Genoarchitectural Definition of the Adult Mouse Mesocortical Ring: A Contribution to Cortical Ring Theory.

Data mining was performed at the databases of the Allen Institute for Brain Science (RRID:SCR_017001) searching for genes expressed selectively throughout the adult mouse mesocortex (transitional cortex ring predicted within the concentric ring theory of mammalian cortical structure, in contrast with central isocortex [ICx] and peripheral allocortex). We aimed to explore a shared molecular profile selective of all or most mesocortex areas. This approach checks and corroborates the precision of other previous definitory criteria, such as poor myelination and high kainate receptor level. Another aim was to examine which cortical areas properly belong to mesocortex. A total of 34 positive adult selective marker genes of mesocortex were identified, jointly with 12 negative selective markers, making a total of 46 markers. All of them identify the same set of cortical areas surrounding the molecularly different ICx as well as excluding adjacent allocortex. Four representative mesocortex markers-Crym, Lypd1, Cdh13, and Smoc2-are amply illustrated, jointly with complementary material including myelin basic protein, to check myelination, and Rorb, to check layer 4 presence. The retrosplenial (ReSp) area, long held to be mesocortical, does not share any of the 46 markers of mesocortex and instead expresses Nr4a2 and Tshz2, selective parahippocampal allocortex markers. Moreover, it is not hypomyelinic and lacks a Rorb-positive layer 4, aspects generally present in mesocortex. Exclusion of the ReSp area from the mesocortex ring reveals the latter to be closed at this locus instead by two adjacent areas previously thought to be associative visual ICx (reidentified here molecularly as postsplenial and parasplenial mesocortex areas). The concepts of ICx, mesocortex, and parahippocampal allocortex are thus subtly modified by substantial molecular evidence.

Tangential Intrahypothalamic Migration of the Mouse Ventral Premamillary Nucleus and Fgf8 Signaling.

The tuberal hypothalamic ventral premamillary nucleus (VPM) described in mammals links olfactory and metabolic cues with mating behavior and is involved in the onset of puberty. We offer here descriptive and experimental evidence on a migratory phase in the development of this structure in mice at E12.5-E13.5. Its cells originate at the retromamillary area (RM) and then migrate tangentially rostralward, eschewing the mamillary body, and crossing the molecularly distinct perimamillary band, until they reach a definitive relatively superficial ventral tuberal location. Corroborating recent transcriptomic studies reporting a variety of adult glutamatergic cell types in the VPM, and different projections in the adult, we found that part of this population heterogeneity emerges already early in development, during tangential migration, in the form of differential gene expression properties of at least 2-3 mixed populations possibly derived from subtly different parts of the RM. These partly distribute differentially in the core and shell parts of the final VPM. Since there is a neighboring acroterminal source of Fgf8, and Fgfr2 is expressed at the early RM, we evaluated a possible influence of Fgf8 signal on VPM development using hypomorphic Fgf8neo/null embryos. These results suggested a trophic role of Fgf8 on RM and all cells migrating tangentially out of this area (VPM and the subthalamic nucleus), leading in hypomorphs to reduced cellularity after E15.5 without alteration of the migrations proper.

Sim1-expressing cells illuminate the origin and course of migration of the nucleus of the lateral olfactory tract in the mouse amygdala.

We focus this report on the nucleus of the lateral olfactory tract (NLOT), a superficial amygdalar nucleus receiving olfactory input. Mixed with its Tbr1-expressing layer 2 pyramidal cell population (NLOT2), there are Sim1-expressing cells whose embryonic origin and mode of arrival remain unclear. We examined this population with Sim1-ISH and a Sim1-tauLacZ mouse line. An alar hypothalamic origin is apparent at the paraventricular area, which expresses Sim1 precociously. This progenitor area shows at E10.5 a Sim1-expressing dorsal prolongation that crosses the telencephalic stalk and follows the terminal sulcus, reaching the caudomedial end of the pallial amygdala. We conceive this Sim1-expressing hypothalamo-amygdalar corridor (HyA) as an evaginated part of the hypothalamic paraventricular area, which participates in the production of Sim1-expressing cells. From E13.5 onwards, Sim1-expressing cells migrated via the HyA penetrate the posterior pallial amygdalar radial unit and associate therein to the incipient Tbr1-expressing migration stream which swings medially past the amygdalar anterior basolateral nucleus (E15.5), crosses the pallio-subpallial boundary (E16.5), and forms the NLOT2 within the anterior amygdala by E17.5. We conclude that the Tbr1-expressing NLOT2 cells arise strictly within the posterior pallial amygdalar unit, involving a variety of required gene functions we discuss. Our results are consistent with the experimental data on NLOT2 origin reported by Remedios et al. (Nat Neurosci 10:1141-1150, 2007), but we disagree on their implication in this process of the dorsal pallium, observed to be distant from the amygdala.

