in the lepidosirenid lungfishes.

Olfactory nerve and olfactory bulb projections in lepidosirenid lungfishes were experimentally determined with neural tracers. Unilateral injections of DiI into the olfactory nerve labeled the accessory and main olfactory bulbs as well as fibers of the anterior root of the terminal nerve, which terminates extensively in cell groups of the medial hemispheric wall, the dorsal and lateral pallia, and the preoptic nuclei and posterior tubercle. Lepidosirenid lungfishes do not exhibit separate vomeronasal nerves, but previous data indicate that calbindin-positive receptors within basal crypts of the olfactory epithelium are homologous to the vomeronasal organ of tetrapods. Unilateral injections of DiI into the accessory olfactory bulb reveal an accessory olfactory tract which terminates primarily if not solely in the ipsilateral medial amygdalar nucleus as in amphibians. Unilateral injections of tracers into the main olfactory bulb reveal extensive projections to all cell groups in the ipsilateral telencephalic hemisphere, except for the medial amygdalar nucleus, as well as secondary olfactory projections (decussating in the habenular commissure) to the contralateral dorsal pallium and main olfactory bulb. Secondary olfactory projections also terminate bilaterally in diencephalic and midbrain centers after partial decussation in the anterior and postoptic commissures, as well as in the ventral hypothalamus and posterior tubercle. Cladistic analysis of the extensive secondary olfactory projections indicates that this pattern is primitive for all bony fishes whereas the reduction in secondary olfactory projections in amphibians, particularly anurans, is a derived, simplified pattern.

Forebrain organization in elasmobranchs.

It has long been known that many elasmobranch fishes have relatively large brains. The telencephalon, in particular, has increased in size in several groups, and as a percent of total brain weight, it is as large as in some mammals. Little is known, however, about the organization, connections, and functions of the telencephalon in elasmobranchs. Early experimental studies indicated that olfaction does not dominate the telencephalon and that other sensory modalities are represented, particularly in the pallium. We have investigated the intrinsic and extrinsic connections of the telencephalon in two elasmobranch species: the thornback guitarfish, Platyrhinoidis triseriata, and the spiny dogfish, Squalus acanthias. Tracers were injected into various parts of the forebrain and olfactory pathways were found to be extensive and were seen to involve the pallium. Injections into various parts of the pallium revealed a major input from the area basalis, which receives secondary and tertiary olfactory fibers. Nonolfactory input from the diencephalon appeared relatively minor and seemed to converge with olfactory information in the dorsal pallium and area superficialis basalis. Major descending projections were seen to originate in the dorsal pallium and terminate in the hypothalamus and - in the case of Platyrhinoidis - massively in the lateral mesencephalic nucleus. Descending pathways appeared mainly crossed in Platyrhinoidis, but not in Squalus. Our data indicate that the concept of the dorsal pallium as a nonolfactory area in elasmobranchs must be reconsidered, and we suggest that many telencephalic centers, including the dorsal pallium, are involved in olfactory orientation.

Connections of the medial telencephalic wall in the spotted African Lungfish.

The extent and boundaries of the roof, or pallium, of the telencephalon in lungfishes have been debated for over 30 years, and two hypotheses exist. Proponents of a restricted pallium claim that the medial border of the pallium occurs in a dorsal position and that the entire medial hemispheric wall is formed by the septal nuclei. Proponents of an extended pallium claim that the medial border of the pallium occurs in a more ventral position and that the medial hemispheric wall is divided into a dorsal medial pallium and ventral septal nuclei, as in amphibians. Immunohistochemical data have generally been interpreted to support the hypothesis of an extended pallium, but disagreement still exists. To clarify the extent of the pallium in lungfishes, the connections of the dorsal and ventral divisions of the medial hemispheric wall in the Spotted African Lungfish were examined using a number of neuronal tracers. In amphibians and other tetrapods, the afferent projections to the medial pallium and the septal nuclei differ extensively, as do the commissural routes taken by decussating interhemispheric connections. Although the descending projections of the medial pallium and septal nuclei are very similar to one another in amphibians and other tetrapods, they do differ in that the septal nuclei and the ventral thalamus are extensively interlinked, whereas the medial pallium lacks such connections. These differences also characterize the connections of the dorsal and ventral divisions of the medial hemispheric wall in the Spotted African Lungfish, which supports the hypothesis of an extended pallium. The telencephalic organization in lungfishes thus appears remarkably similar to that in amphibians and reflects a pattern that almost certainly existed in the last common ancestor of lungfishes and tetrapods.

Immunohistochemical localization of calbindin-D28k and calretinin in the spinal cord of lungfishes.

