T-brain-1: a homolog of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex.

The mechanisms that regulate regional specification and evolution of the cerebral cortex are obscure. To this end, we have identified and characterized a novel murine and human gene encoding a putative transcription factor related to the Brachyury (T) gene that is expressed only in postmitotic cells. T-brain-1 (Tbr-1) mRNA is largely restricted to the cerebral cortex, where during embryogenesis it distinguishes domains that we propose may give rise to paleocortex, limbic cortex, and neocortex. Tbr-1 and Id-2 expression in the neocortex have discontinuities that define molecularly distinct neocortical areas. Tbr-1 expression is analyzed in the context of the prosomeric model. Topological maps are proposed for the organization of the dorsal telencephalon.

A model of early molecular regionalization in the chicken embryonic pretectum.

The pretectal region of the brain is visualized as a dorsal region of prosomere 1 in the caudal diencephalon, including derivatives from both the roof and alar plates. Its neuronal derivatives in the adult brain are known as pretectal nuclei. The literature is inconsistent about the precise anteroposterior delimitation of this region and on the number of specific histogenetic domains and subdomains that it contains. We performed a cross-correlated gene-expression map of this brain area in chicken embryos, with the aim of identifying differently fated pretectal domains on the basis of combinatorial gene expression patterns. We examined in detail Pax3, Pax6, Pax7, Tcf4, Meis1, Meis2, Nkx2.2, Lim1, Dmbx1, Dbx1, Six3, FoxP2, Zic1, Ebf1, and Shh mRNA expression, as well as PAX3 and PAX7 immunoreaction, between stages HH11 and HH28. The patterns analyzed serve to fix the cephalic and caudal boundaries of the pretectum and to define three molecularly distinct anteroposterior pretectal domains (precommissural, juxtacommissural, and commissural) and several dorsoventral subdomains. These molecular specification patterns are established step by step between stages HH10 and HH18, largely before neurogenesis begins. This set of gene-architectonic data constitutes a useful scaffold for correlations with fate maps and other experimental embryologic results and may serve as well for inquiries on homologies in this part of the brain.

Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization.

The molecular mechanisms that control regional specification, morphogenesis and differentiation of the embryonic forebrain are not known, although recently several laboratories have isolated homeobox, Wnt and other genes that are candidates for playing roles in these processes. Most of these genes exhibit temporally and spatially restricted patterns of expression within the forebrain. However, analysis of the spatial patterns has been complicated because an understanding of the organization of the embryonic forebrain has been lacking. This article describes a neuromeric model of the forebrain that is consistent with the expression patterns of these genes, and that provides a framework for understanding the morphological relationships within this complex structure.

Chicken lateral septal organ and other circumventricular organs form in a striatal subdomain abutting the molecular striatopallidal border.

The avian lateral septal organ (LSO) is a telencephalic circumventricular specialization with liquor-contacting neurons (Kuenzel and van Tienhoven [1982] J. Comp. Neurol. 206:293-313). We studied the topological position of the chicken LSO relative to molecular borders defined previously within the telencephalic subpallium (Puelles et al. [2000] J. Comp. Neurol. 424:409-438). Differential expression of Dlx5 and Nkx2.1 homeobox genes, or the Shh gene encoding a secreted morphogen, allows distinction of striatal, pallidal, and preoptic subpallial sectors. The chicken LSO complex was characterized chemoarchitectonically from embryonic to posthatching stages, by using immunohistochemistry for calbindin, tyrosine hydroxylase, NKX2.1, and BEN proteins and in situ hybridization for Nkx2.1, Nkx2.2, Nkx6.1, Shh, and Dlx5 mRNA. Medial and lateral parts of LSO appear, respectively, at the striatal part of the septum and adjacent bottom of the lateral ventricle (accumbens), in lateral continuity with another circumventricular organ that forms along a thin subregion of the entire striatum, abutting the molecular striatopallidal boundary; we called this the "striatopallidal organ" (SPO). The SPO displays associated distal periventricular cells, which are lacking in the LSO. Moreover, the SPO is continuous caudomedially with a thin, linear ependymal specialization found around the extended amygdala and preoptic areas. This differs from SPO and LSO in some molecular aspects. We tentatively identified this structure as being composed of an "extended amygdala organ" (EAO) and a "preoptohypothalamic organ" (PHO). The position of LSO, SPO, EAO, and PHO within a linear Dlx5-expressing ventricular domain that surrounds the Nkx2.1-expressing pallidopreoptic domain provides an unexpected insight into possible common and differential causal mechanisms underlying their formation.

