Effects of blastocyst elongation and implantation chamber formation on the alignment of the embryonic axis and uterine axis in mice.

Embryo implantation involves a series of events that bring the embryo and maternal tissues into contact to support post-implantation development in mammals. During implantation, alignment of the embryonic-abembryonic (E-Ab) axis of the blastocyst with the mesometrial-antimesometrial (M-AM) axis of the uterus precedes post-implantation embryonic development and placentation. In the present study, we observed the morphological changes in blastocysts and the endometrial luminal epithelium (LE) that occur during the alignment of the embryonic and the uterine axes. We found that at the time that the blastocysts attached to the LE at the mural trophectoderm, the embryonic axis was not aligned with the uterine axis. Alignment of the embryonic E-Ab axis with the uterine M-AM axis occurred after E4.0, and the embryo was significantly elongated during the process. The depth of the implantation chamber (IC) correlated with the degree of alignment, suggesting that elongated embryos are oriented along the M-AM axis during IC formation. Transplantation of the Concanavalin A (Con A)-coated beads induced IC formation, and the alignment of two Con A-coated beads present in the same IC in the M-AM direction suggested that elongated materials can align along the M-AM axis. These data suggest that an elongated shape of the embryo and IC formation coordinate the alignment of the embryonic and uterine axes.

Simultaneous visualization of extrinsic and intrinsic axon collaterals in Golgi-like detail for mouse corticothalamic and corticocortical cells: a double viral infection method.

Here we present a novel tracing technique to stain projection neurons in Golgi-like detail by double viral infection. We used retrograde lentiviral vectors and adeno-associated viral vectors (AAV) to drive "TET-ON/TET-OFF system" in neurons connecting two regions. Using this method, we successfully labeled the corticothalamic (CT) cells of the mouse somatosensory barrel field (S1BF) and motor cortex (M1) in their entirety. We also labeled contra- and ipsilaterally-projecting corticocortical (CC) cells of M1 by targeting contralateral M1 or ipsilateral S1 for retrograde infection. The strength of this method is that we can observe the morphology of specific projection neuron subtypes en masse. We found that the group of CT cells extends their dendrites and intrinsic axons extensively below but not within the thalamorecipient layer in both S1BF and M1, suggesting that the primary target of this cell type is not layer 4. We also found that both ipsi- and contralateral targeting CC cells in M1 commonly exhibit widespread collateral extensions to contralateral M1 (layers 1-6), bilateral S1 and S2 (layers 1, 5 and 6), perirhinal cortex (layers 1, 2/3, 5, and 6), striatum and claustrum. These findings not only strengthened the previous findings of single cell tracings but also extended them by enabling cross-area comparison of CT cells or comparison of CC cells of two different labeling.

Characterization of claustral neurons by comparative gene expression profiling and dye-injection analyses.

The identity of the claustrum as a part of cerebral cortex, and in particular of the adjacent insular cortex, has been investigated by connectivity features and patterns of gene expression. In the present paper, we mapped the cortical and claustral expression of several cortical genes in rodent and macaque monkey brains (nurr1, latexin, cux2, and netrinG2) to further assess shared features between cortex and claustrum. In mice, these genes were densely expressed in the claustrum, but very sparsely in the cortex and not present in the striatum. To test whether the cortical vs. claustral cell types can be distinguished by co-expression of these genes, we performed a panel of double ISH in mouse and macaque brain. NetrinG2 and nurr1 genes were co-expressed across entire cortex and claustrum, but cux2 and nurr1 were co-expressed only in the insular cortex and claustrum. Latexin was expressed, in the macaque, only in the claustrum. The nurr1 (+) claustral neurons expressed VGluT1, a marker for cortical glutamatergic cells and send cortical projections. Taken together, our data suggest a partial commonality between claustral neurons and a subtype of cortical neurons in the monkey brain. Moreover, in the embryonic (E110) macaque brain, many nurr1 (+) neurons were scattered in the white matter between the claustrum and the insular cortex, possibly representing their migratory history. In a second set of experiments, we injected Lucifer Yellow intracellularly in mouse and rat slices to investigate whether dendrites of insular and claustral neurons can cross the border of the two brain regions. Dendrites of claustral neurons did not invade the overlying insular territory. In summary, gene expression profile of the claustrum is similar to that of the neocortex, in both rodent and macaque brains, but with modifications in density of expression and cellular co-localization of specific genes.