Development of the mouse anterior amygdalar radial unit marked by Lhx9-expression.

The amygdala in mammals plays a key role in emotional processing and learning, being subdivided in pallial and subpallial derivatives. Recently, the cortical ring model and the pallial amygdalar radial model (Puelles et al. 2019; Garcia-Calero et al. 2020) described the pallial amygdala as an histogenetic field external to the allocortical ring, and subdivided it in five major radial domains called lateral, basal, anterior, posterior and retroendopiriform units. The anterior radial unit, whose cells typically express the Lhx9 gene (see molecular profile in Garcia-Calero et al. 2020), is located next to the pallial/subpallial boundary. This radial domain shows massive radial translocation and accumulation of its derivatives into its intermediate and superficial strata, with only a glial palisade representing its final periventricular domain. To better understand the development of this singular radial domain, not described previously, we followed the expression of Lhx9 during mouse amygdalar development in the context of the postulated radial subdivisions of the pallial amygdala and other telencephalic developmental features.

Histogenetic Radial Models as Aids to Understanding Complex Brain Structures: The Amygdalar Radial Model as a Recent Example.

The radial dimension expands during central nervous system development after the proliferative neuroepithelium is molecularly patterned. The process is associated with neurogenesis, radial glia scaffolding, and migration of immature neurons into the developing mantle stratum. Radial histogenetic units, defined as a delimited neural polyclone whose cells share the same molecular profile, are molded during these processes, and usually become roughly stratified into periventricular, intermediate, and superficial (subpial) strata wherein neuronal cell types may differ and be distributed in various patterns. Cell-cell adhesion or repulsion phenomena together with interaction with local intercellular matrix cues regulate the acquisition of nuclear, reticular, or layer histogenetic forms in such strata. Finally, the progressive addition of inputs and outputs soon follows the purely neurogenetic and radial migratory phase. Frequently there is heterochrony in the radial development of adjacent histogenetic units, apart of peculiarities in differentiation due to non-shared aspects of the respective molecular profiles. Tangential migrations may add complexity to radial unit cytoarchitecture and function. The study of the contributions of such genetically controlled radial histogenetic units to the emerging complex neural structure is a key instrument to understand central nervous system morphology and function. One recent example in this scenario is the recently proposed radial model of the mouse pallial amygdala. This is theoretically valid generally in mammals (Garcia-Calero et al., 2020), and subdivides the nuclear complex of the pallial amygdala into five main radial units. The approach applies a novel ad hoc amygdalar section plane, given the observed obliquity of the amygdalar radial glial framework. The general relevance of radial unit studies for clarifying structural analysis of all complex brain regions such as the pallial amygdala is discussed, with additional examples.

A radial histogenetic model of the mouse pallial amygdala.

Conventional anatomic models of the rodent (mammalian) amygdala are based on section planes oblique to its intrinsic radial glial organization. As a result, we still lack a model of amygdalar histogenesis in terms of radial units (progenitor domains and related radial migration and layering patterns). A radial model of the mouse pallial amygdala is first offered here, based on three logical steps: (1) analysis of amygdalar radial structure in variously discriminative genoarchitectonic material, using an optimal ad hoc section plane; (2) testing preliminary models with experiments labelling at the brain surface single packets of radial glia processes, to be followed into the ventricular surface across intervening predicted elements; (3) selection of 81 differential amygdalar gene markers and checking planar and radial aspects of their distribution across the model elements. This approach shows that subtle changes to the conventional schema of the amygdala allow a radial histogenetic model to be recognized, which is consistent with molecularly coded differential identities of its units and strata. It is expected that this model will help both causal studies of amygdalar developmental patterning and comparative evolutionary studies. It also may have potential impact on hodological and functional studies.

Concentric ring topology of mammalian cortical sectors and relevance for patterning studies.

