Thalamocortical axons control the cytoarchitecture of neocortical layers by area-specific supply of VGF.

Neuronal abundance and thickness of each cortical layer are specific to each area, but how this fundamental feature arises during development remains poorly understood. While some of area-specific features are controlled by intrinsic cues such as morphogens and transcription factors, the exact influence and mechanisms of action by cues extrinsic to the cortex, in particular the thalamic axons, have not been fully established. Here, we identify a thalamus-derived factor, VGF, which is indispensable for thalamocortical axons to maintain the proper amount of layer 4 neurons in the mouse sensory cortices. This process is prerequisite for further maturation of the primary somatosensory area, such as barrel field formation instructed by a neuronal activity-dependent mechanism. Our results provide an actual case in which highly site-specific axon projection confers further regional complexity upon the target field through locally secreting signaling molecules from axon terminals.

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.

Dysfunction of the proteoglycan Tsukushi causes hydrocephalus through altered neurogenesis in the subventricular zone in mice.

The lateral ventricle (LV) is flanked by the subventricular zone (SVZ), a neural stem cell (NSC) niche rich in extrinsic growth factors regulating NSC maintenance, proliferation, and neuronal differentiation. Dysregulation of the SVZ niche causes LV expansion, a condition known as hydrocephalus; however, the underlying pathological mechanisms are unclear. We show that deficiency of the proteoglycan Tsukushi (TSK) in ependymal cells at the LV surface and in the cerebrospinal fluid results in hydrocephalus with neurodevelopmental disorder-like symptoms in mice. These symptoms are accompanied by altered differentiation and survival of the NSC lineage, disrupted ependymal structure, and dysregulated Wnt signaling. Multiple TSK variants found in patients with hydrocephalus exhibit reduced physiological activity in mice in vivo and in vitro. Administration of wild-type TSK protein or Wnt antagonists, but not of hydrocephalus-related TSK variants, in the LV of TSK knockout mice prevented hydrocephalus and preserved SVZ neurogenesis. These observations suggest that TSK plays a crucial role as a niche molecule modulating the fate of SVZ NSCs and point to TSK as a candidate for the diagnosis and therapy of hydrocephalus.

The Pace of Neurogenesis Is Regulated by the Transient Retention of the Apical Endfeet of Differentiating Cells.

The development of the mammalian cerebral cortex involves a variety of temporally organized events such as successive waves of neuronal production and the transition of progenitor competence for each neuronal subtype generated. The number of neurons generated in a certain time period, that is, the rate of neuron production, varies across the regions of the brain and the specific developmental stage; however, the underlying mechanism of this process is poorly understood. We have recently found that nascent neurons communicate with undifferentiated progenitors and thereby regulate neurogenesis, through a transiently retained apical endfoot that signals via the Notch pathway. Here, we report that the retention time length of the neuronal apical endfoot correlates with the rate of neuronal production in the developing mouse cerebral cortex. We further demonstrate that a forced reduction or extension of the retention period through the disruption or stabilization of adherens junction, respectively, resulted in the acceleration or deceleration of neurogenesis, respectively. Our results suggest that the apical endfeet of differentiating cells serve as a pace controller for neurogenesis, thereby assuring the well-proportioned laminar organization of the neocortex.

Developing guinea pig brain as a model for cortical folding.

The cerebral cortex in mammals, the neocortex specifically, is highly diverse among species with respect to its size and morphology, likely reflecting the immense adaptiveness of this lineage. In particular, the pattern and number of convoluted ridges and fissures, called gyri and sulci, respectively, on the surface of the cortex are variable among species and even individuals. However, little is known about the mechanism of cortical folding, although there have been several hypotheses proposed. Recent studies on embryonic neurogenesis revealed the differences in cortical progenitors as a critical factor of the process of gyrification. Here, we investigated the gyrification processes using developing guinea pig brains that form a simple but fundamental pattern of gyri. In addition, we established an electroporation-mediated gene transfer method for guinea pig embryos. We introduce the guinea pig brain as a useful model system to understand the mechanisms and basic principle of cortical folding.

Significance of Stat3 Signaling in Epithelial Cell Differentiation of Fetal Mouse Lungs.

