Populational heterogeneity and partial migratory origin of the ventromedial hypothalamic nucleus: genoarchitectonic analysis in the mouse.

The ventromedial hypothalamic nucleus (VMH) is one of the most distinctive hypothalamic tuberal structures, subject of numerous classic and modern functional studies. Commonly, the adult VMH has been divided in several portions, attending to differences in cell aggregation, cell type, connectivity, and function. Consensus VMH partitions in the literature comprise the dorsomedial (VMHdm), and ventrolateral (VMHvl) subnuclei, which are separated by an intermediate or central (VMHc) population (topographic names based on the columnar axis). However, some recent transcriptome analyses have identified a higher number of different cell types in the VMH, suggesting additional subdivisions, as well as the possibility of separate origins. We offer a topologic and genoarchitectonic developmental study of the mouse VMH complex using the prosomeric axis as a reference. We analyzed genes labeling specific VMH subpopulations, with particular focus upon the Nkx2.2 transcription factor, a marker of the alar-basal boundary territory of the prosencephalon, from where some cells seem to migrate dorsoventrally into VMH. We also identified separate neuroepithelial origins of a Nr2f1-positive subpopulation, and a new Six3-positive component, as well as subtle differences in origin of Nr5a1 positive versus Nkx2.2-positive cell populations entering dorsoventrally the VMH. Several of these migrating cell types are born in the dorsal tuberal domain and translocate ventralwards to reach the intermediate tuberal domain, where the adult VMH mass is located in the adult. This work provides a more detailed area map on the intrinsic organization of the postmigratory VMH complex, helpful for deeper functional studies of this basal hypothalamic entity.

Neurogenetic Heterochrony in Chick, Lizard, and Rat Mapped with Wholemount Acetylcholinesterase and the Prosomeric Model.

In the developing brain, the phenomenon of neurogenesis is manifested heterotopically, that is, much the same neurogenetic steps occur at different places with a different timetable. This is due apparently to early molecular regionalization of the neural tube wall in the anteroposterior and dorsoventral dimensions, in a checkerboard pattern of more or less deformed quadrangular histogenetic areas. Their respective fate is apparently specified by a locally specific combination of active/repressed genes known as "molecular profile." This leads to position-dependent differential control of proliferation, neurogenesis, differentiation, and other aspects, eventually in a heterochronic manner across adjacent areal units with sufficiently different molecular profiles. It is not known how fixed these heterochronic patterns are. We reexamined here comparatively early patterns of forebrain and hindbrain neurogenesis in a lizard (Lacerta gallotia galloti), a bird (the chick), and a mammal (the rat), as demonstrated by activation of acetylcholinesterase (AChE). This is an early marker of postmitotic neurons, which leaves unlabeled the neuroepithelial ventricular cells, so that we can examine cleared wholemounts of the reacted brains to have a birds-eye view of the emergent neuronal pattern at each stage. There is overall heterochrony between the basal and alar plates of the brain, a known fact, but, remarkably, heterochrony occurs even within the precocious basal plate among its final anteroposterior neuromeric subdivisions and their internal microzonal subdivisions. Some neuromeric units or microzones are precocious, while others follow suit without any specific spatial order or gradient; other similar neuromeric units remain retarded in the midst of quite advanced neighbors, though they do produce similar neurogenetic patterns at later stages. It was found that some details of such neuromeric heterochrony are species-specific, possibly related to differential morphogenetic properties. Given the molecular causal underpinning of the updated prosomeric model used here for interpretation, we comment on the close correlation between some genetic patterns and the observed AChE differentiation patterns.

Molecular Segmentation of the Spinal Trigeminal Nucleus in the Adult Mouse Brain.

The trigeminal column is a hindbrain structure formed by second order sensory neurons that receive afferences from trigeminal primary (ganglionic) nerve fibers. Classical studies subdivide it into the principal sensory trigeminal nucleus located next to the pontine nerve root, and the spinal trigeminal nucleus which in turn consists of oral, interpolar and caudal subnuclei. On the other hand, according to the prosomeric model, this column would be subdivided into segmental units derived from respective rhombomeres. Experimental studies have mapped the principal sensory trigeminal nucleus to pontine rhombomeres (r) r2-r3 in the mouse. The spinal trigeminal nucleus emerges as a plurisegmental formation covering several rhombomeres (r4 to r11 in mice) across pontine, retropontine and medullary hindbrain regions. In the present work we reexamined the issue of rhombomeric vs. classical subdivisions of this column. To this end, we analyzed its subdivisions in an AZIN2-lacZ transgenic mouse, known as a reference model for hindbrain topography, together with transgenic reporter lines for trigeminal fibers. We screened as well for genes differentially expressed along the axial dimension of this structure in the adult and juvenile mouse brain. This analysis yielded genes from multiple functional families that display transverse domains fitting the mentioned rhombomeric map. The spinal trigeminal nucleus thus represents a plurisegmental structure with a series of distinct neuromeric units having unique combinatorial molecular profiles.

