The Midbrain Preisthmus: A Poorly Known Effect of the Isthmic Organizer.

This essay reexamines molecular evidence supporting the existence of the 'preisthmus', a caudal midbrain domain present in vertebrates (studied here in the mouse). It is thought to derive from the embryonic m2 mesomere and appears intercalated between the isthmus (caudally) and the inferior colliculus (rostrally). Among a substantial list of gene expression mappings examined from the Allen Developing and Adult Brain Atlases, a number of quite consistent selective positive markers, plus some neatly negative markers, were followed across embryonic stages E11.5, E13.5, E15.5, E18.5, and several postnatal stages up to the adult brain. Both alar and basal subdomains of this transverse territory were explored and illustrated. It is argued that the peculiar molecular and structural profile of the preisthmus is due to its position as rostrally adjacent to the isthmic organizer, where high levels of both FGF8 and WNT1 morphogens must exist at early embryonic stages. Isthmic patterning of the midbrain is discussed in this context. Studies of the effects of the isthmic morphogens usually do not attend to the largely unknown preisthmic complex. The adult alar derivatives of the preisthmus were confirmed to comprise a specific preisthmic sector of the periaqueductal gray, an intermediate stratum represented by the classic cuneiform nucleus, and a superficial stratum containing the subbrachial nucleus. The basal derivatives, occupying a narrow retrorubral domain intercalated between the oculomotor and trochlear motor nuclei, include dopaminergic and serotonergic neurons, as well as a variety of peptidergic neuron types.

Origin of Neuroblasts in the Avian Otic Placode and Their Distributions in the Acoustic and Vestibular Ganglia.

The inner ear is a complex three-dimensional sensorial structure with auditory and vestibular functions. This intricate sensory organ originates from the otic placode, which generates the sensory elements of the membranous labyrinth, as well as all the ganglionic neuronal precursors. How auditory and vestibular neurons establish their fate identities remains to be determined. Their topological origin in the incipient otic placode could provide positional information before they migrate, to later segregate in specific portions of the acoustic and vestibular ganglia. To address this question, transplants of small portions of the avian otic placode were performed according to our previous fate map study, using the quail/chick chimeric graft model. All grafts taking small areas of the neurogenic placodal domain contributed neuroblasts to both acoustic and vestibular ganglia. A differential distribution of otic neurons in the anterior and posterior lobes of the vestibular ganglion, as well as in the proximal, intermediate, and distal portions of the acoustic ganglion, was found. Our results clearly show that, in birds, there does not seem to be a strict segregation of acoustic and vestibular neurons in the incipient otic placode.

CRABP-I Expression Patterns in the Developing Chick Inner Ear.

The vertebrate inner ear is a complex three-dimensional sensorial structure with auditory and vestibular functions, regarded as an excellent system for analyzing events that occur during development, such as patterning, morphogenesis, and cell specification. Retinoic acid (RA) is involved in all these development processes. Cellular retinoic acid-binding proteins (CRABPs) bind RA with high affinity, buffering cellular free RA concentrations and consequently regulating the activation of precise specification programs mediated by particular regulatory genes. In the otic vesicle, strong CRABP-I expression was detected in the otic wall's dorsomedial aspect, where the endolymphatic apparatus develops, whereas this expression was lower in the ventrolateral aspect, where part of the auditory system forms. Thus, CRABP-I proteins may play a role in the specification of the dorsal-to-ventral and lateral-to-medial axe of the otic anlagen. Regarding the developing sensory patches, a process partly involving the subdivision of a ventromedial pro-sensory domain, the CRABP-I gene displayed different levels of expression in the presumptive territory of each sensory patch, which was maintained throughout development. CRABP-I was also relevant in the acoustic-vestibular ganglion and in the periotic mesenchyme. Therefore, CRABP-I could protect RA-sensitive cells in accordance with its dissimilar concentration in specific areas of the developing chick inner ear.

An Update on the Molecular Mechanism of the Vertebrate Isthmic Organizer Development in the Context of the Neuromeric Model.

