Potential protumor function of CD74 in clear cell renal cell carcinoma.

CD74 is a transmembrane protein that functions as a specialized chaperone of HLA class II and CD74 in tumor cells was suggested to be involved in cell proliferation in several kinds of malignant tumors. CD74 is also known to be expressed in macrophages, therefore, we investigated the CD74 expression in clear cell renal cell carcinoma (ccRCC). Immunohistochemistry of CD74 indicated that CD74 was expressed not only in cancer cells but also macrophages. CD74 was detected in surface membrane and cytoplasm of cancer cells in 92 of 94 cases (98%) and of 87 of 94 cases (93%). CD74 was expressed both in cancer cells and TAMs in 86 of 94 cases (91%). In vitro studies using cancer cell lines and monocyte-derived macrophages stimulated by anti-CD74 antibodies showed that CD74 signal accelerated cancer cell proliferation and macrophage activation. However, macrophage activation via CD74 signal did not influence macrophage-mediated cancer cell growth. RNA-sequence of macrophages stimulated by anti-CD74 antibodies indicated that CD74 signal was associated to inflammatory responses in macrophages. In conclusion, we examined the expression and functional significance of CD74 in ccRCC using tissue specimens and cell culture studies. The function of CD74 was suggested to be different in cancer cells and in macrophages, and further studies are necessary to clarify the functional significance of CD74 in ccRCC.

The Anterolateral Barrel Subfield Differs from the Posteromedial Barrel Subfield in the Morphology and Cell Density of Parvalbumin-Positive GABAergic Interneurons.

Layer 4 of the rodent somatosensory cortex has unitary structures called barrels that receive tactile information from individual vibrissae. Barrels in the anterolateral barrel subfield (ALBSF) are much smaller and have gained less attention than larger barrels in the posteromedial barrel subfield (PMBSF), though the former outnumber the latter. We compared the morphological features of barrels between the ALBSF and PMBSF in male mice using deformation-free tangential sections and confocal optical slice-based, precise reconstructions of barrels. The average volume of a single barrel in the ALBSF was 34.7% of that in the PMBSF, but the numerical density of parvalbumin (PV)-positive interneurons in the former was 1.49 times higher than that in the latter. Moreover, PV neuron density in septa was 2.08 times higher in the ALBSF than that in the PMBSF. The proportions of PV neuron number to both all neuron number and all GABAergic neuron number in the ALBSF were also higher than those in the PMBSF. Somata of PV neurons in barrels and septa in the ALBSF received 1.64 and 1.50 times more vesicular glutamate transporter Type 2-labeled boutons than those in the PMBSF, suggesting more potent feedforward inhibitory circuits in the ALBSF. The mode of connectivity through dendritic gap junctions among PV neurons also differed between the ALBSF and PMBSF. Clusters of smaller unitary structures containing a higher density of representative GABAergic interneurons with differential morphological features in the ALBSF suggest a division of functional roles in the two vibrissa-barrel systems, as has been demonstrated by behavioral studies.

Connectivity and molecular profiles of Foxp2- and Dbx1-lineage neurons in the accessory olfactory bulb and medial amygdala.

In terrestrial vertebrates, the olfactory system is divided into main (MOS) and accessory (AOS) components that process both volatile and nonvolatile cues to generate appropriate behavioral responses. While much is known regarding the molecular diversity of neurons that comprise the MOS, less is known about the AOS. Here, focusing on the vomeronasal organ (VNO), the accessory olfactory bulb (AOB), and the medial amygdala (MeA), we reveal that populations of neurons in the AOS can be molecularly subdivided based on their ongoing or prior expression of the transcription factors Foxp2 or Dbx1, which delineate separate populations of GABAergic output neurons in the MeA. We show that a majority of AOB neurons that project directly to the MeA are of the Foxp2 lineage. Using single-neuron patch-clamp electrophysiology, we further reveal that in addition to sex-specific differences across lineage, the frequency of excitatory input to MeA Dbx1- and Foxp2-lineage neurons differs between sexes. Together, this work uncovers a novel molecular diversity of AOS neurons, and lineage and sex differences in patterns of connectivity.