Models aiming to explain causally the evolutionary or ontogenetic emergence of the pallial isocortex and its regional/areal heterogeneity in mammals use simple or complex assumptions about the pallial structure present in basal mammals and nonmammals. The question arises: how complex is the pattern that needs to be accounted for in causal models? This topic is also paramount for comparative purposes, since some topological relationships may be explained as being ancestral, rather than newly emerged. The mouse pallium is apt to be reexamined in this context, due to the breadth of available molecular markers and correlative experimental patterning results. We center the present essay on a recapitulative glance at the classic theory of concentric mammalian allo-, meso-, and neocortex domains. In its simplest terms, this theory postulates a central neocortical island (6 layers) separated by a surrounding mesocortical ring (4-5 layers) from a peripheral allocortical ring (3 layers). These territories show additional partition into regional or areal subdivisions. There are also borderline amygdalar, claustral, and septal areas of the pallium, nuclear in structure. There has been little effort so far to contemplate the full concentric ring model in current "cortex patterning" models. In this essay, we recapitulate the ring idea in mammals (mouse) and consider a potential causal patterning scenario using topologic models. Finally, we briefly explore how far this theory may apply to pallium models proposed recently for sauropsids.

FoxP1 Protein Shows Differential Layer Expression in the Parahippocampal Domain among Bird Species.

Different bird orders show diversity in neural capabilities supported by variations in brain morphology. The parahippocampal domain in the medial pallium, together with the hippocampus proper, plays an important role in memory skills. In the present work, we analyze the expression pattern of the FoxP1 protein in the parahippocampal area of four different bird species: the nonvocal learner birds quail and chicken (Galliformes) and two vocal learner birds, i.e. the zebra finch (Passeriformes) and the budgerigar (Psittaciformes), at different developmental and adult stages. We also analyze the expression of the calbindin protein in quails and zebra finches. We observed differences in the FoxP1 parahippocampal layer among bird species. In quails, chickens, and budgerigar, FoxP1 cells were located in the outer layers of the lateral and caudolateral parahippocampal sectors. In contrast, FoxP1 immunoreactive cells appeared in the inner layer of the same sectors in the zebra finch parahippocampal domain. These differences suggest two possibilities: either the FoxP1-positive cells described in quails, chickens, and budgerigars are a different population than the one described in the zebra finch, or there are changes in the pattern of radial migration in the parahippocampal area among birds. In the present study, we show that FoxP1 expression is more similar between quails, chickens, and budgerigars than between budgerigars and zebra finches in the parahippocampal area. This result contrasts with previous data in other telencephalic structures, like the calbindin-positive projection neurons described in the striatum of budgerigars and zebra finches but not in quails and chickens. All of these data point to diversity in the evolution of different morphological characters and, therefore, a mosaic model for telencephalic evolution in birds.

FoxP2 protein levels regulate cell morphology changes and migration patterns in the vertebrate developing telencephalon.

In the mammalian telencephalon, part of the progenitor cells transition from multipolar to bipolar morphology as they invade the mantle zone. This associates with changing patterns of radial migration. However, the molecules implicated in these morphology transitions are not well known. In the present work, we analyzed the function of FoxP2 protein in this process during telencephalic development in vertebrates. We analyzed the expression of FoxP2 protein and its relation with cell morphology and migratory patterns in mouse and chicken developing striatum. We observed FoxP2 protein expressed in a gradient from the subventricular zone to the mantle layer in mice embryos. In the FoxP2 low domain cells showed multipolar migration. In the striatal mantle layer where FoxP2 protein expression is higher, cells showed locomoting migration and bipolar morphology. In contrast, FoxP2 showed a high and homogenous expression pattern in chicken striatum, thus bipolar morphology predominated. Elevation of FoxP2 in the striatal subventricular zone by in utero electroporation promoted bipolar morphology and impaired multipolar radial migration. In mouse cerebral cortex we obtained similar results. FoxP2 promotes transition from multipolar to bipolar morphology by means of gradiental expression in mouse striatum and cortex. Together these results indicate a role of FoxP2 differential expression in cell morphology control of the vertebrate telencephalon.

Calbindin expression in developing striatum of zebra finches and its relation to the formation of area X.

