Luisa Sánchez-Arrones

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. J. Comp. Neurol. 505:379–403, 2007.

Genoarchitectonic profile of developing nuclear groups in the chicken pretectum.

Earlier results on molecularly coded progenitor domains in the chicken pretectum revealed an anteroposterior subdivision of the pretectum in precommissural (PcP), juxtacommissural (JcP), and commissural (CoP) histogenetic areas, each specified differentially (Ferran et al. [2007] J Comp Neurol 505:379–403). Here we examined the nuclei derived from these areas with regard to characteristic gene expression patterns and gradual histogenesis (eventually, migration patterns). We sought a genoarchitectonic schema of the avian pretectum within the prosomeric model of the vertebrate forebrain (Puelles and Rubenstein [2003] Trends Neurosci 26:469–476; Puelles et al. [2007] San Diego: Academic Press). Transcription‐factor gene markers were used to selectively map derivatives of the three pretectal histogenetic domains: Pax7 and Pax6 (CoP); FoxP1 and Six3 (JcP); and FoxP2, Ebf1, and Bhlhb4 (PcP). The combination of this genoarchitectonic information with additional data on Lim1, Tal2, and Nbea mRNA expression and other chemoarchitectonic results allowed unambiguous characterization of some 30 pretectal nuclei. Apart from grouping them as derivatives of the three early anteroposterior domains, we also assigned them to postulated dorsoventral subdomains (Ferran et al. [2007]). Several previously unknown neuronal populations were detected, thus expanding the list of pretectal structures, and we corrected some apparently confused concepts in the earlier literature. The composite gene expression map represents a substantial advance in anatomical and embryological knowledge of the avian pretectum. Many nuclear primordia can be recognized long before the mature differentiated state of the pretectum is achieved. This study provides fundamental notions for ultimate scientific study of the specification and regionalization processes building up this brain area, both in birds and other vertebrates. J. Comp. Neurol. 517:405–451, 2009.

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.

Ontogenetic Expression of Sonic Hedgehog in the Chicken Subpallium

Sonic hedgehog (SHH) is a secreted signaling factor that is implicated in the molecular patterning of the central nervous system (CNS), somites, and limbs in vertebrates. SHH has a crucial role in the generation of ventral cell types along the entire rostrocaudal axis of the neural tube. It is secreted early in development by the axial mesoderm (prechordal plate and notochord) and the overlying ventral neural tube. Recent studies clarified the impact of SHH signaling mechanisms on dorsoventral patterning of the spinal cord, but the corresponding phenomena in the rostral forebrain are slightly different and more complex. This notably involves separate Shh expression in the preoptic part of the forebrain alar plate, as well as in the hypothalamic floor and basal plates. The present work includes a detailed spatiotemporal description of the singular alar Shh expression pattern in the rostral preoptic forebrain of chick embryos, comparing it with FoxG1, Dlx5, Nkx2.1, and Nkx2.2 mRNA expression at diverse stages of development. As a result of this mapping, we report a subdivision of the preoptic region in dorsal and ventral zones; only the dorsal part shows Shh expression. The positive area impinges as well upon a median septocommissural preoptic domain. Our study strongly suggests tangential migration of Shh-positive cells from the preoptic region into other subpallial domains, particularly into the pallidal mantle and the intermediate septum.

Cdon acts as a Hedgehog decoy receptor during proximal-distal patterning of the optic vesicle

Patterning of the vertebrate optic vesicle into proximal/optic stalk and distal/neural retina involves midline-derived Hedgehog (Hh) signalling, which promotes stalk specification. In the absence of Hh signalling, the stalks are not specified, causing cyclopia. Recent studies showed that the cell adhesion molecule Cdon forms a heteromeric complex with the Hh receptor Patched 1 (Ptc1). This receptor complex binds Hh and enhances signalling activation, indicating that Cdon positively regulates the pathway. Here we show that in the developing zebrafish and chick optic vesicle, in which cdon and ptc1 are expressed with a complementary pattern, Cdon acts as a negative Hh signalling regulator. Cdon predominantly localizes to the basolateral side of neuroepithelial cells, promotes the enlargement of the neuroepithelial basal end-foot and traps Hh protein, thereby limiting its dispersion. This Ptc-independent function protects the retinal primordium from Hh activity, defines the stalk/retina boundary and thus the correct proximo-distal patterning of the eye.

Sharpening of the anterior neural border in the chick by rostral endoderm signalling

The anterior border of the neural plate, presumed to contain the prospective peripheral portion (roof) of the prospective telencephalon, emerges within a vaguely defined proneural ectodermal region. Fate maps carried out at HH4 in the chick reveal that this region still produces indistinctly neural, placodal and non-neural derivatives; it does not express neural markers. We examined how the definitive anterior border domain of the rostral forebrain becomes established and comes to display a neural molecular profile, whereas local non-neural derivatives become separated. The process, interpreted as a border sharpening mechanism via intercalatory cell movements, was studied using fate mapping, time-lapse microscopy and in situ hybridization. Separation of neural and non-neural domains proceeds along stages HH4-HH4+, is well advanced at HH5, and is accompanied by a novel dorsoventral intercalation, oriented orthogonal to the border, that distributes transitional cells into molecularly distinct neural and non-neural fields. Meanwhile, neuroectodermal Sox2 expression spreads peripherally from the neighbourhood of the node, reaching the nascent anterior border domain at HH5. We also show that concurrent signals from the endodermal layer are necessary to position and sharpen the neural border, and suggest that FGF8 might be a component of this signalling.

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. Developmental Dynamics 240:1475–1492, 2011.

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.

03-P087 SHH patterning effects on chicken pretectal region between stages HH13 and HH24

with this construct to obtain a permanent clone cell line in which the Fgf8b-EGFP could be secreted on the extracellular matrix. The inductive activity of Fgf8b-EGFP protein was proved either in vitro (cell cultures) as in ex-vivo (organotypic explant cultures of E9.5 mouse neural tube). Ectopically, Fgf8b-EGFP induces the phosphorilation of the final effector of the FGF signalling cascade (ERK1/2) and also the expression of downstream isthmic genes (Dusp6, Spry, SEF, En1/2). Using passive carriers (loaded heparin beads) or active productive sources (HEK-293-Fgf8b-EGFP cells) we have analysed the gradiental component of FGF8b over the neuroepithelium in time and space. The present study is supported by MEC SAF2008-01004, BFU2008-00588, FPI BES-2006-12420M, EC-CONSOLIDER CSD200700023, Imagenio 2010, D.E. supported by Programa Ramón y Cajal-2004 and the Spanish Ministry of Health, Instituto de Salud Carlos III-CIBERSAM.

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 Hensens 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.