Sylvia M. Bardet

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.

New and Old Thoughts on the Segmental Organization of the Forebrain in Lampreys

Ten years ago, we published the first detailed prosomeric map of the forebrain in lampreys. In the interim, the prosomeric model has been modified and simplified to better explain numerous data on the expression patterns of regulatory genes, as well as data from chemical, hodological and neuroembryological experiments, mostly in amniote vertebrates. In this report we first review the main historical concepts of lamprey forebrain organization, relating them to either columnar- or segmental-influenced models and explicit or implicit axial references. Next, our previous prosomeric model of the lamprey forebrain is updated, postulating some new hypotheses on the organization of the secondary prosencephalon.

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.

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.

Chapter 8 – Hypothalamus

Publisher Summary Neuroanatomical concepts of the hypothalamus, the forebrain territory that controls homeostasis and drives in vertebrates, are over 100 years old. Neuroanatomists of the 19th century generally considered the hypothalamus to consist of the tuberal or infundibular area, a median prominence at the brain ventral surface at the location of the pituitary gland, framed in front and at the sides by the optic chiasm and tracts. The neural plate fate maps of amniotes and anamniotes are strictly comparable from a topological point of view. The relative positions of the prospective brain areas can be extrapolated from one species to another. The prospective longitudinal or axial dimension of the brain is best represented by the neural/non-neural border of the neural plate, which will transform into the longitudinal roof plate of the closed neural tube. This developmental analysis suggests that all adult median forebrain derivatives found between the anterior commissure and the mamillary area are equally rostralmost loci in the brain. At the open neural plate, the rostral forebrain primordium contains the prospective hypothalamus as a large area centered upon the alar, basal, and floor portions of the terminal wall, its caudal part covering as well a rostral part of the lateral wall, whereas the prospective telencephalon forms a thin band at the dorsal periphery of the alar portion of these domains, including the corresponding roof area. A handful of genes, coding either for transcription factors or secreted protein morphogens, participate in early patterning and regional identity specification along the AP and DV dimensions of the whole forebrain, including the hypothalamus. The complex networked interplay of many molecular signals that control brain development represents a dynamic ontogenetic system that tends to reach different equilibrium states in different parts of the neuroepithelial wall.

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.

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

The avian lateral septal organ (LSO) is a telencephalic circumventricular specialization with liquor‐contacting neurons (Kuenzel and van Tienhoven [1982] J. Comp. Neurol. 206:293–313). We studied the topological position of the chicken LSO relative to molecular borders defined previously within the telencephalic subpallium (Puelles et al. [2000] J. Comp. Neurol. 424:409–438). Differential expression of Dlx5 and Nkx2.1 homeobox genes, or the Shh gene encoding a secreted morphogen, allows distinction of striatal, pallidal, and preoptic subpallial sectors. The chicken LSO complex was characterized chemoarchitectonically from embryonic to posthatching stages, by using immunohistochemistry for calbindin, tyrosine hydroxylase, NKX2.1, and BEN proteins and in situ hybridization for Nkx2.1, Nkx2.2, Nkx6.1, Shh, and Dlx5 mRNA. Medial and lateral parts of LSO appear, respectively, at the striatal part of the septum and adjacent bottom of the lateral ventricle (accumbens), in lateral continuity with another circumventricular organ that forms along a thin subregion of the entire striatum, abutting the molecular striatopallidal boundary; we called this the “striatopallidal organ” (SPO). The SPO displays associated distal periventricular cells, which are lacking in the LSO. Moreover, the SPO is continuous caudomedially with a thin, linear ependymal specialization found around the extended amygdala and preoptic areas. This differs from SPO and LSO in some molecular aspects. We tentatively identified this structure as being composed of an “extended amygdala organ” (EAO) and a “preoptohypothalamic organ” (PHO). The position of LSO, SPO, EAO, and PHO within a linear Dlx5‐expressing ventricular domain that surrounds the Nkx2.1‐expressing pallidopreoptic domain provides an unexpected insight into possible common and differential causal mechanisms underlying their formation. J. Comp. Neurol. 499:745–767, 2006.