A common pattern of distribution of neurons and fibers containing the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) in the spinal cord of terrestrial vertebrates has been recently demonstrated. Lungfishes are considered the closest living relatives of tetrapods, but practically no experimental data exist on the organization of their spinal cord. By means of immunohistochemical techniques, the localization of CB and CR was investigated in the spinal cord of the African (Protopterus dolloi) and Australian (Neoceratodus forsteri) lungfishes. Abundant cell bodies and fibers immunoreactive for either CB or CR were widely distributed throughout the spinal cord. A large population of immunoreactive cells was found in the dorsal column of the gray matter in both species, and abundant cells were distributed in the lateral and ventral columns. Ventrolateral motoneurons and multipolar cells were only intensely CB and CR immunoreactive in Neoceratodus. For the most part, separate cell populations contained either CB or CR, but a small subset of dorsally located neurons contained both in the two lungfishes. Colocalization was found in motoneurons and in ventrolaterally located cells only in Neoceratodus. Fiber labeling showed a predominance of CR-containing axons in the lateral and ventral funiculi of presumed supraspinal origin. These results show that lung-fishes and tetrapods have many features in common, suggesting that primitive anatomical, and likely functional, organization of the spinal cord of tetrapods is present in lungfishes.

Telencephalic organization in the spotted African Lungfish, Protopterus dolloi: a new cytological model.

Recent studies have greatly increased our knowledge of telencephalic organization in ray-finned fishes and terrestrial vertebrates, particularly amphibians. In contrast, little new information has been generated on telencephalic organization in lobe-finned fishes. The coelacanth, Latimeria, and three genera of lungfishes constitute the living lobe-finned fishes. Latimeria is extremely rare and critically endangered, so the living lungfishes, therefore, offer the only feasible source of new information on telencephalic organization in lobe-finned fishes. A re-examination of the cytoarchitectonics of the telencephalon in the Spotted African Lungfish has allowed the generation of a new model of telencephalic organization in lungfishes. To begin to test this model, examination was made of the telencephalic distribution of acetylcholinesterase, enkephalin, the neurotensin-related hexapeptide LANT6, nitric oxide synthase (nicotinamide adenine dinucleotide phosphate-diaphorase), substance P, and tyrosine hydroxylase. This distribution supports the new model and suggests that the medial pallial-subpallial border, the striatopallidal systems, and the amygdalar organization in this lungfish are more similar to these features in terrestrial vertebrates than was previously suspected.

An immunohistochemical approach to lungfish telencephalic organization.

The brains of lungfishes have received little attention in comparative neuroanatomy, in spite of the strategic position of these fishes in phylogeny, in that they are currently recognized as the closest living relatives of tetrapods. The neglect has probably been due to the difficulty of obtaining these unique animals, which comprise (along with the coelacanths) the only extant representatives of the lobe-finned fishes. Several previous studies have established the basic anatomy of the telencephalon in lungfishes, but many aspects of the intrinsic organization of the main telencephalic subregions have remained unclear. The immunohistochemical localization of diverse neurotransmitters, neuromodulators, and transcription factors expressed by genes involved in brain regionalization has served to clarify many aspects of the organization of the telencephalon in lungfishes. Here we describe the main immunohistochemical features of the telencephalon in two lungfish species, Protopterus dolloi and Neoceratodus forsteri. Our analysis highlights the common traits shared by lungfishes and tetrapods. These include four pallial regions, distinct striatal and pallidal components of the basal ganglia, specific regionalization of the septum, and the presence of three amygdaloid regions. In general, the use of immunohistochemistry in the study of the telencephalon of lungfishes reveals that this structure is notably more complex than previously thought and that it possesses all major subregions recognized in amphibians and amniotes.

Forebrain evolution in bony fishes.

The bony fishes consist of ray-finned fishes and lobe-finned fishes. In ray-finned fishes, the forebrain forms a morphocline from the cladistian bichirs through teleosts regarding the number and increasing complexity of pallial connections. The nuclei of the posterior tubercle parallel this increase in complexity, but the dorsal thalamic nuclei do not. The primary targets of the dorsal thalamic nuclei are the subpallial nuclei, whereas the primary targets of the posterior tubercle are various pallial divisions. Primitively, nucleus medianus is the primary projection nucleus of the posterior tubercle. It is either reduced or lost in teleosts, and its role is taken over by the preglomerular complex, which appears to develop from proliferative zones in both the thalamic alar plate and the posterior tubercle. Although there are numerous hodological data for the pallium in ray-finned fishes, there is no consensus regarding its homologies with other vertebrates. In contrast to ray-finned fishes, very few experimental data exist for lobe-finned fishes. The coelacanth, Latimeria, is extremely rare, and lungfishes are the best source for new experimental data. At this point, there are sufficient data to suggest that lungfishes are characterized by a pallium that is divided into four components, separate dorsal and ventral striatopallidal systems, and an amygdala that consists of anterior, central, lateral, and medial nuclei. The data suggest that telencephalic organization in lungfishes is far more similar to that in amphibians than was previously suspected.

