Carmen Díaz

Fate-Mapping the Mammalian Hindbrain: Segmental Origins of Vestibular Projection Neurons Assessed Using Rhombomere-Specific Hoxa2 Enhancer Elements in the Mouse Embryo

As a step toward generating a fate map of identified neuron populations in the mammalian hindbrain, we assessed the contributions of individual rhombomeres to the vestibular nuclear complex, a major sensorimotor area that spans the entire rhombencephalon. Transgenic mice harboring either the lacZ or the enhanced green fluorescent protein reporter genes under the transcriptional control of rhombomere-specific Hoxa2 enhancer elements were used to visualize rhombomere-derived domains. We labeled functionally identifiable vestibular projection neuron groups retrogradely with conjugated dextran-amines at successive embryonic stages and obtained developmental fate maps through direct comparison with the rhombomere-derived domains in the same embryos. The fate maps show that each vestibular neuron group derives from a unique rostrocaudal domain that is relatively stable developmentally, suggesting that anteroposterior migration is not a major contributor to the rostrocaudal patterning of the vestibular system. Most of the groups are multisegmental in origin, and each rhombomere is fated to give rise to two or more vestibular projection neuron types, in a complex pattern that is not segmentally iterated. Comparison with studies in the chicken embryo shows that the rostrocaudal patterning of identified vestibular projection neuron groups is generally well conserved between avians and mammalians but that significant species-specific differences exist in the rostrocaudal limits of particular groups. This mammalian hindbrain fate map can be used as the basis for targeting genetic manipulation to specific subpopulations of vestibular projection neurons.

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.

The relationship between hodological and cytoarchitectonic organization in the vestibular complex of the 11‐day chicken embryo

To understand the relationship between structure and function in specific brain regions, it is necessary to ascertain which anatomical features are physiologically relevant. Physiological studies of brain function traditionally have been set in the context of anatomical features based on cytoarchitectonics and myeloarchitectonics, but the relationship between structure and function in this context can be complex. Alternative schemes of anatomical organization, such as that based on hodology (the mapping of projections) may provide greater insight. Here, we make a direct comparison of the hodological and the cytoarchitectonic organization of the vestibular complex in the mid‐term chicken embryo, using retrograde tracing and three‐dimensional reconstruction. In one set of experiments, vestibulospinal and vestibulo‐ocular neuron groups were selectively labeled with biotin dextran‐amines and aligned with the cytoarchitectonically defined vestibular nuclei in alternating sections that were then combined into intercalated three‐dimensional models. This allowed a semiquantitative analysis of the apportionment of individual hodological groups among cytoarchitectonic nuclei. In another set of experiments, vestibulospinal and vestibulo‐ocular neuron groups were labeled differentially with fluorescent dextran‐amines, three‐dimensionally reconstructed, and subjected to a quantitative analysis of spatial overlap. Our results provide the first three‐dimensional representation and quantitative analysis of the hodological compartmentalization of the vestibular complex (the “hodological mosaic”). They also show directly how each hodologically defined neuron group relates to the conventional vestibular nuclei, underscoring the fact that the units of the hodological mosaic do not bear a one‐to‐one correspondence to the cytoarchitectonic nuclear divisions. Some hodologically defined groups are localized to restricted portions of a nucleus, whereas others overlap multiple nuclei. Thus, hodology and cytoarchitectonic features appear to be separately regulated in the vestibular complex of the chicken embryo, possibly through different sets of positional specification mechanisms. The three‐dimensional representations we present here provide a foundation for integrating anatomical, physiological, developmental, and evolutionary studies of the vestibular system. J. Comp. Neurol. 457:87–105, 2003.

Comparative aspects of the hodological organization of the vestibular nuclear complex and related neuron populations.