Salient brain entities labelled in P2rx7-EGFP reporter mouse embryos include the septum, roof plate glial specializations and circumventricular ependymal organs.

The purinergic system is one of the oldest cell-to-cell communication mechanisms and exhibits relevant functions in the regulation of the central nervous system (CNS) development. Amongst the components of the purinergic system, the ionotropic P2X7 receptor (P2X7R) stands out as a potential regulator of brain pathology and physiology. Thus, P2X7R is known to regulate crucial aspects of neuronal cell biology, including axonal elongation, path-finding, synapse formation and neuroprotection. Moreover, P2X7R modulates neuroinflammation and is posed as a therapeutic target in inflammatory, oncogenic and degenerative disorders. However, the lack of reliable technical and pharmacological approaches to detect this receptor represents a major hurdle in its study. Here, we took advantage of the P2rx7-EGFP reporter mouse, which expresses enhanced green fluorescence protein (EGFP) immediately downstream of the P2rx7 proximal promoter, to conduct a detailed study of its distribution. We performed a comprehensive analysis of the pattern of P2X7R expression in the brain of E18.5 mouse embryos revealing interesting areas within the CNS. Particularly, strong labelling was found in the septum, as well as along the entire neural roof plate zone of the brain, except chorioidal roof areas, but including specialized circumventricular roof formations, such as the subfornical and subcommissural organs (SFO; SCO). Moreover, our results reveal what seems a novel circumventricular organ, named by us postarcuate organ (PArcO). Furthermore, this study sheds light on the ongoing debate regarding the specific presence of P2X7R in neurons and may be of interest for the elucidation of additional roles of P2X7R in the idiosyncratic histologic development of the CNS and related systemic functions.

Sim1-expressing cells illuminate the origin and course of migration of the nucleus of the lateral olfactory tract in the mouse amygdala.

We focus this report on the nucleus of the lateral olfactory tract (NLOT), a superficial amygdalar nucleus receiving olfactory input. Mixed with its Tbr1-expressing layer 2 pyramidal cell population (NLOT2), there are Sim1-expressing cells whose embryonic origin and mode of arrival remain unclear. We examined this population with Sim1-ISH and a Sim1-tauLacZ mouse line. An alar hypothalamic origin is apparent at the paraventricular area, which expresses Sim1 precociously. This progenitor area shows at E10.5 a Sim1-expressing dorsal prolongation that crosses the telencephalic stalk and follows the terminal sulcus, reaching the caudomedial end of the pallial amygdala. We conceive this Sim1-expressing hypothalamo-amygdalar corridor (HyA) as an evaginated part of the hypothalamic paraventricular area, which participates in the production of Sim1-expressing cells. From E13.5 onwards, Sim1-expressing cells migrated via the HyA penetrate the posterior pallial amygdalar radial unit and associate therein to the incipient Tbr1-expressing migration stream which swings medially past the amygdalar anterior basolateral nucleus (E15.5), crosses the pallio-subpallial boundary (E16.5), and forms the NLOT2 within the anterior amygdala by E17.5. We conclude that the Tbr1-expressing NLOT2 cells arise strictly within the posterior pallial amygdalar unit, involving a variety of required gene functions we discuss. Our results are consistent with the experimental data on NLOT2 origin reported by Remedios et al. (Nat Neurosci 10:1141-1150, 2007), but we disagree on their implication in this process of the dorsal pallium, observed to be distant from the amygdala.

LacZ-reporter mapping of Dlx5/6 expression and genoarchitectural analysis of the postnatal mouse prethalamus.