A crucial event during the development of the central nervous system (CNS) is the early subdivision of the neural tube along its anterior-to-posterior axis to form neuromeres, morphogenetic units separated by transversal constrictions and programed for particular genetic cascades. The narrower portions observed in the developing neural tube are responsible for relevant cellular and molecular processes, such as clonal restrictions, expression of specific regulatory genes, and differential fate specification, as well as inductive activities. In this developmental context, the gradual formation of the midbrain-hindbrain (MH) constriction has been an excellent model to study the specification of two major subdivisions of the CNS containing the mesencephalic and isthmo-cerebellar primordia. This MH boundary is coincident with the common Otx2-(midbrain)/Gbx2-(hindbrain) expressing border. The early interactions between these two pre-specified areas confer positional identities and induce the generation of specific diffusible morphogenes at this interface, in particular FGF8 and WNT1. These signaling pathways are responsible for the gradual histogenetic specifications and cellular identity acquisitions with in the MH domain. This review is focused on the cellular and molecular mechanisms involved in the specification of the midbrain/hindbrain territory and the formation of the isthmic organizer. Emphasis will be placed on the chick/quail chimeric experiments leading to the acquisition of the first fate mapping and experimental data to, in this way, better understand pioneering morphological studies and innovative gain/loss-of-function analysis.

Cyp1B1 expression patterns in the developing chick inner ear.

BACKGROUND: Retinoic acid (RA) plays an important role in organogenesis as a paracrine signal through transcriptional regulation of an increasing number of known downstream target genes, regulating cell proliferation, and differentiation. During the development of the inner ear, RA directly governs the morphogenesis and specification processes mainly by means of RA-synthesizing retinaldehyde dehydrogenase (RALDH) enzymes. Interestingly, CYP1B1, a cytochrome P450 enzyme, is able to mediate the oxidative metabolisms also leading to RA generation, its expression patterns being associated with many known sites of RA activity. RESULTS: This study describes for the first time the presence of CYP1B1 in the developing chick inner ear as a RALDH-independent RA-signaling mechanism. In our in situ hybridization analysis, Cyp1B1 expression was first observed in a domain located in the ventromedial wall of the otic anlagen, being included within the rostralmost aspect of an Fgf10-positive pan-sensory domain. As development proceeds, all identified Fgf10-positive areas were Cyp1B1 stained, with all sensory patches being Cyp1B1 positive at stage HH34, except the macula neglecta. CONCLUSIONS: Cyp1B1 expression suggested a possible contribution of CYP1B1 action in the specification of the lateral-to-medial and dorsal-to-ventral axes of the developing chick inner ear.

Origin of acoustic-vestibular ganglionic neuroblasts in chick embryos and their sensory connections.

The inner ear is a complex three-dimensional sensory structure with auditory and vestibular functions. It originates from the otic placode, which generates the sensory elements of the membranous labyrinth and all the ganglionic neuronal precursors. Neuroblast specification is the first cell differentiation event. In the chick, it takes place over a long embryonic period from the early otic cup stage to at least stage HH25. The differentiating ganglionic neurons attain a precise innervation pattern with sensory patches, a process presumably governed by a network of dendritic guidance cues which vary with the local micro-environment. To study the otic neurogenesis and topographically-ordered innervation pattern in birds, a quail-chick chimaeric graft technique was used in accordance with a previously determined fate-map of the otic placode. Each type of graft containing the presumptive domain of topologically-arranged placodal sensory areas was shown to generate neuroblasts. The differentiated grafted neuroblasts established dendritic contacts with a variety of sensory patches. These results strongly suggest that, rather than reverse-pathfinding, the relevant role in otic dendritic process guidance is played by long-range diffusing molecules.

Fgf3 and Fgf16 expression patterns define spatial and temporal domains in the developing chick inner ear.

The inner ear is a morphologically complex sensory structure with auditory and vestibular functions. The developing otic epithelium gives rise to neurosensory and non-sensory elements of the adult membranous labyrinth. Extrinsic and intrinsic signals manage the patterning and cell specification of the developing otic epithelium by establishing lineage-restricted compartments defined in turn by differential expression of regulatory genes. FGF3 and FGF16 are excellent candidates to govern these developmental events. Using the chick inner ear, we show that Fgf3 expression is present in the borders of all developing cristae. Strong Fgf16 expression was detected in a portion of the developing vertical and horizontal pouches, whereas the cristae show weaker or undetected Fgf16 expression at different developmental stages. Concerning the rest of the vestibular sensory elements, both the utricular and saccular maculae were Fgf3 positive. Interestingly, strong Fgf16 expression delimited these Fgf16-negative sensory patches. The Fgf3-negative macula neglecta and the Fgf3-positive macula lagena were included within weakly Fgf16-expressing areas. Therefore, different FGF-mediated mechanisms might regulate the specification of the anterior (utricular and saccular) and posterior (neglecta and lagena) maculae. In the developing cochlear duct, dynamic Fgf3 and Fgf16 expression suggests their cooperation in the early specification and later cell differentiation in the hearing system. The requirement of Fgf3 and Fgf16 genes in endolymphatic apparatus development and neurogenesis are discussed. Based on these observations, FGF3 and FGF16 seem to be key signaling pathways that control the inner ear plan by defining epithelial identities within the developing otic epithelium.