Expression of Lrrn1 marks the prospective site of the zona limitans thalami in the early embryonic chicken diencephalon.

An unknown chicken gene selected from a published substractive hybridization screen (GenBank Accession No. ; [Christiansen, J.H., Coles, E.G., Robinson, V., Pasini, A., Wilkinson, D.G., 2001. Screening from a subtracted embryonic chick hindbrain cDNA library: identification of genes expressed during hindbrain, midbrain and cranial neural crest development. Mech. Dev. 102, 119-133.]) was deemed of interest because of its dynamic pattern of expression across the forebrain and midbrain regions. A 528bp fragment cloned from early chick embryo cDNA and used for in situ hybridization corresponded to part of the 3' untranslated region of the chicken gene Leucine-rich repeat neuronal protein 1 (Lrrn1). The expression of this gene, mapped in the embryonic chick brain between stages HH10 and HH26, apparently preconfigures the zona limitans thalami site before overt formation of this boundary structure. Apart of colateral expression in the forebrain, midbrain and hindbrain basal plate, the most significant expression of Lrrn1 was found early on across the entire alar plate of midbrain and forebrain (HH10). This unitary domain soon divides at HH14 into a rostral part, across alar secondary prosencephalon and prospective alar prosomere 3 (prethalamus; caudal limit at the prospective zona limitans), and a caudal part in alar prosomere 1 (pretectum) and midbrain. The rostral forebrain domain later downregulates gradually most extratelencephalic signal of Lrrn1, but the rostral shell of zona limitans retains expression longer. Expression in the caudal alar domain also changes by downregulation within its pretectal subdomain. Caudally, the midbrain domain ends at the isthmo-mesencephalic junction throughout the studied period. Embryonic Lrrn1 signal also appears in the somites and in the otic vesicle.

EphA7 receptor is expressed differentially at chicken prosomeric boundaries.

We reexamined tyrosine-kinase receptor EphA7 RNA signal in embryonic chicken forebrain, to clarify its topographic relationships with early regionalization processes, such as establishment of prosomeric boundaries. After neurulation, uniform alar expression appears across prospective prosomeres prosomere 1, prosomere 2 and prosomere 3 (prethalamus, thalamus and pretectum). This pattern soon changes by differential downregulation at or in between some of the prosomeric boundaries in an individual pattern for each limit, and by expansion of expression into the rostral midbrain. The secondary distribution highlights various transversal and longitudinal domains, notably the zona limitans intrathalamica and the pretectum limits, as well as two longitudinal bands in the basal plate, termed paramedian and parabasal. Strong expression of EphA7 appears at the mammillary pouch and a supramammillary tegmental arch from stage Hamburger and Hamilton stages 14-15 onwards. At the end of the developmental period examined, expression of EphA7 in the ventricular zone decreases generally (with some exceptions) and novel expression domains start to appear in the mantle layer, initiating a third phase of differential expression. Thus, while the expression of EphA7 does not show a fixed functional or topographic relationship to prosomeric boundaries, sequential transcription changes during chicken development are consistent with a differential involvement of the diverse interprosomeric boundaries, as well as dorsoventral patterning organizers, in the regulation of EphA7 expression.

Selective early expression of the orphan nuclear receptor Nr4a2 identifies the claustrum homolog in the avian mesopallium: Impact on sauropsidian/mammalian pallium comparisons.

The transcription factor Nr4a2 was recently revealed as a very early developmental marker of the claustrum (CL) proper in the mouse. The earliest claustral primordium was identified superficially, dorsal to the olfactory cortex, and was subsequently covered by the Nr4a2-negative cells of the insular cortex. Some tangentially migrating claustral derivatives (subplate cells and some endopiriform elements) also expressed this marker. The present study employs the same genetic marker to explore the presence of a comparable pallial division in chicken in which, in principle, the same pallial sectors exist as in mammals. We were indeed able to delineate an early-developing Nr4a2-positive mantle domain at the expected topologic position within the developing chicken lateral pallium. In the chicken as well as in the turtle (from data in the literature), the earliest postmitotic lateropallial cells likewise express Nr4a2 and occupy a corticoid superficial stratum of the mesopallium, which is clearly comparable in spatial and chronological profile to the mouse CL. Other cells produced in this pallial sector include various tangentially migrating Nr4a2-labeled derivatives as well as Nr4a2-negative and Nr4a2-positive local deeper subpopulations that partially interdigitate, forming mesopallial core and shell populations. We hold that the deep avian and reptilian mesopallial formation developing under the superficial corticoid CL homolog represents a field homolog of the insula, although additional studies are required to underpin this hypothesis.