Analysis of the regional anatomy of the retro-oesophageal right subclavian artery and surrounding structures.

BACKGROUND: The retro-oesophageal right subclavian artery (RRSA) is a congenital anomalous branching of the arch of the aorta. Because its incidence is very low, it has not been fully understood how the RRSA develops during embryogenesis, and thus accumulation of observed findings in newly found cases is important to elucidate the aetiology of the RRSA. MATERIALS AND METHODS: We encountered a case of the RRSA during the course of gross anatomy dissection for medical students. RESULTS: The main findings in the present observations are that (a) the RRSA arose from the right side wall of the arch of the aorta as its last branch; (b) the detected RRSA was directed to the right and upward between the oesophagus and vertebral column; (c) the right vertebral artery branched from the RRSA and entered the sixth cervical foramen transversarium; (d) the suprema intercostal artery branched from the costocervical trunk on both sides and its distal branches were distributed to the first and second intercostal spaces; and (e) both sides of bronchial arteries originated from the thoracic aorta. CONCLUSIONS: The present study gives further information about the morphological details of the RRSA leading to better understanding of its developmental process.

Potential involvement of IL-32 in cell-to-cell communication between macrophages and hepatoblastoma.

PURPOSE: This study investigated the expression of interleukin 32 (IL-32) in hepatoblastoma, the most common primary pediatric liver tumor, and its possible roles in tumorigenesis. METHODS: IL-32 expression was investigated in two hepatoblastoma cell lines (Hep G2 and HuH 6) in the steady state and after co-culture with macrophages by RNA-seq analysis and RT-qPCR, and after stimulation with chemotherapy. Cultured macrophages were stimulated by IL-32 isoforms followed by RT-qPCR and western blot analysis. IL-32 immunohistochemical staining (IHC) was performed using specimens from 21 hepatoblastoma patients. Clustering analysis was also performed using scRNA-seq data downloaded from Gene Expression Omnibus. RESULTS: The IL-32 gene is expressed by hepatoblastoma cell lines; expression is upregulated by paracrine cell-cell communication with macrophages, also by carboplatin and etoposide. IL-32 causes protumor activation of macrophages with upregulation of PD-L1, IDO-1, IL-6, and IL-10. In the patient pool, IHC was positive only in 48% of cases. However, in the downloaded dataset, IL-32 gene expression was negative. CONCLUSION: IL-32 was detected in hepatoblastoma cell lines, but not in all hepatoblastoma patients. We hypothesized that stimulation such as chemotherapy might induce expression of IL-32, which might be a critical mediator of chemoresistance in hepatoblastoma through inducing protumor activation in macrophages.

SPP1 Derived from Macrophages Is Associated with a Worse Clinical Course and Chemo-Resistance in Lung Adenocarcinoma.

Osteopontin, also called secreted phosphoprotein 1 (SPP1), is a multifunctional secreted phosphorylated glycoprotein. SPP1 is also expressed in tumor cells, and many studies demonstrated that a high level of circulating SPP1 is correlated with a poor prognosis in various cancers. SPP1 is expressed not only by tumor cells but also by stromal cells, such as macrophages. However, there have been no studies distinguishing the SPP1 expression of cancer cells and tumor-associated macrophages (TAMs). Thus, in this study, we tried to accurately evaluate the SPP1 expression status on cancer cells and TAMs separately in patients with non-small cell lung cancer by using double immunohistochemistry. We demonstrated that high SPP1 expression on TAMs predicted a poor prognosis in lung adenocarcinoma patients. Additionally, we investigated the expression mechanisms related to SPP1 using human-monocyte-derived macrophages and revealed that the SPP1 expression level increased in macrophage differentiation mediated by granulocyte-macrophage colony-stimulating factor. Furthermore, SPP1 contributed to anti-cancer drug resistance in lung cancer cell lines. In conclusion, SPP1 production on TAMs predicted a poor prognosis in lung adenocarcinoma patients, and TAM-derived SPP1's involvement in the chemo-resistance of cancer cells was suggested.