To study the significance of signal transducer and activator of transcription (Stat) 3 in lung epithelial development of fetal mice, we examined fetal mouse lungs, focusing on the expression of Clara cell secretory protein (CCSP), Forkhead box protein J1 (Foxj1), calcitonin gene-related peptide (CGRP), phosphorylated Stat3 (Tyr705), and hairy/enhancer of split (Hes) 1, and observed cultured fetal lungs upon treatment with IL-6, a Stat3 activator, or cucurbitacin I, a Stat3 inhibitor. Moreover, the interaction of Stat3 signaling and Hes1 was studied using Hes1 gene-deficient mice. Phosphorylated Stat3 was detected in fetal lungs and, immunohistochemically, phosphorylated Stat3 was found to be co-localized in developing Clara cells, but not in ciliated cells. In the organ culture studies, upon treatment with IL-6, quantitative RT-PCR revealed that CCSP mRNA increased with increasing Stat3 phosphorylation, while cucurbitacin I decreased Hes1, CCSP, Foxj1 and CGRP mRNAs with decreasing Stat3 phosphorylation. In the lungs of Hes1 gene-deficient mice, Stat3 phosphorylation was not markedly different from wild-type mice, the expression of CCSP and CGRP was enhanced, and the treatment of IL-6 or cucurbitacin I induced similar effects on mouse lung epithelial differentiation regardless of Hes1 expression status. Stat3 signaling acts in fetal mouse lung development, and seems to regulate Clara cell differentiation positively. Hes1 could regulate Clara cell differentiation in a manner independent from Stat3 signaling.

The Retinal Pigment Epithelium Is a Notch Signaling Niche in the Mouse Retina.

Notch signaling in neural progenitor cell is triggered by ligands expressed in adjacent cells. To identify the sources of active Notch ligands in the mouse retina, we negatively regulated Notch ligand activity in various neighbors of retinal progenitor cells (RPCs) by eliminating mindbomb E3 ubiquitin protein ligase 1 (Mib1). Mib1-deficient retinal cells failed to induce Notch activation in intra-lineage RPCs, which prematurely differentiated into neurons; however, Mib1 in post-mitotic retinal ganglion cells was not important. Interestingly, Mib1 in the retinal pigment epithelium (RPE) also contributed to Notch activation in adjacent RPCs by supporting the localization of active Notch ligands at RPE-RPC contacts. Combining this RPE-driven Notch signaling and intra-retinal Notch signaling, we propose a model in which one RPC daughter receives extra Notch signals from the RPE to become an RPC, whereas its sister cell receives only a subthreshold level of intra-retinal Notch signal and differentiates into a neuron.

Optimising aroma quality in curry sauce products using in vivo aroma release measurements.

Reducing fat content in foods to meet consumers' preferences and to address the obesity issue is a key task for food manufacturers but simply reducing fat content affects aroma quality adversely. Measuring the aroma release from regular and low-fat samples during eating to rebalance the aroma release has proved successful in model systems. Here, the reformulation of the spice content in a low fat curry sauce is described. Volatile markers of the key spices (coriander, cumin and turmeric) were selected and used to measure aroma release in regular (10 g oil/100 g) and low (2.5 or 5 g oil/100 g) fat sauces. Regression models were used to adjust the ingredient formulation so that the aroma release profiles in vivo were the same for the regular and reduced oil curry sauces and sensory analysis showed no significant difference between these samples. Despite the complexity of spice aromas, rebalancing was successful.

Reconstruction of a seminiferous tubule-like structure in a 3 dimensional culture system of re-aggregated mouse neonatal testicular cells within a collagen matrix.