Conserved pattern of OTP-positive cells in the paraventricular nucleus and other hypothalamic sites of tetrapods.

The paraventricular nucleus complex (Pa) is a component of central neural circuitry that regulates several homeostatic variables. The paraventricular nucleus is composed of magnocellular neurons that project to the posterior pituitary and parvicellular neurons that project to numerous sites in the central nervous system. According to the revised prosomeric model, the paraventricular nucleus is located caudal to the eye stalk along the rostrocaudal dimension of the dorsal hypothalamic alar plate. Caudally, the paraventricular nucleus abuts the prethalamus (prosomere 3), and the entire complex is flanked ventrally and dorsally by Dlx5-expressing domains of the alar plate. The homeodomain transcription factor Orthopedia (Otp) is expressed in several separate hypothalamic sites: the paraventricular nucleus, perimammillary region and arcuate nucleus. In this study, we compared Otp expression in the hypothalamus of mouse (Mus musculus), chick (Gallus gallus), frog (Rana perezi) and axolotol (Ambystoma mexicanum), using immunohistochemical and in situ hybridization techniques. In all cases, Otp-positive cells in the paraventricular nucleus were excluded from Dlx5-expressing adjacent domains. Other positive neuronal populations were observed in the arcuate nucleus and oblique perimammillary band. Expression in the medial amygdala appears to be continuous with the Otp-expressing paraventricular nucleus complex. This area is relatively unevaginated in the amphibian brains, barely evaginated in the chick, and fully evaginated in the mouse. These data led us to conclude that the expression pattern of Otp is topologically highly conserved in tetrapods and is plesiomorphic among chordates.

Organization of major telencephalic pathways in an elasmobranch, the thornback ray Platyrhinoidis triseriata.

The forebrain of elasmobranchs is well developed, and in some species the relative brain/body weight is comparable to that in mammals. However, little is known about the organization of major telencephalic pathways. We injected biotinylated dextran amines into the olfactory bulb, lateral pallium, dorsomedial pallium, and the forebrain bundles of the thornback ray, Platyrhinoidis triseriata. Secondary olfactory fibers from the bulb innervate the lateral pallium, the ventral division of the rostral telencephalon and area superficialis basalis. Retrogradely labeled cells were seen exclusively in the lateral periventricular area. The projections of the lateral pallium appeared basically similar to those of the olfactory bulb, but labeling was much denser in the superficial part of area basalis. Some fibers were also seen to innervate the posterior tuberal nucleus. Injections into the dorsomedial pallium revealed a major input from area basalis. Only a few cells were retrogradely labeled in the dorsal thalamus and posterior lateral thalamic nucleus. Major efferents of the dorsomedial pallium appear to reach the contralateral inferior lobe of the hypothalamus and the lateral mesencephalic nucleus. Tracer injections into the forebrain bundles retrogradely labeled many cells in the diencephalon and the mesencephalon and also revealed terminal fields in area superficialis basalis. In addition, a large number of cells were labeled in the dorsomedial pallium. Descending telencephalic fibers innervate heavily the inferior lobes and the lateral mesencephalic nucleus. Our results show that higher order olfactory pathway courses from the lateral pallium through area basalis to the dorsomedial pallium and that ascending non-olfactory input is integrated in area superficialis basalis and the dorsal pallium along with olfactory information, rather than being processed in separate, non-olfactory centers.

Regional chemoarchitecture of the brain of lungfishes based on calbindin D-28K and calretinin immunohistochemistry.

Lungfishes are the closest living relatives of land vertebrates, and their neuroanatomical organization is particularly relevant for deducing the neural traits that have been conserved, modified, or lost with the transition from fishes to land vertebrates. The immunohistochemical localization of calbindin (CB) and calretinin (CR) provides a powerful method for discerning segregated neuronal populations, fiber tracts, and neuropils and is here applied to the brains of Neoceratodus and Protopterus, representing the two extant orders of lungfishes. The results showed abundant cells containing these proteins in pallial and subpallial telencephalic regions, with particular distinct distribution in the basal ganglia, amygdaloid complex, and septum. Similarly, the distribution of CB and CR containing cells supports the division of the hypothalamus of lungfishes into neuromeric regions, as in tetrapods. The dense concentrations of CB and CR positive cells and fibers highlight the extent of the thalamus. As in other vertebrates, the optic tectum is characterized by numerous CB positive cells and fibers and smaller numbers of CR cells. The so-called cerebellar nucleus contains abundant CB and CR cells with long ascending axons, which raises the possibility that it could be homologized to the secondary gustatory nucleus of other vertebrates. The corpus of the cerebellum is devoid of CB and CR and cells positive for both proteins are found in the cerebellar auricles and the octavolateralis nuclei. Comparison with other vertebrates reveals that lungfishes share most of their features of calcium binding protein distribution with amphibians, particularly with salamanders.