The Tail of the Mouse Striatum Contains a Novel Large Type of GABAergic Neuron Incorporated in a Unique Disinhibitory Pathway That Relays Auditory Signals to Subcortical Nuclei.

The most caudal part of the striatum in rodents, the tail of the striatum (TS), has many features that distinguish it from the rostral striatum, such as its biased distributions of dopamine receptor subtypes, lack of striosomes and matrix compartmentalization, and involvement in sound-driven behaviors. However, information regarding the TS is still limited. We demonstrate in this article that the TS of the male mouse contains GABAergic neurons of a novel type that were detected immunohistochemically with the neurofilament marker SMI-32. Their somata were larger than cholinergic giant aspiny neurons, were located in a narrow space adjacent to the globus pallidus (GP), and extended long dendrites laterally toward the intermediate division (ID) of the trilaminar part of the TS, the region targeted by axons from the primary auditory cortex (A1). Although vesicular glutamate transporter 1-positive cortical axon terminals rarely contacted these TS large (TSL) neurons, glutamic acid decarboxylase-immunoreactive and enkephalin-immunoreactive boutons densely covered somata and dendrites of TSL neurons, forming symmetrical synapses. Analyses of GAD67-CrePR knock-in mice revealed that these axonal boutons originated from nearby medium spiny neurons (MSNs) in the ID. All MSNs examined in the ID in turn received inputs from the A1. Retrograde tracers injected into the rostral zona incerta and ventral medial nucleus of the thalamus labeled somata of TSL neurons. TSL neurons share many morphological features with GP neurons, but their strategically located dendrites receive inputs from closely located MSNs in the ID, suggesting faster responses than distant GP neurons to facilitate auditory-evoked, prompt disinhibition in their targets.SIGNIFICANCE STATEMENT This study describes a newly found population of neurons in the mouse striatum, the brain region responsible for appropriate behaviors. They are large GABAergic neurons located in the most caudal part of the striatum [tail of the striatum (TS)]. These TS large (TSL) neurons extended dendrites toward a particular region of the TS where axons from the primary auditory cortex (A1) terminated. These dendrites received direct synaptic inputs heavily from nearby GABAergic neurons of the striatum that in turn received inputs from the A1. TSL neurons sent axons to two subcortical regions outside basal ganglia, one of which is related to arousal. Specialized connectivity of TSL neurons suggests prompt disinhibitory actions on their targets to facilitate sound-evoked characteristic behaviors.

M-CSFR expression in the embryonal component of hepatoblastoma and cell-to-cell interaction between macrophages and hepatoblastoma.

Tumor-associated macrophages (TAMs) have protumor functions in various cancers. However, their significance in hepatoblastoma, the most common liver tumor in children, remains unclear. The aim of this study was to explore the potential roles of TAMs in hepatoblastoma. Immunohistochemical analysis revealed that the density of CD204-positive TAMs was significantly higher in the embryonal component than in other histological subtypes of hepatoblastoma. An in vitro co-culture study with Huh6 cells and human monocyte-derived macrophages (HMDMs) showed that macrophage-colony-stimulating factor receptor (M-CSFR) was strongly up-regulated in the Huh6 cells that were directly co-cultured with HMDMs. The expressions of M-CSFR ligands (interleukin-34 and M-CSF) were also increased by co-culture with HMDMs. The proliferation of HepG2 cells (another hepatoblastoma cell line expressing M-CSFR) was inhibited by an M-CSFR inhibitor. M-CSFR was found to be highly expressed in the embryonal component and in recurrent lesions. The number of CD204-positive macrophages was also higher in the M-CSFR-positive areas than in the M-CSFR-negative areas. Thus, M-CSFR expression appeared to be induced by cell-cell contact with macrophages in hepatoblastoma cells, and M-CSFR inhibitor is potentially effective against M-CSFR-positive hepatoblastoma, especially recurrent cases.