Male gonad development is initiated by the aggregation of pre-Sertoli cells (SCs), which surround germ cells to form cords. Several attempts to reconstruct testes from dissociated testicular cells have been made; however, only very limited morphogenesis beyond seminiferous cord formation has been achieved. Therefore, we aimed to reconstruct seminiferous tubules using a 3-dimensional (D) re-aggregate culture of testicular cells, which were dissociated from 6-dpp neonatal mice, inside a collagen matrix. We performed a short-term culture (for 3 days) and a long-term culture (up to 3 wks). The addition of KnockOut Serum Replacement (KSR) promoted (1) the enlargement of SC re-aggregates; (2) the attachment of peritubular myoid (PTM) cells around the SC re-aggregates; (3) the sorting of germ cells inside, and Leydig cells outside, seminiferous cord-like structures; (4) the alignment of SC polarity inside a seminiferous cord-like structure relative to the basement membrane; (5) the differentiation of SCs (the expression of the androgen receptor); (6) the formation of a blood-testis-barrier between the SCs; (7) SC elongation and lumen formation; and (8) the proliferation of SCs and spermatogonia, as well as the differentiation of spermatogonia into primary spermatocytes. Eventually, KSR promoted the formation of seminiferous tubule-like structures, which accompanied germ cell differentiation. However, these morphogenetic events did not occur in the absence of KSR. This in vitro system presents an excellent model with which to identify the possible factors that induce these events and to analyze the mechanisms that underlie cellular interactions during testicular morphogenesis and germ cell differentiation.

Cadherin-based adhesions in the apical endfoot are required for active Notch signaling to control neurogenesis in vertebrates.

The development of the vertebrate brain requires an exquisite balance between proliferation and differentiation of neural progenitors. Notch signaling plays a pivotal role in regulating this balance, yet the interaction between signaling and receiving cells remains poorly understood. We have found that numerous nascent neurons and/or intermediate neurogenic progenitors expressing the ligand of Notch retain apical endfeet transiently at the ventricular lumen that form adherens junctions (AJs) with the endfeet of progenitors. Forced detachment of the apical endfeet of those differentiating cells by disrupting AJs resulted in precocious neurogenesis that was preceded by the downregulation of Notch signaling. Both Notch1 and its ligand Dll1 are distributed around AJs in the apical endfeet, and these proteins physically interact with ZO-1, a constituent of the AJ. Furthermore, live imaging of a fluorescently tagged Notch1 demonstrated its trafficking from the apical endfoot to the nucleus upon cleavage. Our results identified the apical endfoot as the central site of active Notch signaling to securely prohibit inappropriate differentiation of neural progenitors.

Hes repressors are essential regulators of hematopoietic stem cell development downstream of Notch signaling.

Previous studies have identified Notch as a key regulator of hematopoietic stem cell (HSC) development, but the underlying downstream mechanisms remain unknown. The Notch target Hes1 is widely expressed in the aortic endothelium and hematopoietic clusters, though Hes1-deficient mice show no overt hematopoietic abnormalities. We now demonstrate that Hes is required for the development of HSC in the mouse embryo, a function previously undetected as the result of functional compensation by de novo expression of Hes5 in the aorta/gonad/mesonephros (AGM) region of Hes1 mutants. Analysis of embryos deficient for Hes1 and Hes5 reveals an intact arterial program with overproduction of nonfunctional hematopoietic precursors and total absence of HSC activity. These alterations were associated with increased expression of the hematopoietic regulators Runx1, c-myb, and the previously identified Notch target Gata2. By analyzing the Gata2 locus, we have identified functional RBPJ-binding sites, which mutation results in loss of Gata2 reporter expression in transgenic embryos, and functional Hes-binding sites, which mutation leads to specific Gata2 up-regulation in the hematopoietic precursors. Together, our findings show that Notch activation in the AGM triggers Gata2 and Hes1 transcription, and next HES-1 protein represses Gata2, creating an incoherent feed-forward loop required to restrict Gata2 expression in the emerging HSCs.

Method for electroporation for the early chick embryo.

In vitro whole-embryo culture of chick embryos, originally invented by New, has been widely used for studies of early embryogenesis. Here, a method for electroporation using the New culture and its derivatives is described, to achieve misexpression of exogenous gene in a temporally and spatially controlled manner in gastrulating chick embryos. Detailed information for the devices and procedures, and some experimental examples are presented.

Zebrafish relatively relaxed mutants have a ryanodine receptor defect, show slow swimming and provide a model of multi-minicore disease.