Anatomical and gene expression mapping of the ventral pallium in a three-dimensional model of developing human brain.

Combining gene expression data with morphological information has revolutionized developmental neuroanatomy in the last decade. Visualization and interpretation of complex images have been crucial to these advances in our understanding of mechanisms underlying early brain development, as most developmental processes are spatially oriented, in topologically invariant patterns that become overtly distorted during brain morphogenesis. It has also become clear that more powerful methodologies are needed to accommodate the increasing volume of data available and the increasingly sophisticated analyses that are required, for example analyzing anatomy and multiple gene expression patterns at individual developmental stages, or identifying and analyzing homologous structures through time and/or between species. Three-dimensional models have long been recognized as a valuable way of providing a visual interpretation and overview of complex morphological data. We have used a recently developed method, optical projection tomography, to generate digital three-dimensional models of early human brain development. These models can be used both as frameworks, onto which normal or experimental gene expression data can be mapped, and as objects, within which topological morphological relationships can be investigated in silico. Gene expression patterns and selected morphological structures or boundaries can then be visualized individually or in different combinations in order to study their respective morphogenetic significance. Here, we review briefly the optical projection tomography method, placing it in the context of other methods used to generate developmental three dimensional models, and show the definition of some CNS anatomical domains within a Carnegie stage 19 human model. We also map the telencephalic EMX1 and PAX6 gene expression patterns to this model, corroborating for the first time the existence of a ventral pallium primordium in the telencephalon of human embryos, a distinct claustroamygdaloid histogenetic area comparable to the recently defined mouse primordium given that name [Puelles L, Kuwana E, Puelles E, Bulfone A, Shimamura K, Keleher J, Smiga S, Rubenstein JLR (2000) Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1. J Comp Neurol 424:409-438; Puelles L, Martínez S, Martínez-de-la-Torre M, Rubenstein JLR (2004) Gene maps and related histogenetic domains in the forebrain and midbrain. In: The rat nervous system, 3rd ed (Paxinos G, ed), pp 3-25. San Diego: Academic Press].

Expression pattern of cSix3, a member of the Six/sine oculis family of transcription factors.

We describe the expression pattern of cSix3, a chick homologue of the murine Six3. cSix3 transcripts are expressed from presomitic stages in the most anterior portion of the neural plate. As the neural tube folds and the optic vesicles evaginate, cSix3 is expressed in the optic vesicle and the rostroventral forebrain. At later stages, cSix3 is found in most of the structures derived from the anterior neural plate, i.e. olfactory epithelium, septum, adenohypophysis, hypothalamus and preoptic areas. During eye development, cSix3 expression is first found in the entire optic vesicle and the overlying ectoderm but soon becomes restricted to the prospective neural retina and to the lens placode. In the developing neural retina, cSix3 is expressed in the entire undifferentiated neuroepithelium but is rapidly downregulated, first in the postmitotic photoreceptors and later in the majority of retinal ganglion cells.

Chicken Nkx6.1 expression at advanced stages of development identifies distinct brain nuclei derived from the basal plate.

This study of the embryonic chicken central nervous system defines previously unknown domains of neuroepithelial Nkx6.1 expression in neuroepithelial progenitors and identifies nuclei that express Nkx6.1 at progressively more advanced stages of central nervous system development.

Induction of ectopic engrailed expression and fate change in avian rhombomeres: intersegmental boundaries as barriers.

We tested the possibility of inducing ectopic expression of the gene Engrailed-2 (En-2) in the rhombomeres of the 2-days-old chick embryo. The experimental procedure consisted of grafting En-2-expressing neuroepithelium (prospective isthmocerebellum) from a quail or a mouse embryo into different rostrocaudal levels in the hindbrain of a host chick embryo. The graft replaced a given excised host rhombomere, the rostral and caudal limits of which were either also excised or left intact in different experiments. Induction of En-2 occurred in the host hindbrain, but only when the graft did not contact with host interrhombomeric boundaries and only in the alar plate of the rhombomeres immediately contacting the graft. Long survival experimental embryos showed that induced rhombomeres give rise to a cerebellar phenotype in their transformed alar plates. We thus demonstrate here a pluripotential state of the early rhombomeres as well as a possible role of the interrhombomeric limits as barriers to morphogenetic influences.