Immunohistochemical Analysis of Neurotransmitters in Neurosecretory Protein GL-Producing Neurons of the Mouse Hypothalamus.

We recently discovered a novel neuropeptide of 80 amino acid residues: neurosecretory protein GL (NPGL), in the hypothalamus of birds and rodents. NPGL is localized in the lateral posterior part of the arcuate nucleus (ArcLP), and it enhances feeding behavior and fat accumulation in mice. Various neurotransmitters, such as catecholamine, glutamate, and γ-aminobutyric acid (GABA), produced in the hypothalamus are also involved in energy metabolism. The colocalization of neurotransmitters and NPGL in neurons of the ArcLP leads to the elucidation of the regulatory mechanism of NPGL neurons. In this study, we performed double immunofluorescence staining to elucidate the relationship between NPGL and neurotransmitters in mice. The present study revealed that NPGL neurons did not co-express tyrosine hydroxylase as a marker of catecholaminergic neurons and vesicular glutamate transporter-2 as a marker of glutamatergic neurons. In contrast, NPGL neurons co-produced glutamate decarboxylase 67, a marker for GABAergic neurons. In addition, approximately 50% of NPGL neurons were identical to GABAergic neurons. These results suggest that some functions of NPGL neurons may be related to those of GABA. This study provides insights into the neural network of NPGL neurons that regulate energy homeostasis, including feeding behavior and fat accumulation.

Macrophage/microglia-derived IL-1ß induces glioblastoma growth via the STAT3/NF-κB pathway.

Glioblastoma is a glioma characterized by highly malignant features. Numerous studies conducted on the relationship between glioblastoma and the microenvironment have indicated the significance of tumor-associated macrophages/microglia (TAMs) in glioblastoma progression. Since interleukin (IL)-1ß secreted by TAMs has been suggested to promote glioblastoma growth, we attempted to elucidate the detailed mechanisms of IL-1ß in glioblastoma growth in this study. A phospho-receptor tyrosine kinase array and RNA-sequencing studies indicated that IL-1ß induced the activation of signal transducer and activator of transcription-3 and nuclear factor-kappa B signaling. Glioblastoma cells stimulated by IL-1ß induced the production of IL-6 and CXCL8, which synergistically promoted glioblastoma growth via signal transducer and activator of transcription-3 and nuclear factor-kappa B signaling. By immunohistochemistry, IL-1ß expression was seen on TAMs, especially in perinecrotic areas. These results suggest that IL-1ß might be a useful target molecule for anti-glioblastoma therapy.

α1-Acid Glycoprotein Enhances the Immunosuppressive and Protumor Functions of Tumor-Associated Macrophages.

Blood levels of acute-phase protein α1-acid glycoprotein (AGP, orosmucoid) increase in patients with cancer. Although AGP is produced from hepatocytes following stimulation by immune cell-derived cytokines under conditions of inflammation and tumorigenesis, the functions of AGP in tumorigenesis and tumor progression remain unknown. In the present study, we revealed that AGP contributes directly to tumor development by induction of programmed death ligand 1 (PD-L1) expression and IL6 production in macrophages. Stimulation of AGP induced PD-L1 expression in both human monocyte-derived macrophages through STAT1 activation, whereas AGP had no direct effect on PD-L1 expression in tumor cells. AGP also induced IL6 production from macrophages, which stimulated proliferation in tumor cells by IL6R-mediated activation of STAT3. Furthermore, administration of AGP to AGP KO mice phenocopied effects of tumor-associated macrophages (TAM) on tumor progression. AGP decreased IFNγ secretion from T cells and enhanced STAT3 activation in subcutaneous tumor tissues. In addition, AGP regulated PD-L1 expression and IL6 production in macrophages by binding with CD14, a coreceptor for Toll-like receptor 4 (TLR4), and inducing TLR4 signaling. These results provide the first evidence that AGP is directly involved in tumorigenesis by interacting with TAMs and that AGP might be a target molecule for anticancer therapy. SIGNIFICANCE: AGP-mediated suppression of antitumor immunity contributes to tumor progression by inducing PD-L1 expression and IL6 production in TAMs.