Immunohistochemical organization of the forebrain in the white sturgeon, Acipenser transmontanus.

The distribution of substance P (SP), leucine-enkephalin (LENK), serotonin (5HT), dopamine (DA), and tyrosine hydroxylase (TH) was examined in the forebrain of the white sturgeon in order to evaluate several anatomical hypotheses based on cytoarchitectonics, and to gain a better understanding of the evolution of the forebrain in ray-finned fishes. The subpallium of the telencephalon has the highest concentration of the neuropeptides SP and LENK, allowing the pallial-subpallial border to be easily distinguished. The distribution of dopamine is similar to that of serotonin in the subpallium, fibers positive for these transmitters are particularly dense in the dorsal and ventral divisions of the subpallium. In addition, a small population of DA- and 5HT-positive cell bodies--which appear to be unique to sturgeons--was identified at the level of the anterior commissure. The internal granular layer of the olfactory bulbs had large numbers of TH-positive cell bodies and fibers, as did the rostral subpallium. The occurrence of cell bodies positive for LENK in the dorsal nucleus of the rostral subpallium supports the hypothesis that this nucleus is homologous to the striatum in other vertebrates. This is further reinforced by the apparent origin of an ascending dopaminergic pathway from cells in the posterior tubercle that are likely homologous to the ventral tegmental area/substantia nigra in land vertebrates. Finally, the differential distribution of SP and TH in the pallium supports the hypothesis that the pallium, or area dorsalis, can be divided medially into a rostral division (Dm), a caudal division (Dp) that is the main pallial target of secondary olfactory projections, and a narrow lateral division (Dd+Dl) immediately adjacent to the attachment of the tela choroidea along the entire rostrocaudal length of the telencephalic hemisphere.

Connections of the lateral and medial divisions of the goldfish telencephalic pallium.

Biotinylated dextran amine and fluorescent carbocyanine dye (DiI) were used to examine connections of the lateral (Dl) and medial (Dm) divisions of the goldfish pallium. Besides numerous intrinsic telencephalic connections to Dl and Dm, major ascending projections to these pallial divisions arise in the preglomerular complex of the posterior tuberculum, rather than in the dorsal thalamus. The rostral subnucleus of the lateral preglomerular nucleus receives auditory input via the medial pretoral nucleus, lateral line input via the ventrolateral toral nucleus, and visual input via the optic tectum, and it projects to both Dl and Dm. The anterior preglomerular nucleus and caudal subnucleus of the lateral preglomerular nucleus receive auditory input via the central toral nucleus and project to Dm. This pallial division also receives chemosensory information via the medial preglomerular nucleus. The central posterior (CP) nucleus, which receives both auditory and visual inputs, also projects to Dm and is the only dorsal thalamic nucleus projecting to the pallium. Thus, both Dl and Dm clearly receive multisensory inputs. Major projections of CP and projections of all other dorsal thalamic nuclei are to the subpallium, however. Descending projections of Dl are primarily to the preoptic area and the caudal hypothalamus, whereas descending projections of Dm are more extensive and particularly heavy to the anterior tuber and nucleus diffusus of the hypothalamus. The topography and connections of Dl are remarkably similar to those of the hippocampus of tetrapods, whereas the topography and connections of Dm are similar to those of the amygdala.

Taste bud development in the channel catfish.

Taste bud formation in channel catfish is first seen to occur in stage 39 embryos, when taste bud primordia (stage 1), consisting of three to five cells, including a single calretinin-positive cell, can be recognized within the oropharyngeal cavity and maxillary barbels. Within a short time (stage 40), stage 2 taste bud primordia are apparent and include two or three calretinin-positive cells. The number of calretinin-positive cells continues to increase (stage 3), and the primordia begin to erupt as mature taste buds (stage 4) by embryonic stage 48. This same pattern of taste bud development characterizes other regions of the head, with calretinin-positive cells first detected around the mouth and on the other barbels by stage 41 and on the rest of the head by stage 48. The development of trunk taste buds lags far behind that of the head, with the first calretinin-positive cells occurring on the lobes of the caudal fin by stage 48 and on the remaining fins by stage 50. Taste bud primordia on the trunk proper do not begin to appear until stage 53, when the larvae begin to feed, and these receptors begin to erupt only in 1-week-old larvae. Fibers of the facial nerve, which innervate all external taste buds, ramify within the ectoderm prior to the first appearance of taste bud primordia or their precursors.