Differential distribution of competence for panplacodal and neural crest induction to non-neural and neural ectoderm.

It is still controversial whether cranial placodes and neural crest cells arise from a common precursor at the neural plate border or whether placodes arise from non-neural ectoderm and neural crest from neural ectoderm. Using tissue grafting in embryos of Xenopus laevis, we show here that the competence for induction of neural plate, neural plate border and neural crest markers is confined to neural ectoderm, whereas competence for induction of panplacodal markers is confined to non-neural ectoderm. This differential distribution of competence is established during gastrulation paralleling the dorsal restriction of neural competence. We further show that Dlx3 and GATA2 are required cell-autonomously for panplacodal and epidermal marker expression in the non-neural ectoderm, while ectopic expression of Dlx3 or GATA2 in the neural plate suppresses neural plate, border and crest markers. Overexpression of Dlx3 (but not GATA2) in the neural plate is sufficient to induce different non-neural markers in a signaling-dependent manner, with epidermal markers being induced in the presence, and panplacodal markers in the absence, of BMP signaling. Taken together, these findings demonstrate a non-neural versus neural origin of placodes and neural crest, respectively, strongly implicate Dlx3 in the regulation of non-neural competence, and show that GATA2 contributes to non-neural competence but is not sufficient to promote it ectopically.

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.

Cloning of two tryptophan hydroxylase genes expressed in the diencephalon of the developing zebrafish brain.

The monoamine serotonin (5-HT) exerts key neuromodulatory activities in all animal phyla, but the development and function of the serotonergic system is still incompletely understood. The zebrafish Danio rerio is an excellent model to approach this question since it is amenable to a combination of genetic, molecular and embryological studies. In order to characterize the organization of serotonergic neurons in the zebrafish we cloned two cDNAs encoding distinct forms of tryptophan hydroxylase (Tph), the rate-limiting enzyme in serotonin synthesis. We report here the pattern of expression of these two genes in relation with immunoreactive TH and 5-HT nuclei in the developing zebrafish embryo and early larva. tphD1 expression starts at 22 h post-fertilization (hpf) in the epiphysis and in basal spinal cells. Expression persists in the epiphysis until at least 4 days (dpf). Between 48 hpf and 3 dpf, tphD1 expression is initiated in retinal amacrine cells and in restricted preoptic and posterior tubercular nuclei within the basal diencephalon. At 3 and 4 dpf, tphD1 expression is newly initiated in the caudal hypothalamus and in branchial arches-associated neurons. tphD2 mRNA is detected transiently (between 30 somites and 32 hpf) in a restricted preoptic nucleus. All sites of tphD1 or D2 expression within the anterior central nervous system are also immunoreactive for 5-HT, but are not positive for TH. However, neither tphD gene is expressed in raphe nuclei, suggesting that additional tph gene(s) exist in zebrafish to account for 5-HT synthesis in that location. The co-expression of tphD1, tphD2 and 5-HT in the zebrafish diencephalon appears in striking contrast to the situation in mammals, where diencephalic serotonin results from re-uptake rather than from local production.

Cloning of two tryptophan hydroxylase genes expressed in the diencephalon of the developing zebrafish brain.

The monoamine serotonin (5-HT) exerts key neuromodulatory activities in all animal phyla, but the development and function of the serotonergic system is still incompletely understood. The zebrafish Danio rerio is an excellent model to approach this question since it is amenable to a combination of genetic, molecular and embryological studies. In order to characterize the organization of serotonergic neurons in the zebrafish we cloned two cDNAs encoding distinct forms of tryptophan hydroxylase (Tph), the rate-limiting enzyme in serotonin synthesis. We report here the pattern of expression of these two genes in relation with immunoreactive TH and 5-HT nuclei in the developing zebrafish embryo and early larva. tphD1 expression starts at 22 h post-fertilization (hpf) in the epiphysis and in basal spinal cells. Expression persists in the epiphysis until at least 4 days (dpf). Between 48 hpf and 3 dpf, tphD1 expression is initiated in retinal amacrine cells and in restricted preoptic and posterior tubercular nuclei within the basal diencephalon. At 3 and 4 dpf, tphD1 expression is newly initiated in the caudal hypothalamus and in branchial arches-associated neurons. tphD2 mRNA is detected transiently (between 30 somites and 32 hpf) in a restricted preoptic nucleus. All sites of tphD1 or D2 expression within the anterior central nervous system are also immunoreactive for 5-HT, but are not positive for TH. However, neither tphD gene is expressed in raphe nuclei, suggesting that additional tph gene(s) exist in zebrafish to account for 5-HT synthesis in that location. The co-expression of tphD1, tphD2 and 5-HT in the zebrafish diencephalon appears in striking contrast to the situation in mammals, where diencephalic serotonin results from re-uptake rather than from local production.