A sexually dimorphic network of brain regions controls learning and production of song in zebra finches. How this specialized song system evolved is unknown. To start addressing this question, we focused on developmental differences between the sexes, using the expression of the calcium-binding protein calbindin (CB) during embryonic to adult stages to map out the early development of Area X, a male-specific striatal structure. We related this pattern to the expression of three transcription factors, Pax6 and Islet1 to delineate the striatal radial domains, and Nkx2.1 as a marker for cells of pallidal origin. An incipient Area X-CB+ domain became discernable at embryonic day 13 in the Islet1-ventral striatal field. This region contained many Nkx2.1-expressing cells with a morphology characteristic of migrating cells. Eight days after hatching (PHD) CB staining clearly delineated Area X. Another CB+ structure formed around PHD5 at the subpallial/pallial boundary. We call it the CB+striatal capsule (CB-StC) and discuss its relation with the previously described striatal capsule in vertebrates. The CB cell population in both Area X and CB-StC, but not in the surrounding striatum, colocalized with the striatal medium spiny neurons (MSNs) marker, D1-receptor associated signaling protein dopamine-and-cAMP-regulated phosphoprotein of 32 kDa, DARPP32. In females, CB-positive cells were also present in the rostral striatum but did not coalesce into an Area X-like structure. We discuss possible reasons for CB expression in MSNs in songbirds and mammals, but not described in chicken striatum.

Differences in number and distribution of striatal calbindin medium spiny neurons between a vocal-learner (Melopsittacus undulatus) and a non-vocal learner bird (Colinus virginianus).

Striatal projecting neurons, known as medium spiny neurons (MSNs), segregate into two compartments called matrix and striosome in the mammalian striatum. The matrix domain is characterized by the presence of calbindin immunopositive (CB+) MSNs, not observed in the striosome subdivision. The existence of a similar CB+ MSN population has recently been described in two striatal structures in male zebra finch (a vocal learner bird): the striatal capsule and the Area X, a nucleus implicated in song learning. Female zebra finches show a similar pattern of CB+ MSNs than males in the developing striatum but loose these cells in juveniles and adult stages. In the present work we analyzed the existence and allocation of CB+ MSNs in the striatal domain of the vocal learner bird budgerigar (representative of psittaciformes order) and the non-vocal learner bird quail (representative of galliformes order). We studied the co-localization of CB protein with FoxP1, a transcription factor expressed in vertebrate striatal MSNs. We observed CB+ MSNs in the medial striatal domain of adult male and female budgerigars, although this cell type was missing in the potentially homologous nucleus for Area X in budgerigar. In quail, we observed CB+ cells in the striatal domain at developmental and adult stages but they did not co-localize with the MSN marker FoxP1. We also described the existence of the CB+ striatal capsule in budgerigar and quail and compared these results with the CB+ striatal capsule observed in juvenile zebra finches. Together, these results point out important differences in CB+ MSN distribution between two representative species of vocal learner and non-vocal learner avian orders (respectively the budgerigar and the quail), but also between close vocal learner bird families.

A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

Enc1 expression in the chick telencephalon at intermediate and late stages of development.

In this work we studied the regional expression pattern of the Enc1 gene in the chick embryo telencephalon at intermediate and late stages of development, bearing on architectonic groupings and boundaries of current interest. In general, the Enc1 signal shows a markedly heterogeneous areal pattern of expression throughout the telencephalon; this corroborates data on new pallial and subpallial structures defined recently in the stereotaxic chick brain atlas of Puelles et al. (2007. The chick brain in stereotaxic coodinates. San Diego, CA: Academic Press). For example: a periventricular/central domain is Enc1-negative in the ventral pallium or nidopallium; core and shell nuclei appear in the mesopallium; the redefined caudodorsolateral area shows a characteristic pattern; the limits of the densocellular hyperpallium in the dorsal pallium are illuminated; and the postulated entorhinal cortex area is distinct at the posterior telencephalic pole. Interestingly, Enc1 transcripts are distinctly present in the piriform cortex at the surface of the ventral pallium throughout its longitudinal extent, as well as in the most rostral part of the lateral pallium, implying a layout of this cortex more similar to the situation in mammals than was assumed previously. Separate corticoid superficial strata are labeled by the Enc1 probe in the lateral and dorsal pallial regions. In the subpallium, the expression of Enc1 agrees with the new radial subdivisions defined by Puelles et al. (2007).

Early mammillary pouch specification in the course of prechordal ventralization of the forebrain tegmentum.