Comparison of Pretectal Genoarchitectonic Pattern between Quail and Chicken Embryos

Regionalization of the central nervous system is controlled by local networks of transcription factors that establish and maintain the identities of neuroepithelial progenitor areas and their neuronal derivatives. The conserved cerebral Bauplan of vertebrates must result essentially from conserved patterns of developmentally expressed transcription factors. We have previously produced detailed molecular maps for the alar plate of prosomere 1 (the pretectal region) in chicken (Ferran et al., 2007, 2008, 2009). Here we compare the early molecular signature of the pretectum of two closely related avian species of the family Phasianidae, Coturnix japonica (Japanese quail) and Gallus gallus (chicken), aiming to test conservation of the described pattern at a microevolutionary level. We studied the developmental pretectal expression of Bhlhb4, Dbx1, Ebf1, Gata3, Gbx2, Lim1, Meis1, Meis2, Pax3, Pax6, Six3, Tal2, and Tcf7l2 (Tcf4) mRNA, using in situ hybridization, and PAX7 immunohistochemistry. The genoarchitectonic profile of individual pretectal domains and strata was produced, using comparable section planes. Remarkable conservation of the combinatorial genoarchitectonic code was observed, fundamented in a tripartite anteroposterior subdivision. However, we found that at corresponding developmental stages the pretectal region of G. gallus was approximately 30% larger than that of C. japonica, but seemed relatively less mature. Altogether, our results on a conserved genoarchitectonic pattern highlight the importance of early developmental gene networks that causally underlie the production of homologous derivatives in these two evolutionarily closely related species. The shared patterns probably apply to sauropsids in general, as well as to more distantly related vertebrate species.

Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells

High powered, nanosecond duration, pulsed electric fields (nsPEF) cause cell death by a mechanism that is not fully understood and have been proposed as a targeted cancer therapy. Numerous chemotherapeutics work by disrupting microtubules. As microtubules are affected by electrical fields, this study looks at the possibility of disrupting them electrically with nsPEF. Human glioblastoma cells (U87-MG) treated with 100, 10 ns, 44 kV/cm pulses at a frequency of 10 Hz showed a breakdown of their interphase microtubule network that was accompanied by a reduction in the number of growing microtubules. This effect is temporally linked to loss of mitochondrial membrane potential and independent of cellular swelling and calcium influx, two factors that disrupt microtubule growth dynamics. Super-resolution microscopy revealed microtubule buckling and breaking as a result of nsPEF application, suggesting that nsPEF may act directly on microtubules.

Electromagnetic Analysis of an Aperture Modified TEM Cell Including an ITO Layer for Real-Time Observation of Biological Cells Exposed to Microwaves

We propose to analyze the aperture and ITO layer presence of a modified transverse electromagnetic (TEM) cell. This TEM cell can be used to study the potential effects of microwave electromagnetic fields on biological cells. This modified delivery device allows real-time observation of biological cells during exposure. Microscopic observation is achieved through an aperture in the lower wall of the TEM cell that is sealed with a 700-nm film of the transparent conducting material Indium tin oxide (ITO). To determine the device efficiency, numerical and experimental electromagnetic dosimetry was conducted. For assessing the effect of the aperture on the specific absorption rate (SAR) in the exposed sample, a plastic Petri dish containing cell culture medium, full-wave 3-D electromagnetic simulations and temperature measurements were performed. For 1-W input power, the SAR values obtained at 1.8 GHz in the sample exposed in the TEM cell with the sealed or non-sealed aperture of 20-mm diameter were 1.1 W/kg and 23.6 W/kg, respectively. An excellent homogeneity of the SAR distribution was achieved when the aperture was sealed with the ITO layer. The performance of the delivery system was confirmed by microwave exposure and simultaneous observation of living cells.