Area-specific substratification of deep layer neurons in the rat cortex.

Gene markers are useful tools to identify cell types for fine mapping of neuronal circuits. Here we report area-specific sublamina structure of the rat cerebral cortex using cholecystokinin (cck) and purkinje cell protein4 (pcp4) mRNAs as the markers for excitatory neuron subtypes in layers 5 and 6. We found a segregated expression, especially pronounced in layer 6, where corticothalamic and corticocortical projecting neurons reside. To examine the relationship between gene expression and projection target, we injected retrograde tracers into several thalamic subnuclei, ventral posterior (VP), posterior (PO), mediodorsal (MD), medial and lateral geniculate nuclei (MGN and LGN); as well as into two cortical areas (M1 and V1). This combination of tracer-in situ hybridization (ISH) experiments revealed that corticocortical neurons predominantly express cck and corticothalamic neurons predominantly express pcp4 mRNAs in all areas tested. In general, cck(+) and pcp4(+) cells occupied the upper and lower compartment of layer 6a, respectively. However, the sublaminar distribution and the relative abundance of cck(+) and pcp4(+) cells were quite distinctive across areas. For example, layer 6 of the prelimbic cortex was almost devoid of cck(+) neurons, and was occupied instead by corticothalamic pcp4(+) neurons. In the lateral areas, such as S2, there was an additional layer of cck(+) cells positioned below the pcp4(+) compartment. The claustrum, which has a tight relationship with the cortex, mostly consisted of cck(+)/pcp4(-) cells. In summary, the combination of gene markers and retrograde tracers revealed a distinct sublaminar organization, with conspicuous cross-area variation in the arrangement and relative density of corticothalamic connections.

Fluorescent in situ hybridization technique for cell type identification and characterization in the central nervous system.

Central nervous system consists of a myriad of cell types. In particular, many subtypes of neuronal cells, which are interconnected with each other, form the basis of functional circuits. With the advent of genomic era, there have been systematic efforts to map gene expression profiles by in situ hybridization (ISH) and enhancer-trapping strategy. To make full use of such information, it is important to correlate "cell types" to gene expression. Toward this end, we have developed highly sensitive method of fluorescent dual-probe ISH, which is essential to distinguish two cell types expressing distinct marker genes. Importantly, we were able to combine ISH with retrograde tracing and antibody staining including BrdU staining that enables birthdating. These techniques should prove useful in identifying and characterizing the cell types of the neural tissues. In this article, we describe the methodology of these techniques, taking examples from our analyses of the mammalian cerebral cortex.

Analysis of area-specific expression patterns of RORbeta, ER81 and Nurr1 mRNAs in rat neocortex by double in situ hybridization and cortical box method.