The mammillary body, a ventral specialization of the caudal hypothalamus, lies close to the transition between epichordal and prechordal parts of the forebrain (Puelles and Rubenstein, 2003). This report examines its presumed causal connection with either prechordal or notochordal mesodermal induction, as well as the timing of its specification, in the context of early ventral forebrain patterning. It was recently found that the ephrin receptor gene EphA7 is selectively expressed in the mammillary pouch from early stages of development (HH14: García-Calero et al., 2006). We used mammillary EphA7 expression as well as ventral hypothalamic expression of the gene markers Nkx2.1 and Shh to analyze experimental effects on mammillary specification and morphogenesis after axial mesoderm ablation at stages HH4+ to HH6. Progressively delayed ablation of the prechordal plate revealed its sequential implication in molecular specification of the entire ventral forebrain, including the mammillary and tuberal regions of the hypothalamus. We observed differential contact requirements for induction by the prechordal plate of all the forebrain regions expressing Shh and Nkx2.1, including distant subpallial ones. In contrast, ablation of the anterior notochordal tip at these stages did not elicit significant patterning changes, particularly no effects on mammillary EphA7 expression or mammillary pouch development.

Pallial expression of Enc1 RNA in postnatal mouse telencephalon.

We analysed the pallial expression pattern of Enc1 (a member of the kelch family of genes) in postnatal mice (P1-P10). At early developmental stages this gene plays a role in the histogenesis of cortical structures [M.C. Hernández, P.J. Andrés-Barquin, S. Martínez, A. Bulfone, J.L.R. Rubenstein, M.A. Israel, Enc1: novel mammalian kelch-related gene specifically expressed in the nervous system encodes an actino-binding protein, J. Neurosci. 17 (1997) 3038-3051]. A restricted expression of Enc1 was found in the mouse pallium, notably within claustroamygdaloid derivatives of the lateral pallium and in some cortical layers in the lateral, dorsal and medial pallium sectors, with distinct regional differences. The strongest cortical expression was found in isocortical layer II and in the piriform cortex, anterior olfactory area and olfactory bulb mitral cells. The lowest signal occurred in the retrosplenial cortex. The subgranular layers V/VI were also positive, particularly layer V, with clearcut areal differences. The hippocampal CA3/CA4 areas and the dentate gyrus were strongly positive. The dorsolateral (core) portion of the claustrum and dorsal endopiriform nucleus were moderately positive, as were the amygdaloid lateral and basolateral nuclei.

Correlation of a chicken stage 4 neural plate fate map with early gene expression patterns.

A number of gene markers are currently claimed to allow positive or negative visualization of the early chick neural plate at stages 3d/4, when its fate becomes determined. Some markers labeled by various authors as either "neural" or "non-neural" indeed show ectodermal expression patterns roughly correlative with widespread yet vague ideas on the shape and size of the early neural plate, based on previous fate maps. However, for technical reasons, it is not clear how precisely these expression patterns correlate with any experimentally determined fate boundaries. An eventual mismatch between fate and marker interpretation might bear importantly on ideas about gene functions and causal hypotheses in issues such as the establishment of the neural/non-neural border or the earliest mechanisms of neural regionalization. In this review, we correlated a set of epiblastic and mesendodermal gene expression patterns with the novel neuroectoderm proportions suggested by our recent fate map of the chick neural plate at stages HH 3d/4 [P. Fernández-Garre, L. Rodriguez-Gallardo, V. Gallego-Diaz, I.S. Alvarez, L. Puelles, Fate map of the chicken neural plate at stage 4, Development 129 (2002) 2807-2822.]. This analysis suggests the existence of various nested subregions of the epiblast with boundaries codefined by given sets of gene patterns. No gene expression studied reproduces exactly or even approximately the entire neural plate shape, leading to a combinatorial hypothesis on its specification. This kind of analysis (fate and molecular maps), jointly with competence maps, provides the basis for understanding gene functions and the mechanisms of neural induction, specification and regionalization. Several gene patterns observed are consistent with precocious incipient regionalization of the neural plate along the dorsoventral and anteroposterior axes.

The avian griseum tectale: cytoarchitecture, NOS expression and neurogenesis.

The griseum tectale (GT) is a retinorecipient layered formation located in the rostral alar midbrain, just behind the constriction that separates it from the diencephalon (pretectum). Tritiated-thymidine autoradiographic data on neuronal birthdates show that the GT cell population starts to be generated at HH21 and most neurons are born by stage HH25, accumulating within a primordial periventricular layer, which shows strong nitric oxide synthase immunoreactivity at later stages of development. There is a barely noticeable rostrocaudal neurogenetic and differentiation gradient across the GT, which seems to continue into that of the neighboring optic tectum. The GT layering develops gradually by radial migration of its postmitotic neurons between stages HH26 and HH35. The structure of the mature GT can be divided into periventricular, central, and superficial layers, similarly to the adjacent optic tectum, but it shows different layering aspects, particularly in the retinorecipient superficial layer.