The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos.

The pattern of RNA expression of the murine Dlx-2 (Tes-1) homeobox gene is described in embryos ranging in age from E8.5 through E11.5. Dlx-2 is a vertebrate homologue of the Drosophila Distal-less (Dll) gene. Dll expression in the Drosophila embryo is principally limited to the primordia of the brain, head and limbs. Dlx-2 is also expressed principally in the primordia of the forebrain, head and limbs. Within these regions it is expressed in spatially restricted domains. These include two discontinuous regions of the forebrain (basal telencephalon and ventral diencephalon), the branchial arches, facial ectoderm, cranial ganglia and limb ectoderm. Several mouse and human disorders have phenotypes which potentially are the result of mutations in the Dlx genes.

Patterning signals acting in the spinal cord override the organizing activity of the isthmus.

The regionalization of the neural tube along the anteroposterior axis is established through the action of patterning signals from the endomesoderm including the organizer. These signals set up a pre-pattern which is subsequently refined through local patterning events. The midbrain-hindbrain junction, or isthmus, is endowed with such an organizing activity. It is able to induce graded expression of the Engrailed protein in the adjacent mesencephalon and rhombencephalon, and subsequently elicits the development of tectal and cerebellar structures. Ectopically grafted isthmus was also shown to induce Engrailed expression in diencephalon and otic and pre-otic rhombencephalon. Fgf8 is a signalling protein which is produced by the isthmus and which is able to mimic most isthmic properties. We show here that the isthmus, when transposed to the level of either rhombomere 8 or the spinal cord, loses its ability to induce Engrailed and cerebellar development in adjacent tissues. This is accompanied by the down-regulation of fgf8 expression in the grafted isthmus and by the up-regulation of a marker of the recipient site, Hoxb-4. Moreover, these changes in gene activity in the transplant are followed by a transformation of the fate of the grafted cells which adjust to their novel environment. These results show that the fate of the isthmus is not determined at 10-somite stage and that the molecular loop of isthmic maintenance can be disrupted by exogenous signals.

Early neuromeric distribution of tyrosine-hydroxylase-immunoreactive neurons in human embryos.

A segmental mapping of brain tyrosine-hydroxylase-immunoreactive (TH-IR) neurons in human embryos between 4.5 and 6 weeks of gestation locates with novel precision the dorsoventral and anteroposterior topography of the catecholamine-synthetizing primordia relative to neuromeric units. The data support the following conclusions. (1) All transverse sectors of the brain (prosomeres in the forebrain, midbrain, rhombomeres in the hindbrain, spinal cord) produce TH-IR neuronal populations. (2) Each segment shows peculiarities in its contribution to the catecholamine system, but there are some overall regularities, which reflect that some TH-IR populations develop similarly in different segments. (3) Dorsoventral topology of the TH-IR neurons indicates that at least four separate longitudinal zones (in the floor and basal plates and twice in the alar plate) found across most segments are capable of producing the TH-IR phenotype. (4) Basal plate TH-IR neurons tend to migrate intrasegmentally to a ventrolateral superficial position, although some remain periventricular; those in the brainstem are related to motoneurons of the oculomotor and branchiomotor nuclei. (5) Some alar TH-IR populations migrate superficially within the segmental boundaries. (6) Most catecholaminergic anatomical entities are formed as fusions of smaller segmental components, each of which show similar histogenetic patterns. A nomenclature is proposed that partly adheres to previous terminology but introduces the distinction of embryologically different cell populations and unifies longitudinally analogous entities. Such a model, as presented in the present study, is convenient for resolving problems of homology of the catecholamine system across the diversity of vertebrate forms.

Rostrocaudal nuclear relationships in the avian medulla oblongata: a fate map with quail chick chimeras.