We present here a thorough and complete analysis of mouse P0-P140 prethalamic histogenetic subdivisions and corresponding nuclear derivatives, in the context of local tract landmarks. The study used as fundamental material brains from a transgenic mouse line that expresses LacZ under the control of an intragenic enhancer of Dlx5 and Dlx6 (Dlx5/6-LacZ). Subtle shadings of LacZ signal, jointly with pan-DLX immunoreaction, and several other ancillary protein or RNA markers, including Calb2 and Nkx2.2 ISH (for the prethalamic eminence, and derivatives of the rostral zona limitans shell domain, respectively) were mapped across the prethalamus. The resulting model of the prethalamic region postulates tetrapartite rostrocaudal and dorsoventral subdivisions, as well as a tripartite radial stratification, each cell population showing a characteristic molecular profile. Some novel nuclei are proposed, and some instances of potential tangential cell migration were noted.

A radial histogenetic model of the mouse pallial amygdala.

Conventional anatomic models of the rodent (mammalian) amygdala are based on section planes oblique to its intrinsic radial glial organization. As a result, we still lack a model of amygdalar histogenesis in terms of radial units (progenitor domains and related radial migration and layering patterns). A radial model of the mouse pallial amygdala is first offered here, based on three logical steps: (1) analysis of amygdalar radial structure in variously discriminative genoarchitectonic material, using an optimal ad hoc section plane; (2) testing preliminary models with experiments labelling at the brain surface single packets of radial glia processes, to be followed into the ventricular surface across intervening predicted elements; (3) selection of 81 differential amygdalar gene markers and checking planar and radial aspects of their distribution across the model elements. This approach shows that subtle changes to the conventional schema of the amygdala allow a radial histogenetic model to be recognized, which is consistent with molecularly coded differential identities of its units and strata. It is expected that this model will help both causal studies of amygdalar developmental patterning and comparative evolutionary studies. It also may have potential impact on hodological and functional studies.

Concentric ring topology of mammalian cortical sectors and relevance for patterning studies.

Models aiming to explain causally the evolutionary or ontogenetic emergence of the pallial isocortex and its regional/areal heterogeneity in mammals use simple or complex assumptions about the pallial structure present in basal mammals and nonmammals. The question arises: how complex is the pattern that needs to be accounted for in causal models? This topic is also paramount for comparative purposes, since some topological relationships may be explained as being ancestral, rather than newly emerged. The mouse pallium is apt to be reexamined in this context, due to the breadth of available molecular markers and correlative experimental patterning results. We center the present essay on a recapitulative glance at the classic theory of concentric mammalian allo-, meso-, and neocortex domains. In its simplest terms, this theory postulates a central neocortical island (6 layers) separated by a surrounding mesocortical ring (4-5 layers) from a peripheral allocortical ring (3 layers). These territories show additional partition into regional or areal subdivisions. There are also borderline amygdalar, claustral, and septal areas of the pallium, nuclear in structure. There has been little effort so far to contemplate the full concentric ring model in current "cortex patterning" models. In this essay, we recapitulate the ring idea in mammals (mouse) and consider a potential causal patterning scenario using topologic models. Finally, we briefly explore how far this theory may apply to pallium models proposed recently for sauropsids.

The Postmigratory Alar Topography of Visceral Cranial Nerve Efferents Challenges the Classical Model of Hindbrain Columns.

The classic columnar model of cranial nerve central representation assumes that all motor and sensory hindbrain neurons develop within four radial migration domains, held to be separated by a sulcal alar-basal boundary (sulcus limitans). This essay reviews a number of developmental data that challenge these concepts. These results are interpreted within the framework of present day neuromeric conception of the brainstem (the prosomeric model). Advances in dorsoventral patterning of the spinal cord and hindbrain now show that there exist up to eight alar microzones and five basal microzones (molecularly and histogenetically distinct longitudinal progenitor domains). This reveals that the classic tetracolumnar model is excessively simplistic. There is both older and recent data revealing that the visceral efferent neurons of the cranial nerves (preganglionic and branchiomotor neurons) are generated next to the floor plate and later migrate dorsalwards before adopting their final topography in the mantle, contrary to the purely radial migration assumed in the classic model. Moreover, various results support the conclusion that at least the branchiomotor neurons end their migration and mature within the alar region of the mantle. Evidence on this point obtained in chick embryos is reviewed in detail, and novel evidence in mouse embryos is presented. Anat Rec, 302:485-504, 2019. © 2018 Wiley Periodicals, Inc.