Characterization and Stage-Dependent Lineage Analysis of Intermediate Progenitors of Cortical GABAergic Interneurons.

Intermediate progenitors of both excitatory and inhibitory neurons, which can replenish neurons in the adult brain, were recently identified. However, the generation of intermediate progenitors of GABAergic inhibitory neurons (IPGNs) has not been studied in detail. Here, we characterized the spatiotemporal distribution of IPGNs in mouse cerebral cortex. IPGNs generated neurons during both embryonic and postnatal stages, but the embryonic IPGNs were more proliferative. Our lineage tracing analyses showed that the embryonically proliferating IPGNs tended to localize to the superficial layers rather than the deep cortical layers at 3 weeks after birth. We also found that embryonic IPGNs derived from the medial and caudal ganglionic eminence (CGE) but more than half of the embryonic IPGNs were derived from the CGE and broadly distributed in the cerebral cortex. Taken together, our data indicate that the broadly located IPGNs during embryonic and postnatal stages exhibit a different proliferative property and layer distribution.

Two-Phase Lineage Specification of Telencephalon Progenitors Generated From Mouse Embryonic Stem Cells.

Proper brain development requires precisely controlled phases of stem cell proliferation, lineage specification, differentiation, and migration. Lineage specification depends partly on concentration gradients of chemical cues called morphogens. However, the rostral brain (telencephalon) expands prominently during embryonic development, dynamically altering local morphogen concentrations, and telencephalic subregional properties develop with a time lag. Here, we investigated how progenitor specification occurs under these spatiotemporally changing conditions using a three-dimensional in vitro differentiation model. We verified the critical contributions of three signaling factors for the lineage specification of subregional tissues in the telencephalon, ventralizing sonic hedgehog (Shh) and dorsalizing bone morphogenetic proteins (BMPs) and WNT proteins (WNTs). We observed that a short-lasting signal is sufficient to induce subregional progenitors and that the timing of signal exposure for efficient induction is specific to each lineage. Furthermore, early and late progenitors possess different Shh signal response capacities. This study reveals a novel developmental mechanism for telencephalon patterning that relies on the interplay of dose- and time-dependent signaling, including a time lag for specification and a temporal shift in cellular Shh sensitivity. This delayed fate choice through two-phase specification allows tissues with marked size expansion, such as the telencephalon, to compensate for the changing dynamics of morphogen signals.

Sequential pattern of sublayer formation in the paleocortex and neocortex.

The piriform cortex (paleocortex) is the olfactory cortex or the primary cortex for the sense of smell. It receives the olfactory input from the mitral and tufted cells of the olfactory bulb and is involved in the processing of information pertaining to odors. The piriform cortex and the adjoining neocortex have different cytoarchitectures; while the former has a three-layered structure, the latter has a six-layered structure. The regulatory mechanisms underlying the building of the six-layered neocortex are well established; in contrast, less is known about of the regulatory mechanisms responsible for structure formation of the piriform cortex. The differences as well as similarities in the regulatory mechanisms between the neocortex and the piriform cortex remain unclear. Here, the expression of neocortical layer-specific genes in the piriform cortex was examined. Two sublayers were found to be distinguished in layer II of the piriform cortex using Ctip2/Bcl11b and Brn1/Pou3f3. The sequential expression pattern of Ctip2 and Brn1 in the piriform cortex was similar to that detected in the neocortex, although the laminar arrangement in the piriform cortex exhibited an outside-in arrangement, unlike that observed in the neocortex.

Formation of dorsal-ventral axis of the pallium derived from mouse embryonic stem cells.