We present a correlative fate map of the nonsegmented caudal hindbrain down to the medullospinal boundary (medulla oblongata), as a companion to a previous fate mapping study of the hindbrain rhombomeres r2-r6 in quail chick chimeras at stages HH10/11 [Marín and Puelles (1995) Eur J Neurosci 7:1714-1738]. For reproducibility and equivalent precision of analysis, successive portions of the medulla-called pseudorhombomeres "r7" to "r11"-were delimited by transverse planes through the center of adjacent somites at stages HH10/11. These units were each grafted homotopically and isochronically from quail donors into chick hosts. The chimeric specimens were fixed at stages HH35/36 and alternate Nissl-stained sagittal sections were compared to adjacent sections in which quail cells were detected immunocytochemically. This analysis in general showed that there is little intermixing between adjacent pseudorhombomeric domains, although some neuronal populations in the vestibular and trigeminal columns, as well as in the reticular formation and pontine nuclei, do migrate selectively into the host hindbrain. Contralateral migration was scarce up to the stages examined. Several motor nuclei, i.e., the vagal motor complex, or sensory nuclei, i.e., the medial vestibular nucleus, show cytoarchitectonic limits that coincide with pseudorhombomeric ones; however, most conventional grisea were found to originate across several pseudorhombomeres. The inferior olivary complex originated between "r8" and "r11" (between the centers of somites 1 and 5). The medullospinal boundary coincided precisely with the center of the fifth somite, slightly caudal to the obex and the end of the choroidal roof, and correlated with the end of many medullary cytoarchitectonic units. In contrast, the dorsal column nuclei and the caudal subnucleus of the descending trigeminal column fell within the spinal cord. On the whole, the patterns observed were very similar to those found before within the overtly segmented part of the hindbrain, suggesting that some underlying common mechanism may account for the transverse cytoarchitectonic boundaries.

Cadherin expression by embryonic divisions and derived gray matter structures in the telencephalon of the chicken.

The expression of three cadherins (cadherin-6B, cadherin-7, and R-cadherin) was studied by immunohistochemistry in the telencephalon of chicken embryos at intermediate stages of development (11 and 15 days of incubation). Expression patterns were related to cytoarchitecture and to previously published data on functional connections and on the expression of gene regulatory proteins. Our results indicate that, like in other regions of the embryonic chicken brain, the expression of each cadherin is restricted to parts of embryonic divisions as well as to particular nuclei, areas or their subdivisions. The expression patterns are largely complementary with partial overlap. The regional expression of the cadherins respects the boundary between the pallium and the subpallium as well as between various pallial and subpallial subdivisions. Novel subdivisions were found in several telencephalic areas. For example, subjacent to the hyperstriatum, the neostriatum contains multiple islands of cells with a profile of cadherin expression that differs from the surrounding matrix ("island fields"). Moreover, the expression of each cadherin is apparently associated with parts of intratelencephalic neural circuits and of thalamopallial and basal ganglia pathways. These results support a role for cadherins in the aggregation and differentiation of gray matter structures within embryonic brain divisions. The cadherin immunostaining patterns are interpreted in the context of a recently proposed divisional scheme of the avian pallium that postulates medial, dorsal, lateral, and ventral divisions as complete radial histogenetic units (Puelles et al. [2000]).

Patterns of calretinin, calbindin, and tyrosine-hydroxylase expression are consistent with the prosomeric map of the frog diencephalon.

This paper re-examines a previously published segmental map of the frog diencephalon (Puelles et al. [1996] Brain Behav.Evol. 47:279-310) by means of immunocytochemical mapping of calretinin, calbindin, and tyrosine hydroxylase. The distribution of neuronal populations, axon tracts, and neuropils immunoreactive for these markers was studied in adult specimens of Rana perezi and Xenopus laevis sectioned sagittally or horizontally. Emphasis was placed on study of the relationship of observed chemoarchitectural boundaries with the postulated overall prosomeric organization and the schema of nuclear subdivisions we reported previously, based on acetylcholinesterase histochemistry and Nissl pattern in Rana. The data reveal a large-scale correspondence with the segmental map in both species, although some differences were noted between Rana and Xenopus. Notably, retinorecipient neuropils were generally immunoreactive for calretinin only in Rana. Importantly, calretinin immunostaining underlines particularly well the transverse prosomeric boundaries of the dorsal thalamus. A number of nuclear subdivisions noted before with AChE were corroborated, and some novel subdivisions became apparent, particularly in the anterior nucleus of the dorsal thalamus and in the habenular complex. The mapping of tyrosine hydroxylase clarified the segmental distribution of the catecholaminergic cell groups in the frog forebrain, which is comparable to that observed in other vertebrates.

Prosomeric map of the lamprey forebrain based on calretinin immunocytochemistry, Nissl stain, and ancillary markers.