Connections of the ventral telencephalon (subpallium) in the zebrafish (Danio rerio).

Connections of the medial precommissural subpallial ventral telencephalon, i.e., dorsal (Vd, interpreted as part of striatum) and ventral (Vv, interpreted as part of septum) nuclei of area ventralis telencephali, were studied in the zebrafish (Danio rerio) using two tracer substances (DiI or biocytin). The following major afferent nuclei to Vd/Vv were identified: medial and posterior pallial zones of dorsal telencephalic area, and the subpallial supracommissural and postcommissural nuclei of the ventral telencephalic area, the olfactory bulb, dorsal entopeduncular, anterior and posterior parvocellular preoptic and suprachiasmatic nuclei, anterior, dorsal and central posterior dorsal thalamic, as well as rostrolateral nuclei, periventricular nucleus of the posterior tuberculum, posterior tuberal nucleus, various tuberal hypothalamic nuclei, dorsal tegmental nucleus, superior reticular nucleus, locus coeruleus, and superior raphe nucleus. Efferent projections of the ventral telencephalon terminate in the supracommissural nucleus of area ventralis telencephali, the posterior zone of area dorsalis telencephali, habenula, periventricular pretectum, paracommissural nucleus, posterior dorsal thalamus, preoptic region, midline posterior tuberculum (especially the area dorsal to the posterior tuberal nucleus), tuberal (midline) hypothalamus and interpeduncular nucleus. Strong reciprocal interconnections likely exist between septum and preoptic region/midline hypothalamus and between striatum and dorsal thalamus (dopaminergic) posterior tuberculum. Regarding ascending activating/modulatory systems, the pallium shares with the subpallium inputs from the (noradrenergic) locus coeruleus, and the (serotoninergic) superior raphe, while the subpallium additionally receives such inputs from the (dopaminergic) posterior tuberculum, the (putative cholinergic) superior reticular nucleus, and the (putative histaminergic) caudal hypothamalic zone.

Development of the catecholaminergic system in the early zebrafish brain: an immunohistochemical study.

Tyrosine hydroxylase-containing cells (TH cells) were investigated immunohistochemically in early and late postembryonic zebrafish brain sections (at 2 and 5 days postfertilization [dpf]) yielding an improved neuroanatomical resolution of spatiotemporal developmental dynamics of the catecholaminergic system. Additionally, double-immunolabel preparations for visualizing TH cells and cells containing the proliferating cell nuclear antigen (PCNA cells) were carried out allowing for a prosomeric interpretation of early forebrain TH cell clusters. Many TH cell populations recently described in the adult zebrafish brain could be identified in the present study by location and cell type already in the 5 dpf (e.g. eight of 12 adult diencephalic TH cell populations) and 2 dpf (e.g. five of 12 adult TH cell populations) zebrafish brain. Early and adult diencephalic TH cells are restricted to the pretectum (P1) and ventral thalamus (P3) in the alar plate, and to various TH groups in the basal plate posterior tuberculum (P3), as well as to various populations in the hypothalamus (secondary prosencephalon). The alar plate ventral thalamic and most anterodorsal posterior tubercular TH cell populations range among the earliest detectable ones. There was no indication of migration of TH cells from the midbrain-hindbrain boundary or anterior neural ridge into the diencephalon.