The telencephalon is one of the most-elaborated tissues. A broad variety of cell types is produced by spatiotemporally regulated mechanisms and is involved, in different combinations, in subregional formation. The dorsal half of the telencephalon, the pallium or cerebral cortex, is subdivided along the dorsal-ventral (D-V) axis into the medial, dorsal, lateral, and ventral pallium (MP, DP, LP and VP, respectively). An in vitro differentiation system has been achieved using mouse embryonic stem cells, and major telencephalic neurons can be obtained in this way; however, in using the in vitro differentiation system, many telencephalic neuron subtypes remain undifferentiated, although some of them are related to neuronal diseases. In the current study, we found that inhibiting the TGFbeta signal was efficient for neural induction. A continuous arrangement of Emx1+/Pax6-, Emx1+/Pax6+, and Emx1-/Pax6+ cells was achieved in Foxg1+ neuroepithelia, corresponding approximately to cortical progenitors derived from MP, DP/LP, and VP, respectively. A small portion of Dbx1+ cells resided in the VP fraction. These findings suggested that the D-V axis of the pallium was recapitulated in the in vitro-derived pallium.

ß-N-methylamino-L-alanine (BMAA) suppresses cell cycle progression of non-neuronal cells.

ß-N-methylamino-L-alanine (BMAA), a natural non-proteinaceous amino acid, is a neurotoxin produced by a wide range of cyanobacteria living in various environments. BMAA is a candidate environmental risk factor for neurodegenerative diseases such as amyotrophic lateral sclerosis and Parkinson-dementia complex. Although BMAA is known to exhibit weak neuronal excitotoxicity via glutamate receptors, the underlying mechanism of toxicity has yet to be fully elucidated. To examine the glutamate receptor-independent toxicity of BMAA, we investigated the effects of BMAA in non-neuronal cell lines. BMAA potently suppressed the cell cycle progression of NIH3T3 cells at the G1/S checkpoint without inducing plasma membrane damage, apoptosis, or overproduction of reactive oxygen species, which were previously reported for neurons and neuroblastoma cells treated with BMAA. We found no evidence that activation of glutamate receptors was involved in the suppression of the G1/S transition by BMAA. Our results indicate that BMAA affects cellular functions, such as the division of non-neuronal cells, through glutamate receptor-independent mechanisms.

Embryonic transcription factor expression in mice predicts medial amygdala neuronal identity and sex-specific responses to innate behavioral cues.

The medial subnucleus of the amygdala (MeA) plays a central role in processing sensory cues required for innate behaviors. However, whether there is a link between developmental programs and the emergence of inborn behaviors remains unknown. Our previous studies revealed that the telencephalic preoptic area (POA) embryonic niche is a novel source of MeA destined progenitors. Here, we show that the POA is comprised of distinct progenitor pools complementarily marked by the transcription factors Dbx1 and Foxp2. As determined by molecular and electrophysiological criteria this embryonic parcellation predicts postnatal MeA inhibitory neuronal subtype identity. We further find that Dbx1-derived and Foxp2+ cells in the MeA are differentially activated in response to innate behavioral cues in a sex-specific manner. Thus, developmental transcription factor expression is predictive of MeA neuronal identity and sex-specific neuronal responses, providing a potential developmental logic for how innate behaviors could be processed by different MeA neuronal subtypes.

Molecular and behavioral profiling of Dbx1-derived neurons in the arcuate, lateral and ventromedial hypothalamic nuclei.