Expression patterns of Irx genes in the developing chick inner ear.

The vertebrate inner ear is a complex three-dimensional sensorial structure with auditory and vestibular functions. The molecular patterning of the developing otic epithelium creates various positional identities, consequently leading to the stereotyped specification of each neurosensory and non-sensory element of the membranous labyrinth. The Iroquois (Iro/Irx) genes, clustered in two groups (A: Irx1, Irx2, and Irx4; and B: Irx3, Irx5, and Irx6), encode for transcriptional factors involved directly in numerous patterning processes of embryonic tissues in many phyla. This work presents a detailed study of the expression patterns of these six Irx genes during chick inner ear development, paying particular attention to the axial specification of the otic anlagen. The Irx genes seem to play different roles at different embryonic periods. At the otic vesicle stage (HH18), all the genes of each cluster are expressed identically. Both clusters A and B seem involved in the specification of the lateral and posterior portions of the otic anlagen. Cluster B seems to regulate a larger area than cluster A, including the presumptive territory of the endolymphatic apparatus. Both clusters seem also to be involved in neurogenic events. At stages HH24/25-HH27, combinations of IrxA and IrxB genes participate in the specification of most sensory patches and some non-sensory components of the otic epithelium. At stage HH34, the six Irx genes show divergent patterns of expression, leading to the final specification of the membranous labyrinth, as well as to cell differentiation.

piRNA-associated proteins and retrotransposons are differentially expressed in murine testis and ovary of aryl hydrocarbon receptor deficient mice.

Previous studies suggested that the aryl hydrocarbon receptor (AhR) contributes to mice reproduction and fertility. However, the mechanisms involved remain mostly unknown. Retrotransposon silencing by Piwi-interacting RNAs (piRNAs) is essential for germ cell maturation and, remarkably, AhR has been identified as a regulator of murine B1-SINE retrotransposons. Here, using littermate AhR+/+ and AhR-/- mice, we report that AhR regulates the general course of spermatogenesis and oogenesis by a mechanism likely to be associated with piRNA-associated proteins, piRNAs and retrotransposons. piRNA-associated proteins MVH and Miwi are upregulated in leptotene to pachytene spermatocytes with a more precocious timing in AhR-/- than in AhR+/+ testes. piRNAs and transcripts from B1-SINE, LINE-1 and IAP retrotransposons increased at these meiotic stages in AhR-null testes. Moreover, B1-SINE transcripts colocalize with MVH and Miwi in leptonema and pachynema spermatocytes. Unexpectedly, AhR-/- males have increased sperm counts, higher sperm functionality and enhanced fertility than AhR+/+ mice. In contrast, piRNA-associated proteins and B1-SINE and IAP-derived transcripts are reduced in adult AhR-/- ovaries. Accordingly, AhR-null female mice have lower numbers of follicles when compared with AhR+/+ mice. Thus, AhR deficiency differentially affects testis and ovary development possibly by a process involving piRNA-associated proteins, piRNAs and transposable elements.

Origin and early development of the chicken adenohypophysis.

The adenohypophysis (ADH) is an important endocrine organ involved in the regulation of many physiological processes. The late morphogenesis of this organ at neural tube stages is well known: the epithelial ADH primordium is recognized as an invagination of the stomodeal roof (Rathke's pouch), whose walls later thicken and differentiate as the primordium becomes pediculated, and then fully separated from the stomodeum. The primordium attaches to the pial surface of the basal hypothalamus, next to the neurohypophyseal field (NH; future posterior pituitary), from which it was previously separated by migrating prechordal plate (pp) cells. Once the NH evaginates, the ADH surrounds it and jointly forms with it the pituitary gland. In contrast, little is known about the precise origin of the ADH precursors at neural plate stages and how the primordium reaches the stomodeum. For that reason, we produced in the chicken a specific ADH fate map at early neural plate stages, which was amplified with gene markers. By means of experiments labeling the mapped presumptive ADH, we were able to follow the initial anlage into its transformation into Rathke's pouch. The ADH origin was corroborated to be strictly extraneural, i.e., to lie at stage HH4/5 outside of the anterior neural plate (anp) within the pre-placodal field. The ADH primordium is fully segregated from the anterior neural border cells and the neighboring olfactory placodes both in terms of precursor cells and molecular profile from head fold stages onwards. The placode becomes visible as a molecularly characteristic ectodermal thickening from stage HH10 onwards. The onset of ADH genoarchitectonic regionalization into intermediate and anterior lobes occurs at closed neural tube stages.