An exercise in brain genoarchitectonics: Analysis of AZIN2-Lacz expressing neuronal populations in the mouse hindbrain.

We examined in detail the distribution of AZIN2 (antizyme inhibitor 2) expression in the adult mouse hindbrain and neighboring spinal cord. AZIN2, similar to previously known AZIN1, is a recently-discovered, a functional paralog of ornithine decarboxylase (ODC). Due to their structural similarity to ODC, both AZIN1 and AZIN2 counteract the inhibitory action of 3 known antizymes (AZ1-3) on the ODC synthesis of polyamines, thus increasing intracytoplasmic levels of polyamines. AZIN2 is strongly, but heterogeneously, expressed in the brain. Our study uses a mouse line carrying an AZIN2-LacZ construct, and, in our topographic analysis of AZIN2-positive structures, we intend to share new knowledge about the rhombomeric segmentation of the hindbrain (a function of Hox paralogs and other genes). The observed labeled cell populations predominantly coincide with known cholinergic and glutamatergic cells, but occasionally also correspond to GABAergic, and possibly glycinergic cells. Some imperfectly known hindbrain populations stood out in unprecedented detail, and some axonal tracts were also differentially stained. © 2017 Wiley Periodicals, Inc.

Regionalized differentiation of CRH, TRH, and GHRH peptidergic neurons in the mouse hypothalamus.

According to the updated prosomeric model, the hypothalamus is subdivided rostrocaudally into terminal and peduncular parts, and dorsoventrally into alar, basal, and floor longitudinal zones. In this context, we examined the ontogeny of peptidergic cell populations expressing Crh, Trh, and Ghrh mRNAs in the mouse hypothalamus, comparing their distribution relative to the major progenitor domains characterized by molecular markers such as Otp, Sim1, Dlx5, Arx, Gsh1, and Nkx2.1. All three neuronal types originate mainly in the peduncular paraventricular domain and less importantly at the terminal paraventricular domain; both are characteristic alar Otp/Sim1-positive areas. Trh and Ghrh cells appeared specifically at the ventral subdomain of the cited areas after E10.5. Additional Ghrh cells emerged separately at the tuberal arcuate area, characterized by Nkx2.1 expression. Crh-positive cells emerged instead in the central part of the peduncular paraventricular domain at E13.5 and remained there. In contrast, as development progresses (E13.5-E18.5) many alar Ghrh and Trh cells translocate into the alar subparaventricular area, and often also into underlying basal neighborhoods expressing Nkx2.1 and/or Dlx5, such as the tuberal and retrotuberal areas, becoming partly or totally depleted at the original birth sites. Our data correlate a topologic map of molecularly defined hypothalamic progenitor areas with three types of specific neurons, each with restricted spatial origins and differential migratory behavior during prenatal hypothalamic development. The study may be useful for detailed causal analysis of the respective differential specification mechanisms. The postulated migrations also contribute to our understanding of adult hypothalamic complexity.

Development of the serotonergic cells in murine raphe nuclei and their relations with rhombomeric domains.

The raphe nuclei represent the origin of central serotonergic projections. The literature distinguishes seven nuclei grouped into rostral and caudal clusters relative to the pons. The boundaries of these nuclei have not been defined precisely enough, particularly with regard to developmental units, notably hindbrain rhombomeres. We hold that a developmental point of view considering rhombomeres may explain observed differences in connectivity and function. There are twelve rhombomeres characterized by particular genetic profiles, and each develops between one and four distinct serotonergic populations. We have studied the distribution of the conventional seven raphe nuclei among these twelve units. To this aim, we correlated 5-HT-immunoreacted neurons with rhombomeric boundary landmarks in sagittal mouse brain sections at different developmental stages. Furthermore, we performed a partial genoarchitectonic analysis of the developing raphe nuclei, mapping all known serotonergic differentiation markers, and compared these results, jointly with others found in the literature, with our map of serotonin-containing populations, in order to examine regional variations in correspondence. Examples of regionally selective gene patterns were identified. As a result, we produced a rhombomeric classification of some 45 serotonergic populations, and suggested a corresponding modified terminology. Only a minor rostral part of the dorsal raphe nucleus lies in the midbrain. Some serotonergic neurons were found in rhombomere 4, contrary to the conventional assumption that it lacks such neurons. We expect that our reclassification of raphe nuclei may be useful for causal analysis of their differential molecular specification, as well as for studies of differential connectivity and function.