BACKGROUND: Neurons in the hypothalamus function to regulate the state of the animal during both learned and innate behaviors, and alterations in hypothalamic development may contribute to pathological conditions such as anxiety, depression or obesity. Despite many studies of hypothalamic development and function, the link between embryonic development and innate behaviors remains unexplored. Here, focusing on the embryonically expressed homeodomain-containing gene Developing Brain Homeobox 1 (Dbx1), we explored the relationship between embryonic lineage, post-natal neuronal identity and lineage-specific responses to innate cues. We found that Dbx1 is widely expressed across multiple developing hypothalamic subdomains. Using standard and inducible fate-mapping to trace the Dbx1-derived neurons, we identified their contribution to specific neuronal subtypes across hypothalamic nuclei and further mapped their activation patterns in response to a series of well-defined innate behaviors. RESULTS: Dbx1-derived neurons occupy multiple postnatal hypothalamic nuclei including the lateral hypothalamus (LH), arcuate nucleus (Arc) and the ventral medial hypothalamus (VMH). Within these nuclei, Dbx1 (+) progenitors generate a large proportion of the Pmch-, Nesfatin-, Cart-, Hcrt-, Agrp- and ERα-expressing neuronal populations, and to a lesser extent the Pomc-, TH- and Aromatase-expressing populations. Inducible fate-mapping reveals distinct temporal windows for development of the Dbx1-derived LH and Arc populations, with Agrp(+) and Cart(+) populations in the Arc arising early (E7.5-E9.5), while Pmch(+) and Hcrt(+) populations in the LH derived from progenitors expressing Dbx1 later (E9.5-E11.5). Moreover, as revealed by c-Fos labeling, Dbx1-derived cells in male and female LH, Arc and VMH are responsive during mating and aggression. In contrast, Dbx1-lineage cells in the Arc and LH have a broader behavioral tuning, which includes responding to fasting and predator odor cues. CONCLUSION: We define a novel fate map of the hypothalamus with respect to Dbx1 expression in hypothalamic progenitor zones. We demonstrate that in a temporally regulated manner, Dbx1-derived neurons contribute to molecularly distinct neuronal populations in the LH, Arc and VMH that have been implicated in a variety of hypothalamic-driven behaviors. Consistent with this, Dbx1-derived neurons in the LH, Arc and VMH are activated during stress and other innate behavioral responses, implicating their involvement in these diverse behaviors.

Specification of select hypothalamic circuits and innate behaviors by the embryonic patterning gene dbx1.

The hypothalamus integrates information required for the production of a variety of innate behaviors such as feeding, mating, aggression, and predator avoidance. Despite an extensive knowledge of hypothalamic function, how embryonic genetic programs specify circuits that regulate these behaviors remains unknown. Here, we find that in the hypothalamus the developmentally regulated homeodomain-containing transcription factor Dbx1 is required for the generation of specific subclasses of neurons within the lateral hypothalamic area/zona incerta (LH) and the arcuate (Arc) nucleus. Consistent with this specific developmental role, Dbx1 hypothalamic-specific conditional-knockout mice display attenuated responses to predator odor and feeding stressors but do not display deficits in other innate behaviors such as mating or conspecific aggression. Thus, activity of a single developmentally regulated gene, Dbx1, is a shared requirement for the specification of hypothalamic nuclei governing a subset of innate behaviors. VIDEO ABSTRACT.

Amygdala kindling induces nestin expression in the leptomeninges of the neocortex.

Nestin is an intermediate filament found in neurogenic progenitors and non-neuronal cells. Nestin-immunoreactivity (IR) in the brain often increases after brain damage. Here we show that amygdala kindling, which mimics the epileptic seizures, also induces nestin expression in the brain. Nestin-IR was greatly enhanced in the leptomeninges (pia and arachnoid maters) and neocortical parenchyma, but not much in the SVZ around the lateral ventricle, SGZ in the dentate gyrus, or the endothelial progenitor cells of blood vessels, fimbria, or choroid plexus after kindling. Electron microscopy revealed that nestin-IR in the leptomeninges was localized to granule cells, where it co-localized with GAD67-IR after electrical stimulation. The nestin-positive granule cells in the leptomeninges, especially around the emissary vein, were proliferative. However, nestin-IR in the neocortical parenchyma was expressed in NG2 glia and did not co-localize with GAD67-IR. Deletion of nestin-positive cells resulted in a high susceptibility to electrical stimulation. Consequently, almost all of the mice died or dropped out during kindling progression in 20 days, from naturally generated epileptic seizure or exhaustion. We speculate that the nestin-positive cells activated by amygdala kindling may involve in the protection of the brain from epilepsy.