Fate map of the chicken otic placode.

The inner ear is an intricate three-dimensional sensory organ that arises from a flat, thickened portion of the ectoderm termed the otic placode. There is evidence that the ontogenetic steps involved in the progressive specification of the highly specialized inner ear of vertebrates involve the concerted actions of diverse patterning signals that originate from nearby tissues, providing positional identity and instructive context. The topology of the prospective inner ear portions at placode stages when such patterning begins has remained largely unknown. The chick-quail model was used to perform a comprehensive fate mapping study of the chick otic placode, shedding light on the precise topological position of each presumptive inner ear component relative to the dorsoventral and anteroposterior axes of the otic placode and, implicitly, to the possible sources of inducing signals. The findings reveal the existence of three dorsoventrally arranged anteroposterior domains from which the endolymphatic system, the maculae and basilar papilla, and the cristae develop. This study provides new bases for the interpretation of earlier and future descriptive and experimental studies that aim to understand the molecular genetic mechanisms involved in otic placode patterning.

Fgf10 expression patterns in the developing chick inner ear.

The inner ear is a complex three-dimensional sensorial structure with auditory and vestibular functions. It originates from the otic placode, which invaginates, forming the otic vesicle; the latter gives rise to neurosensory and nonsensory elements of the adult membranous labyrinth. A hypothesis based on descriptive and experimental evidence suggests that the acquisition of discrete sensory patches during evolution of this primordium may be related to subdivision of an early pansensory domain. In order to gain insight into this developmental mechanism, we carried out a detailed analysis of the spatial and temporal expression pattern of the gene Fgf10, by comparing different markers of otic patterning and hair cell differentiation. Fgf10 expression labels a sensory-competent domain included in a Serrate-positive territory from which most of the sensory epithelia arise. Our data show that Fgf10 transcripts are present initially in a narrow ventromedial band of the rudimentary otocyst, extending between its rostral and caudal poles. During development, this Fgf10-expressing area splits repetitively into several separate subareas, creating six of the eight sensory organs present in birds. Only the lateral crista and the macula neglecta were initially Fgf10 negative, although they activated Fgf10 expression after their specification as sensory elements. These results allowed us to determine a timetable of sensory specification in the developing chick inner ear. The comparison of the expression pattern of Fgf10 with those of other markers of sensory differentiation contributes to our understanding of the mechanism by which vertebrate inner ear prosensory domains have arisen during evolution.

Origin and plasticity of the subdivisions of the inferior olivary complex.

The precerebellar nuclei (PCN) originate from the rhombic lip, a germinal neuroepithelium adjacent to the roof plate of the fourth ventricle. We first report here that, in chicken, the Brn3a-expressing postmitotic medullary cells that produce the inferior olive (ION, the source of cerebellar climbing fibres) originate from a dorso-ventral domain roughly coinciding with the hindbrain vestibular column. Whereas Foxd3 expression labels the whole mature ION but is only detected in a subpopulation of ION neuroblasts initiating their migration, we report that Brn3a allows the visualization of the whole population of ION neurons from the very beginning of their migration. We show that Brn3a-positive neurons migrate tangentially ventralwards through a characteristic dorso-ventral double submarginal stream. Cath1 expressing progenitors lying just dorsal to the ION origin correlated dorso-ventral topography with the prospective cochlear column (caudal to it) and generate precerebellar nuclei emitting mossy-fiber cerebellar afferents. We used the chick-quail chimaera technique with homotopic grafts at HH10 to determine the precise fate map of ION precursors across the caudal cryptorhombomeric subdivisions of the medullary hindbrain (r8-r11). We demonstrate that each crypto-rhombomere contributes to two lamellae of the ION, while each ION sub-nucleus originates from at least two contiguous crypto-rhombomeres. We then questioned how rhombomere identity is related to the plasticity of cell type specification in the dorsal hindbrain. The potential plasticity of ectopically HH10 grafted ION progenitors to change their original fate in alternative rostrocaudal environments was examined. Heterotopic grafts from the presumptive ION territory to the pontine region (r4-r5) caused a change of fate, since the migrated derivatives adopted a pontine phenotype. The reverse experiment caused pontine progenitors to produce derivatives appropriately integrated into the ION complex. Grafts of ION progenitor domains to myelomeres (my) 2-3 also showed complete fate regulation, reproducing spinal cord-like structures, whereas the reverse experiment revealed the inability of my2-3 to generate ION cell types. This was not the case with more caudal, relatively less specified myelomeres (my5-6). Interestingly, when heterotopically grafted cells are integrated dorsally, they do not change their phenotype. Our results support the hypothesis that positional information present in the hindbrain and spinal cord at early neural tube stages controls the specific fates of ventrally migrating PCN precursors.