In recent years, axonal tracing and fate mapping studies in avian embryos have revealed a mosaic pattern of hodologically defined neuron groups within the vestibular nuclear complex and related nuclei. Specific vestibular neuron clusters projecting to different targets (spinal, oculomotor, cerebellar) reside within largely segregated neuroepithelial domains. The close relationship between this pattern and the neuromeric organization of the hindbrain suggests a strong link between the expression of specific developmental patterning genes (such as Hox and Pax genes) and the specification of the individual neuron groups. Earlier tracing studies in mammals and more recent tracing studies in anamniote species performed by other workers indicate that many of the hodological features seen in avians are highly conserved in the vertebrate line. Here, we compare and contrast hodological patterns in birds and other vertebrate classes in an attempt to elucidate common denominators that may represent an evolutionary bauplan for vestibular connectivity.

Cytoarchitectonic subdivisions in the subtectal midbrain of the lizard Gallotia galloti.

Contemporary study of molecular patterning in the vertebrate midbrain is handicapped by the lack of a complete topological map of the diverse neuronal complexes differentiated in this domain. The relatively less deformed reptilian midbrain was chosen for resolving this fundamental issue in a way that can be extrapolated to other tetrapods. The organization of midbrain centers was mapped topologically in terms of longitudinal columns and cellular strata on transverse, Nissl-stained sections in the lizard Gallotia galloti. Four columns extend along the whole length of the midbrain. In dorsoventral order: 1) the dorsal band contains the optic tectum, surrounded by three ventricularly prominent subdivisions, named griseum tectale, intermediate area and torus semicircularis, in rostrocaudal order; 2) a subjacent region is named here the lateral band, which forms the ventral margin of the alar plate and also shows three rostrocaudal divisions; 3) the basal band forms the basal plate or tegmentum proper; it appears subdivided into medial and lateral parts: the medial part contains the oculomotor and accessory efferent neurons and the medial basal part of the reticular formation, which includes the red nucleus rostrally; the lateral part contains the lateral basal reticular formation, and includes the substantia nigra caudally; 4) the median band contains the ventral tegmental area, representing the mesencephalic floor plate. The alar regions (dorsal and lateral) show an overall cellular stratification into periventricular, central and superficial strata, with characteristic cytoarchitecture for each part. The lateral band contains two well developed superficial nuclei, one of which is commonly misidentified as an isthmic formation. The basal longitudinal subdivisions are simpler and basically consist of periventricular and central strata.

Ontogenesis of peptidergic neurons within the genoarchitectonic map of the mouse hypothalamus

During early development, the hypothalamic primordium undergoes anteroposterior and dorsoventral regionalization into diverse progenitor domains, each characterized by a differential gene expression code. The types of neurons produced selectively in each of these distinct progenitor domains are still poorly understood. Recent analysis of the ontogeny of peptidergic neuronal populations expressing Sst, Ghrh, Crh and Trh mRNAs in the mouse hypothalamus showed that these cell types originate from particular dorsoventral domains, characterized by specific combinations of gene markers. Such analysis implies that the differentiation of diverse peptidergic cell populations depends on the molecular environment where they are born. Moreover, a number of these peptidergic neurons were observed to migrate radially and/or tangentially, invading different adult locations, often intermingled with other cell types. This suggests that a developmental approach is absolutely necessary for the understanding of their adult distribution. In this essay, we examine comparatively the ontogenetic hypothalamic topography of twelve additional peptidergic populations documented in the Allen Developmental Mouse Brain Atlas, and discuss shared vs. variant aspects in their apparent origins, migrations and final distribution, in the context of the respective genoarchitectonic backgrounds. This analysis should aid ulterior attempts to explain causally the development of neuronal diversity in the hypothalamus, and contribute to our understanding of its topographic complexity in the adult.

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.

Radial and tangential migration of telencephalic somatostatin neurons originated from the mouse diagonal area.