Expression of antizyme inhibitor 2 in male haploid germinal cells suggests a role in spermiogenesis.

Recently, we have found that the antizyme inhibitor 2, a novel member of the antizyme binding proteins related to polyamine metabolism, was expressed mainly in the adult testes, although its function in testicular physiology is completely unknown. Therefore, in the present work, the spatial and temporal expression of antizyme inhibitor 2, and other genes related to polyamine metabolism were studied in the mouse testis, in an attempt to understand the role of antizyme inhibitor 2 in testicular functions. For that purpose, the temporal expression of different genes, during the first wave of spermatogenesis in postnatal mice, was studied by real-time RT-PCR, and the spatial distribution of transcripts and protein in the adult testis was examined by both RNA in situ hybridization and immunocytochemistry. The results indicated that antizyme inhibitor 2 was specifically expressed in the haploid germinal cells, similarly to antizyme 3, the testis specific antizyme. Conversely, ornithine decarboxylase mRNA was mainly found in the outer part of the seminiferous tubules where spermatogonia and spermatocytes are located. Functional transfection assays and co-immunoprecipitation experiments corroborated that antizyme inhibitor 2 counteracts the negative action of antizyme 3 on polyamine biosynthesis and uptake. All these results indicate that the expression of antizyme inhibitor 2 is postnatally regulated and strongly suggest that antizyme inhibitor 2 may have a role in spermiogenesis.

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.

Early pretectal gene expression pattern shows a conserved anteroposterior tripartition in mouse and chicken.

A changing network of gene activity settles the molecular basis of regionalization in the nervous system. As a consequence, analysis of combined gene expressions patterns represents a powerful initial approach to decode the complex process that drives neurohistogenesis and generates distinct morphological features. We have started to do a comparative screening of molecular regionalization in the mouse and chicken pretectal region at selected developmental stages. The pretectal region is composed of alar and roof plate derivatives of prosomere 1. This is a poorly understood region, best characterized in avian embryos and adults because nuclear cytoarchitectonic delimitation is clearer in these animals. During the early regionalization process the main pretectal boundaries and histogenetic/progenitor domains are established. We explore here Pax3, Pax6 and Six3 mRNA expression (and PAX3 immunoreactivity) in both chicken and mice, with the aim to compare their respective patterns. Our focus is centered on stages HH22-HH24 in chicken and embryonic days E11.5-E12.5 in mice. We found that, in both vertebrates, the same three main anteroposterior subdivisions are distinguished by these markers. They were defined as precommissural, juxtacommissural and commissural pretectal domains. These preliminary data represent an initial scaffold to explore more detailed pretectal regionalization processes and provide an important new key to approach unresolved pretectal homologies between vertebrates.

Lbx1 acts as a selector gene in the fate determination of somatosensory and viscerosensory relay neurons in the hindbrain.

Distinct types of relay neurons in the hindbrain process somatosensory or viscerosensory information. How neurons choose between these two fates is unclear. We show here that the homeobox gene Lbx1 is essential for imposing a somatosensory fate on relay neurons in the hindbrain. In Lbx1 mutant mice, viscerosensory relay neurons are specified at the expense of somatosensory relay neurons. Thus Lbx1 expression distinguishes between the somatosensory or viscerosensory fate of relay neurons.

A distinct preisthmic histogenetic domain is defined by overlap of Otx2 and Pax2 gene expression in the avian caudal midbrain.

Correlative in situ hybridization of Otx2, Pax2, Gbx2, and Fgf8 mRNA probes in adjacent serial sections through the chicken midbrain and isthmus at early to intermediate stages of development served to map in detail the area of overlap of Otx2 and Pax2 transcripts in the caudal midbrain. The neuronal populations developing within this preisthmic domain made up a caudal part of the midbrain reticular formation, the interfascicular nucleus, and the magnocellular (pre)isthmic nucleus, plus the corresponding part of the periaqueductal gray. The torus semicircularis-the inferior colliculus homolog-expressed Otx2 in its ventricular lining exclusively, but it never expressed Pax2. The parvicellular isthmic nucleus, although placed inside the midbrain lobe, never expressed Otx2, and its cells rapidly down-regulated an early transient Pax2 signal; this pattern is consistent with its reported isthmic origin and forward tangential translocation. This analysis reveals the existence of four distinct midbrain histogenetic domains along the longitudinal axis, at least for the alar plate. These presumably result from step-like isthmic organizer effects on Otx2-expressing midbrain neuroepithelium at different distances from a caudal FGF8 morphogen source (isthmic Fgf8-positive domain). The final phenotypes of these domains are histologically diverse and make up the griseum tectale (rostrally), the optic tectum, the torus semicircularis, and the presently characterized preisthmic domain (lying closest to the isthmic organizer). Available comparative data for reptiles and mammals suggest the general validity of this scheme.