The teleostean forebrain: a comparative and developmental view based on early proliferation, Pax6 activity and catecholaminergic organization.

An improved comparative interpretation of the teleostean forebrain suggests that the dorsal tier (Vd,Vc) and ventral tier (Vv,Vl) nuclei of the ventral telencephalic area (subpallium) represent the striatum and septum, respectively. Among other arguments, a dopaminergic innervation originating in the diencephalic posterior tubercle reaches Vd and dense efferents of Vv project to the midline hypothalamus in the adult zebrafish subpallium. The adult area dorsalis telencephali represents the teleostean pallium. Regulatory genes typically expressed in the early amniote subpallium (e.g., Dlx-1) are also restricted to the presumptive zebrafish ventral telencephalic area. Further, early Pax6 protein distribution in the zebrafish telencephalon corresponds to the migrating stream noted at the pallial-subpallial boundary in amniotes, but a ventricular, radial glia-based expression in the pallium is absent. The peripherally migrated, adult diencephalic preglomerular complex of the basal plate posterior tubercle (early: M2) provides sensory inputs to the pallium. Early Pax6 protein distribution indicates that at least part of M2 may directly originate from alar plate ventral thalamic Pax6-expressing cells. Dopaminergic cells of the basal plate posterior zebrafish forebrain (P1-P3) are restricted to the ventral thalamic prosomere (P3), including those forming the adult ascending dopaminergic system. Moreover, the latter likely depend developmentally on the dorsally adjacent alar plate Pax6-expressing cells.

Connections of the ventral telencephalon and tyrosine hydroxylase distribution in the zebrafish brain (Danio rerio) lead to identification of an ascending dopaminergic system in a teleost.

We studied the connections and catecholaminergic organization of the subpallium in the zebrafish, in particular to demonstrate the origin of the ascending dopaminergic system of teleosts, by using the tracers DiI or biocytin in combination with tyrosine hydroxylase (TH) immunohistochemistry. Retrogradely labeled cells were found in the olfactory bulb, the area dorsalis telencephali, the preoptic region, the dorsal and ventral thalamus, the posterior tubercle, the preglomerular region, and the medulla oblongata. Moreover, the zebrafish subpallium has strong reciprocal connections with the tuberal hypothalamus. Double-labeled cells (for TH and tracer) were identified in two locations of the rostral posterior tubercle: small round neurons in its periventricular nucleus and large pear-shaped cells adjacent to it. These double-labeled cells of the posterior tubercle presumably represent the teleostean dopaminergic system ascending to the striatum.

Secondary neurogenesis in the brain of the African clawed frog, Xenopus laevis, as revealed by PCNA, Delta-1, Neurogenin-related-1, and NeuroD expression.

After primary neurogenesis in the Xenopus laevis embryo, a massive new surge of neurogenesis and related neurogenic and proneural gene expression occurs in the spinal cord at the beginning of the larval period (starting at Stage 46), which corresponds to well-documented secondary neurogenesis in larval zebrafish central nervous system development. Here, we document related neural proliferation and gene expression patterns in the brain of Xenopus, in various embryonic and larval stages, showing the distribution of proliferative cells (immunostaining of cells containing the proliferating cell nuclear antigen; the auxiliary protein of DNA polymerase delta; PCNA), and the activity of some critical genes expressed during neurogenesis (i.e., Delta-1, Neurogenin-related-1, NeuroD). This study reveals that the early larval stage in Xenopus (Stage 48) displays patterns of proliferation (PCNA), as well as of neurogenic (Delta-1) and proneural (Ngnr-1; NeuroD) gene expression that are qualitatively almost identical to those seen in the 3-day postembryonic zebrafish or the 12.5/13.5-day embryonic mouse. Furthermore, a comparable bauplan of early proliferation zones (including their neuromeric organization) as described in the postembryonic zebrafish apparently exists in tetrapods (Xenopus). Altogether, the data presented suggest a common brain bauplan on the level of early proliferation patterns and neurogenic/proneural gene activity in anamniotes, if not vertebrates.