Multiple origins, migratory paths and molecular profiles of cells populating the avian interpeduncular nucleus.

The interpeduncular nucleus (IP) is a key limbic structure, highly conserved evolutionarily among vertebrates. The IP receives indirect input from limbic areas of the telencephalon, relayed by the habenula via the fasciculus retroflexus. The function of the habenulo-IP complex is poorly understood, although there is evidence that in rodents it modulates behaviors such as learning and memory, avoidance, reward and affective states. The IP has been an important subject of interest for neuroscientists, and there are multiple studies about the adult structure, chemoarchitecture and its connectivity, with complex results, due to the presence of multiple cell types across a variety of subnuclei. However, the ontogenetic origins of these populations have not been examined, and there is some controversy about its location in the midbrain-anterior hindbrain area. To address these issues, we first investigated the anteroposterior (AP) origin of the IP complex by fate-mapping its neuromeric origin in the chick, discovering that the IP develops strictly within isthmus and rhombomere 1. Next, we studied the dorsoventral (DV) positional identity of subpopulations of the IP complex. Our results indicate that there are at least four IP progenitor domains along the DV axis. These specific domains give rise to distinct subtypes of cell populations that target the IP with variable subnuclear specificity. Interestingly, these populations can be characterized by differential expression of the transcription factors Pax7, Nkx6.1, Otp, and Otx2. Each of these subpopulations follows a specific route of migration from its source, and all reach the IP roughly at the same stage. Remarkably, IP progenitor domains were found both in the alar and basal plates. Some IP populations showed rostrocaudal restriction in their origins (isthmus versus anterior or posterior r1 regions). A tentative developmental model of the structure of the avian IP is proposed. The IP emerges as a plurisegmental and developmentally heterogeneous formation that forms ventromedially within the isthmus and r1. These findings are relevant since they help to understand the highly complex chemoarchitecture, hodology and functions of this important brainstem structure.

Contrasting 5' and 3' evolutionary histories and frequent evolutionary convergence in Meis/hth gene structures.

Organisms show striking differences in genome structure; however, the functional implications and fundamental forces that govern these differences remain obscure. The intron-exon organization of nuclear genes is involved in a particularly large variety of structures and functional roles. We performed a 22-species study of Meis/hth genes, intron-rich homeodomain-containing transcription factors involved in a wide range of developmental processes. Our study revealed three surprising results that suggest important and very different functions for Meis intron-exon structures. First, we find unexpected conservation across species of intron positions and lengths along most of the Meis locus. This contrasts with the high degree of structural divergence found in genome-wide studies and may attest to conserved regulatory elements residing within these conserved introns. Second, we find very different evolutionary histories for the 5' and 3' regions of the gene. The 5'-most 10 exons, which encode the highly conserved Meis domain and homeodomain, show striking conservation. By contrast, the 3' of the gene, which encodes several domains implicated in transcriptional activation and response to cell signaling, shows a remarkably active evolutionary history, with diverse isoforms and frequent creation and loss of new exons and splice sites. This region-specific diversity suggests evolutionary "tinkering," with alternative splicing allowing for more subtle regulation of protein function. Third, we find a large number of cases of convergent evolution in the 3' region, including 1) parallel losses of ancestral coding sequence, 2) parallel gains of external and internal splice sites, and 3) recurrent truncation of C-terminal coding regions. These results attest to the importance of locus-specific splicing functions in differences in structural evolution across genes, as well as to commonalities of forces shaping the evolution of individual genes along different lineages.

Distinct and redundant expression and transcriptional diversity of MEIS gene paralogs during chicken development.