The telencephalic subpallium is the source of various GABAergic interneuron cohorts that invade the pallium via tangential migration. Based on genoarchitectonic studies, the subpallium has been subdivided into four major domains: striatum, pallidum, diagonal area and preoptic area (Puelles et al. 2013; Allen Developing Mouse Brain Atlas), and a larger set of molecularly distinct progenitor areas (Flames et al. 2007). Fate mapping, genetic lineage-tracing studies, and other approaches have suggested that each subpallial subdivision produces specific sorts of inhibitory interneurons, distinguished by differential peptidic content, which are distributed tangentially to pallial and subpallial target territories (e.g., olfactory bulb, isocortex, hippocampus, pallial and subpallial amygdala, striatum, pallidum, septum). In this report, we map descriptively the early differentiation and apparent migratory dispersion of mouse subpallial somatostatin-expressing (Sst) cells from E10.5 onward, comparing their topography with the expression patterns of the genes Dlx5, Gbx2, Lhx7-8, Nkx2.1, Nkx5.1 (Hmx3), and Shh, which variously label parts of the subpallium. Whereas some experimental results suggest that Sst cells are pallidal, our data reveal that many, if not most, telencephalic Sst cells derive from de diagonal area (Dg). Sst-positive cells initially only present at the embryonic Dg selectively populate radially the medial part of the bed nucleus striae terminalis (from paraseptal to amygdaloid regions) and part of the central amygdala; they also invade tangentially the striatum, while eschewing the globus pallidum and the preoptic area, and integrate within most cortical and nuclear pallial areas between E10.5 and E16.5.

Development of glutamate receptors in auditory neurons from long-term organotypic cultures of the embryonic chick hindbrain.

We used long‐range organotypic cultures of auditory nuclei in the chick hindbrain to test the development of glutamate receptor activity in auditory neurons growing in a tissue environment that includes early deprivation of peripheral glutamatergic input, subsequent to removal of the otocyst. Cultures started at embryonic day (E)5, and lasted from 6 h to 15 days. Neuronal migration, clustering and axonal extension from the nucleus magnocellularis (NM) to the nucleus laminaris (NL) partially resembled events in vivo. However, the distinctive laminar organization of the NL was not observed. Glutamate receptor (GluR) activity was tested with optical recordings of intracellular Ca2+ in the NM. α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionic acid (AMPA)/kainate receptors had Ca2+ responses with a time course similar to that in control slices. Peak amplitude, however, was significantly lower. N‐methyl‐d‐aspartate (NMDA)‐mediated Ca2+ responses were higher in 2‐day cultures (E5 + 2d) than in E7 explant controls, returning later to control values. Metabotropic GluRs did not elicit Ca2+ responses at standard agonist doses. Blocking NMDA or AMPA/kainate receptors with specific antagonists for 10 days in culture did not limit neuronal survival. Blocking metabotropic GluRs resulted in complete neuronal loss. Thus, ionotropic GluRs are not required for NM neuronal survival. However, their activity during development is affected when neurons grow in an in vitro environment that includes prevention of arrival of peripheral glutamatergic input.

Segmental Analysis of the Vestibular Nerve and the Efferents of the Vestibular Complex: Efferents of the Vestibular Complex

Use of a segmental approach in the study of vestibular centers in the hindbrain improves morphological and functional understanding of this region controlled by Hox genes, among other molecular determinants. Here, we review accrued data about segmental organization of vestibular afferents and efferents. Inner ear‐originated vestibular fibers enter the hindbrain, together with auditory ones, through the alar plate of rhombomere 4, then branch into descending and ascending branches to reach appropriate vestibular nuclei along the vestibular column. Classical vestibular nuclei (superior, lateral, medial, and inferior) originate in eight successive rhombomeric segments, which suggests internal subdivisions correlated with distinct connections and functions. The vestibular projection neurons identified for various targets aggregate in discrete groups, which correlate topographically either with rhombomeric units, or with internal subdivisions within them. Each vestibular projection system (e.g., vestibulo‐spinal, vestibulo‐ocular, vestibulocerebellar) has a characteristic ipsilateral/contralateral organization. Comparing them as a connective mosaic in different species shows that various aspects of this segmental connective organization are conserved throughout evolution in vertebrates. Furthermore, certain genes that control the development of the rhombomeric units in the hindbrain may determine, among other aspects, the specific properties of the different neuronal subpopulations related to their axonal navigation and synaptogenesis. Anat Rec, 302:472–484, 2019.