José Luis Ferran

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.

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

BackgroundAs 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.ResultsResolution 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 (~300 μm by ~150 μm) to the rootlets of cranial nerve XII (~20 μm in diameter) were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica (core is ~30 μm 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.ConclusionIn the CS17 model considerable detail is visible within the developing nervous system at a minimum resolution of ~20 μm 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.

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.

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.

Concept of neural genoarchitecture and its genomic fundament

The recent concept of neural genoarchitecture (or genoarchitectonics) is examined from several angles, aiming to clarify the rationale for this new approach in causal and descriptive neuroanatomy. Gene expression patterns can be used as topographic stains revealing architectonic borders that may clarify, dispute, or complicate existing brain anatomical subdivisions based on other methods, while increasing our understanding of how they arise in ontogenesis and evolution. A section of the text deals with differential regulation of gene expression in an ontogenetic causal network, attending to the structure of the genome and the functional peculiarities of enhancer and repressor regulatory regions that modulate gene transcription. The emergence of regionally characteristic sets of active transcription factors represents a critical concept, molecular identity, which can be applied to discrete brain territories and neuronal populations. Gene regulation is tied to positional effects, that is, topologically invariant domains of gene expression and natural boundaries, which can be correlated with anatomic ones. The large-scale stability of these patterns among vertebrates underpins molecularly the structural brain Bauplan, and is the fundament of field homology. The study of genoarchitectonic boundaries is presented as a crucial objective of modern neuroanatomic research. At most brain regions, new neuronal populations are being detected thanks to their differential genoarchitectonic features.

Topography of Somatostatin Gene Expression Relative to Molecular Progenitor Domains during Ontogeny of the Mouse Hypothalamus

The hypothalamus comprises alar, basal, and floor plate developmental compartments. Recent molecular data support a rostrocaudal subdivision into rostral (terminal) and caudal (peduncular) halves. In this context, the distribution of neuronal populations expressing somatostatin (Sst) mRNA was analyzed in the developing mouse hypothalamus, comparing with the expression pattern of the genes Orthopedia (Otp), Distal-less 5 (Dlx5), Sonic Hedgehog (Shh), and Nk2 homeobox 1 (Nkx2.1). At embryonic day 10.5 (E10.5), Sst mRNA was first detectable in the anterobasal nucleus, a Nkx2.1-, Shh-, and Otp-positive basal domain. By E13.5, nascent Sst expression was also related to two additional Otp-positive domains within the alar plate and one in the basal plate. In the alar plate, Sst-positive cells were observed in rostral and caudal ventral subdomains of the Otp-positive paraventricular complex. An additional basal Sst-expressing cell group was found within a longitudinal Otp-positive periretromamillary band that separates the retromamillary area from tuberal areas. Apart of subsequent growth of these initial populations, at E13.5 and E15.5 some Sst-positive derivatives migrate tangentially into neighboring regions. A subset of cells produced at the anterobasal nucleus disperses ventralward into the shell of the ventromedial hypothalamic nucleus and the arcuate nucleus. Cells from the rostroventral paraventricular subdomain reach the suboptic nucleus, whereas a caudal contingent migrates radially into lateral paraventricular, perifornical, and entopeduncular nuclei. Our data provide a topologic map of molecularly defined progenitor areas originating a specific neuron type during early hypothalamic development. Identification of four main separate sources helps to understand causally its complex adult organization.

Embryonic genoarchitecture of the pretectum in Xenopus laevis: a conserved pattern in tetrapods.

Networked gene activities control the evolutionarily conserved histogenetic organization of the central nervous system of vertebrates. Genoarchitectonic studies contribute to the analysis of each morphogenetic field by identifying distinct progenitor domains and corresponding derivatives whose pattern of gene expression shows a unique combinatory code. Previous studies in the pretectal region (caudal diencephalon) have defined three anteroposterior genoarchitectonic domains that are conserved in birds and mammals. Here, we have studied the embryonic pretectal genoarchitecture in the amphibian Xenopus laevis, in order to determine whether it is possible to define a comparable anteroposterior tripartition of the amphibian pretectal area. The expression patterns of 14 genes mapped from early embryonic stages to metamorphic climax allowed us to define the boundaries of the pretectum, the expected precommissural, juxtacommissural, and commissural anteroposterior domains, and some dorsoventral subdivisions. Taken together, our data provide evidence for a conserved pattern of pretectal domains and subdomains, shared by amniotes and amphibian anamniotes (tetrapods), understandable as part of a general Bauplan in vertebrates. J. Comp. Neurol. 519:1024–1050, 2011.

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. J. Comp. Neurol. 524:665–703, 2016.

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.