Members of the Meis family of TALE homeobox transcription factors are involved in many processes of vertebrate development and morphogenesis, showing extremely complex transcriptional and spatiotemporal expression patterns. In this work, we performed a comprehensive study of chicken Meis genes using multiple approaches. First, we assessed whether the chicken genome contains a Meis3 ortholog or harbors only two Meis genes; we gathered several lines of evidence pointing to a specific loss of the Meis3 ortholog in an early ancestor of birds. Next, we studied the transcriptional diversity generated from chicken Meis genes through alternative splicing during development. Finally, we performed a detailed analysis of chick Meis1/2 expression patterns during early embryogenesis and organogenesis. We show that the expression of both Meis genes begins at the gastrulation stage in the three embryonic layers, presenting highly dynamic patterns with overlapping as well as distinct expression domains throughout development.

Meis gene expression patterns in the developing chicken inner ear.

We are interested in stable gene network activities operating sequentially during inner ear specification. The implementation of this patterning process is a key event in the generation of functional subdivisions of the otic vesicle during early embryonic development. The vertebrate inner ear is a complex sensory structure that is a good model system for characterization of developmental mechanisms controlling patterning and specification. Meis genes, belonging to the TALE family, encode homodomain-containing transcription factors remarkably conserved during evolution, which play a role in normal and neoplastic development. To gain understanding of the possible role of homeobox Meis genes in the developing chick inner ear, we comprehensively analyzed their spatiotemporal expression patterns from early otic specification stages onwards. In the invaginating otic placode, Meis1/2 transcripts were observed in the borders of the otic cup, being absent in the portion of otic epithelium closest to the hindbrain. As development proceeds, Meis1 and Meis2 expressions became restricted to the dorsomedial otic epithelium. Both genes were strongly expressed in the entire presumptive domain of the semicircular canals, and more weakly in all associated cristae. The endolymphatic apparatus was labeled in part by Meis1/2. Meis1 was also expressed in the lateral wall of the growing cochlear duct, while Meis2 expression was detected in a few cells of the developing acoustic-vestibular ganglion. Our results suggest a possible role of Meis assigning regional identity in the morphogenesis, patterning, and specification of the developing inner ear.

Raldh3 gene expression pattern in the developing chicken inner ear.

Retinoic acid (RA), an active metabolite of vitamin A, is a diffusible molecule that regulates the expression of several families of genes, playing a key role in specification processes during chordate development. With the aim of defining its possible role in the developing chick inner ear, we obtained in this work a detailed spatiotemporal distribution of the enzymes involved in its synthesis, the retinaldehyde dehydrogenases (RALH1-4). Our results showed that, in contrast to the mouse inner ear, Raldh3 expression was the only Raldh gene detected in the developing chick inner ear, where it appears as early as stage 18. During inner ear morphogenesis, Raldh3 expression was predominantly observed in the endolymphatic system. The Raldh3 expression pattern delimited totally or partially the Bmp4-positive presumptive territories of vestibular sensory epithelia by stage 24 and the basilar papilla at stage 34, suggesting a possible involvement of RA in their specification. In addition, several vestibular sensory areas showed some Raldh3-expressing cells close to the Raldh3-positive domain. These results suggest that the RA signaling pathway may play a role in the initial patterning of the otic epithelium and cell differentiation therein, providing local positional information. Having in mind this Raldh3 expression pattern, we discuss the regulatory interactions among the RA, bone morphogenetic protein, and fibroblast growth factor signaling pathways in the specification of otic sensory elements. Our investigation may underpin further experimental studies aimed at understanding the possible role of signaling pathways in patterning of the developing chick inner ear.

Quantitative analysis of neural plate thickness and cell density during gastrulation in the chick embryo.

We quantitatively analyzed the developing prospective neural and non-neural ectoderm during chicken gastrulation on semithin transverse sections. At stage PS8 (primitive streak stage 8 of Lopez-Sanchez et al. [C. Lopez-Sanchez, L. Puelles, V. Garcia-Martinez, L. Rodriguez-Gallardo, Morphological and molecular analysis of the early developing chick requires an expanded series of primitive streak stages, J. Morphol. 264 (2005) 105-116.], equivalent to stage HH4), the thickest area of the ectoderm agrees in extent with the fate-mapped neural plate we had reported previously. The thickness of the median ectoderm is constantly higher up to a distance of 250mum from Hensen's node, and thickness decreases along a mediolateral gradient with a further drop at the prospective lateral border of the neural plate. A higher cell density of the developing ectoderm also coincided with the prospective neural plate. We observed that cell death does not play an important role in the spatial definition of the neural plate.