3 dimensional modelling of early human brain development using optical projection tomography.

BACKGROUND: As development proceeds the human embryo attains an ever more complex three dimensional (3D) structure. Analyzing the gene expression patterns that underlie these changes and interpreting their significance depends on identifying the anatomical structures to which they map and following these patterns in developing 3D structures over time. The difficulty of this task greatly increases as more gene expression patterns are added, particularly in organs with complex 3D structures such as the brain. Optical Projection Tomography (OPT) is a new technology which has been developed for rapidly generating digital 3D models of intact specimens. We have assessed the resolution of unstained neuronal structures within a Carnegie Stage (CS)17 OPT model and tested its use as a framework onto which anatomical structures can be defined and gene expression data mapped. RESULTS: Resolution of the OPT models was assessed by comparison of digital sections with physical sections stained, either with haematoxylin and eosin (H&E) or by immunocytochemistry for GAP43 or PAX6, to identify specific anatomical features. Despite the 3D models being of unstained tissue, peripheral nervous system structures from the trigeminal ganglion (approximately 300 microm by approximately 150 microm) to the rootlets of cranial nerve XII (approximately 20 microm in diameter) were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica (core is approximately 30 microm thick). Fourteen anatomical domains have been identified and visualised within the CS17 model. Two 3D gene expression domains, known to be defined by Pax6 expression in the mouse, were clearly visible when PAX6 data from 2D sections were mapped to the CS17 model. The feasibility of applying the OPT technology to all stages from CS12 to CS23, which encompasses the major period of organogenesis for the human developing central nervous system, was successfully demonstrated. CONCLUSION: In the CS17 model considerable detail is visible within the developing nervous system at a minimum resolution of approximately 20 microm and 3D anatomical and gene expression domains can be defined and visualised successfully. The OPT models and accompanying technologies for manipulating them provide a powerful approach to visualising and analysing gene expression and morphology during early human brain development.

The avian griseum tectale: cytoarchitecture, NOS expression and neurogenesis.

The griseum tectale (GT) is a retinorecipient layered formation located in the rostral alar midbrain, just behind the constriction that separates it from the diencephalon (pretectum). Tritiated-thymidine autoradiographic data on neuronal birthdates show that the GT cell population starts to be generated at HH21 and most neurons are born by stage HH25, accumulating within a primordial periventricular layer, which shows strong nitric oxide synthase immunoreactivity at later stages of development. There is a barely noticeable rostrocaudal neurogenetic and differentiation gradient across the GT, which seems to continue into that of the neighboring optic tectum. The GT layering develops gradually by radial migration of its postmitotic neurons between stages HH26 and HH35. The structure of the mature GT can be divided into periventricular, central, and superficial layers, similarly to the adjacent optic tectum, but it shows different layering aspects, particularly in the retinorecipient superficial layer.

Gbx2 expression in the late embryonic chick dorsal thalamus.

The expression pattern of the transcription factor gene Gbx2 in the forebrain of chicken embryos (embryonic day 14) was mapped using digoxigenin-labeled riboprobes and compared with the expression of the transcription factors Pax6 and Nkx2.2. The topographic analysis of Gbx2 expression on coronal and sagittal sections discriminated the positions and boundaries of diverse neuronal nuclei belonging to the dorsal thalamus from neighboring territories (the epithalamus, ventral thalamus, pretectum, and the underlying basal plate). The differential expression of Gbx2 within the dorsal thalamus clearly corresponds with the existence of four primary subdivisions identified in a previous study from this laboratory [13]: the anteroventral region and dorsal, intermediate, and ventral tiers. The subhabenular region turned out not to express Gbx2; this possibly implies it needs to be distinguished as a fifth separate dorsal thalamus subdivision.