BACKGROUND: The mammalian neocortex is subdivided into many areas, each of which exhibits distinctive lamina architecture. To investigate such area differences in detail, we chose three genes for comparative analyses, namely, RORbeta, ER81 and Nurr1, mRNAs of which have been reported to be mainly expressed in layers 4, 5 and 6, respectively. To analyze their qualitative and quantitative coexpression profiles in the rat neocortex, we used double in situ hybridization (ISH) histochemistry and cortical box method which we previously developed to integrate the data of different staining and individuals in a standard three-dimensional space. PRINCIPAL FINDINGS: Our new approach resulted in three main observations. First, the three genes showed unique area distribution patterns that are mostly complementary to one another. The patterns revealed by cortical box method matched well with the cytoarchitectonic areas defined by Nissl staining. Second, at single cell level, RORbeta and ER81 mRNAs were coexpressed in a subpopulation of layer 5 neurons, whereas Nurr1 and ER81 mRNAs were not colocalized. Third, principal component analysis showed that the order of hierarchical processing in the cortex correlates well with the expression profiles of these three genes. Based on this analysis, the dysgranular zone (DZ) in the somatosensory area was considered to exhibit a profile of a higher order area, which is consistent with previous proposal. CONCLUSIONS/SIGNIFICANCE: The tight relationship between the expression of the three layer specific genes and functional areas were revealed, demonstrating the usefulness of cortical box method in the study on the cerebral cortex. In particular, it allowed us to perform statistical evaluation and pattern matching, which would become important in interpreting the ever-increasing data of gene expression in the cortex.

Comparative analysis of layer-specific genes in Mammalian neocortex.

We examined the expression patterns of 4 layer-specific genes in monkey and mouse cortices by fluorescence double in situ hybridization. Based on their coexpression profiles, we were able to distinguish several subpopulations of deep layer neurons. One group was characterized by the expression of ER81 and the lack of Nurr1 mRNAs and mainly localized to layer 5. In monkeys, this neuronal group was further subdivided by 5-HT2C receptor mRNA expression. The 5-HT2C(+)/ER81(+) neurons were located in layer 5B in most cortical areas, but they intruded layer 6 in the primary visual area (V1). Another group of neurons, in monkey layer 6, was characterized by Nurr1 mRNA expression and was further subdivided as Nurr1(+)/connective tissue growth factor (CTGF)(-) and Nurr1(+)/CTGF(+) neurons in layers 6A and 6B, respectively. The Nurr1(+)/CTGF(+) neurons coexpressed ER81 mRNA in monkeys but not in mice. On the basis of tracer injections in 3 monkeys, we found that the Nurr1(+) neurons in layer 6A send some corticocortical, but not corticopulvinar, projections. Although the Nurr1(+)/CTGF(-) neurons were restricted to lateral regions in the mouse cortex, they were present throughout the monkey cortex. Thus, an architectonic heterogeneity across areas and species was revealed for the neuronal subpopulations with distinct gene expression profiles.

Binding and complementary expression patterns of semaphorin 3E and plexin D1 in the mature neocortices of mice and monkeys.

Although axon guidance molecules play critical roles in neural circuit formation during development, their roles in the adult circuit are not well understood. In this study we examined the expression patterns of Semaphorin 3E (Sema3E), a member of the semaphorin family, in the mature neocortices of monkeys and mice by in situ hybridization (ISH). We found that Sema3E mRNA is highly specific to layer VI throughout the macaque monkey neocortex. We further examined the ratio of Sema3E+ cells among the layer VI excitatory neurons in areas M1, S1, TE, and V1 by fluorescence double ISH, using the vesicular glutamate transporter 1 (VGluT1) gene as a specific marker for excitatory neurons. Among these areas, 34-63% of the VGluT1+ neurons expressed Sema3E mRNA. In the mouse cortex, two significant differences were observed in the pattern of Sema3E mRNA distribution. 1) Sema3E mRNA was expressed in layer Vb, in addition to layer VI in mice. 2) A subset of GABAergic interneurons expressed Sema3E mRNA in mice. By an in vitro binding experiment, we provide evidence that Plexin D1 is the specific receptor for Sema3E. Plexin D1 mRNA was preferentially expressed in layers II-V in both monkey and mouse cortices. The detailed lamina analysis by double ISH, however, revealed that Plexin D1 mRNA is expressed in layers II-Va, but not in layer Vb in the mouse cortex. Thus, the Plexin D1 and Sema3E mRNAs exhibit conserved complementary lamina patterns in mice and monkeys, despite the species differences in the pattern of each gene.