The structural organization of the lamprey extratelencephalic forebrain is re-examined from the perspective of the prosomeric segmental paradigm. The question asked was whether the prosomeric forebrain model used for gnathostomes is of material advantage for interpreting subdivisions in the lamprey forebrain. To this aim, the main longitudinal and transverse landmarks recognized by the prosomeric model in other vertebrates were identified in Nissl-stained lamprey material. Lines of cytoarchitectural discontinuity and contours of migrated neuronal groups were mapped in a two-dimensional sagittal representation and were also classified according to their radial position. Immunocytochemical mapping of calretinin expression in adjacent sections served to define particular structural units better, in particular, the dorsal thalamus. These data were complemented by numerous other chemoarchitectonic observations obtained with ancillary markers, which identified additional specific formations, subdivisions, or boundaries. Emphasis was placed on studying whether such chemically defined neuronal groups showed boundaries aligned with the postulated inter- or intraprosomeric boundaries. The course of diverse axonal tracts was studied also with regard to their prosomeric topography. This analysis showed that the full prosomeric model applies straightforwardly to the lamprey forebrain. This finding implies that a common segmental and longitudinal organization of the neural tube may be primitive for all vertebrates. Interesting novel aspects appear in the interpretation of the lamprey pretectum, the dorsal and ventral thalami, and the hypothalamus. The topologic continuity of the prosomeric forebrain regions with evaginated or non-evaginated portions of the telencephalon was also examined.

Structure of longitudinal brain zones that provide the origin for the substantia nigra and ventral tegmental area in human embryos, as revealed by cytoarchitecture and tyrosine hydroxylase, calretinin, calbindin, and GABA immunoreactions.

In a previous work, mapping early tyrosine hydroxylase (TH) expressing primordia in human embryos, the tegmental origin of the substantia nigra (SN) and ventral tegmental area (VTA) was located across several neuromeric domains: prosomeres 1-3, midbrain, and isthmus (Puelles and Verney, [1998] J. Comp. Neurol. 394:283-308). The present study examines in detail the architecture of the neural wall along this tegmental continuum in 6-7 week human embryos, to better define the development of the SN and VTA. TH-immunoreactive (TH-IR) structures were mapped relative to longitudinal subdivisions (floor plate, basal plate, alar plate), as well as to radially superposed strata of the neural wall (periventricular, intermediate, and superficial strata). These morphologic entities were delineated at each relevant segmental level by using Nissl-stained sections and immunocytochemical mapping of calbindin, calretinin, and GABA in adjacent sagittal or frontal sections. A numerous and varied neuronal population originates in the floor plate area, and some of its derivatives become related through lateral tangential migration with other neuronal populations born in distinct medial and lateral portions of the basal plate and in a transition zone at the border with the alar plate. Some structural differences characterize each segmental domain within this common schema. The TH-IR neuroblasts arise predominantly within the ventricular zone of the floor plate and, more sparsely, within the adjacent medial part of the basal plate. They first migrate radially from the ventricular zone to the pia and then apparently move laterally and slightly rostralward, crossing the superficial stratum of the basal plate. Several GABA-IR cell populations are present in this region. One of them, which might represent the anlage of the SN pars reticulata, is generated in the lateral part of the basal plate.

Development of catecholamine systems in the brain of the lizard Gallotia galloti.

For a better insight into general and derived traits of developmental aspects of catecholaminergic (CA) systems in amniotes, we have studied the development of these systems in the brain of a lizard, Gallotia galloti, with tyrosine hydroxylase (TH)- and dopamine (DA) immunohistochemical techniques. Two main groups of TH-immunoreactive (THi) perikarya appear very early in development: one group in the midbrain which gives rise to the future ventral tegmental area, substantia nigra and retrorubral cell groups, and another group in the tuberomammillary hypothalamus. Somewhat later in development, TH/DA-immunoreactive cells are observed in the thalamus, rostrodorsal hypothalamus and spinal cord, and, with another delay, in the suprachiasmatic nucleus, the periventricular organ, and the pretectal posterodorsal nucleus. CA cell groups that appear rather late in development include the cells in the olfactory bulb, the locus coeruleus and the caudal brainstem. As expected, the development of immunoreactive fibers stays behind that of the cell bodies, but reaches the adult-like pattern just prior to hatching. The present study revealed considerable variation in the relation between the state of cytodifferentiation and first expression of TH/DA immunoreactivity between CA cell groups. Catecholamine cells in the midbrain and tuberomammillary hypothalamus are still migrating, immature (absence of dendrites) and express only TH immunoreactivity at the time of first detection. Cells which appear at later developmental stages lie already further away from the ventricle, possess two or more dendritic processes, and generally express both TH- and DA immunoreactivity.