Wild-type zebrafish embryos swim away in response to tactile stimulation. By contrast, relatively relaxed mutants swim slowly due to weak contractions of trunk muscles. Electrophysiological recordings from muscle showed that output from the CNS was normal in mutants, suggesting a defect in the muscle. Calcium imaging revealed that Ca(2+) transients were reduced in mutant fast muscle. Immunostaining demonstrated that ryanodine and dihydropyridine receptors, which are responsible for Ca(2+) release following membrane depolarization, were severely reduced at transverse-tubule/sarcoplasmic reticulum junctions in mutant fast muscle. Thus, slow swimming is caused by weak muscle contractions due to impaired excitation-contraction coupling. Indeed, most of the ryanodine receptor 1b (ryr1b) mRNA in mutants carried a nonsense mutation that was generated by aberrant splicing due to a DNA insertion in an intron of the ryr1b gene, leading to a hypomorphic condition in relatively relaxed mutants. RYR1 mutations in humans lead to a congenital myopathy, multi-minicore disease (MmD), which is defined by amorphous cores in muscle. Electron micrographs showed minicore structures in mutant fast muscles. Furthermore, following the introduction of antisense morpholino oligonucleotides that restored the normal splicing of ryr1b, swimming was recovered in mutants. These findings suggest that zebrafish relatively relaxed mutants may be useful for understanding the development and physiology of MmD.

Notch1 expression is spatiotemporally correlated with neurogenesis and negatively regulated by Notch1-independent Hes genes in the developing nervous system.

In the developing nervous system, neural stem cells initially proliferate by a symmetric cell division and then undergo an asymmetric cell division, which makes one neuron (or neuronal precursor) and one progenitor. It remains to be determined how the switch from the symmetric to asymmetric cell divisions is regulated. Here, we found that Notch1 is expressed in the regions where neurogenesis occurs actively but not in the regions where neurogenesis does not yet occur. Furthermore, in Hes-mutant mice where neurogenesis is accelerated, Notch1 expression is also accelerated. Thus, Notch1 expression is negatively regulated by Hes genes and is spatiotemporally correlated with neurogenesis, suggesting that the neural stem cells that undergo asymmetric cell divisions express Notch1, whereas those that undergo symmetric cell divisions do not. We propose that initiation of Notch1 expression is one of the key features for switch from the symmetric to asymmetric cell divisions of neural stem cells and that this process is negatively regulated by Notch1-independent Hes genes.

Persistent and high levels of Hes1 expression regulate boundary formation in the developing central nervous system.

The developing central nervous system is partitioned into compartments by boundary cells, which have different properties than compartment cells, such as forming neuron-free zones, proliferating more slowly and acting as organizing centers. We now report that in mice the bHLH factor Hes1 is persistently expressed at high levels by boundary cells but at variable levels by non-boundary cells. Expression levels of Hes1 display an inverse correlation to those of the proneural bHLH factor Mash1, suggesting that downregulation of Hes1 leads to upregulation of Mash1 in non-boundary regions, whereas persistent and high Hes1 expression constitutively represses Mash1 in boundary regions. In agreement with this notion, in the absence of Hes1 and its related genes Hes3 and Hes5, proneural bHLH genes are ectopically expressed in boundaries, resulting in ectopic neurogenesis and disruption of the organizing centers. Conversely, persistent Hes1 expression in neural progenitors prepared from compartment regions blocks neurogenesis and reduces cell proliferation rates. These results indicate that the mode of Hes1 expression is different between boundary and non-boundary cells, and that persistent and high levels of Hes1 expression constitutively repress proneural bHLH gene expression and reduce cell proliferation rates, thereby forming boundaries that act as the organizing centers.

Hes1 and Hes5 regulate the development of the cranial and spinal nerve systems.

The basic helix-loop-helix genes Hes1 and Hes5, known Notch effectors, regulate the maintenance of neural stem cells and the development of the central nervous system (CNS). In the absence of Hes1 and Hes5, the size, shape and cytoarchitecture of the CNS are severely disorganized, but the development of the peripheral nervous system remains to be analyzed. Here, we found that in Hes1;Hes5 double-mutant mice, the cranial and spinal nerve systems are also severely disorganized. In these mutant mice, axonal projections from the mesencephalic neurons to the trigeminal (V) ganglion become aberrant and the proximal parts of the glossopharyngeal (IX) and vagus (X) nerves are fused. The hypoglossal (XII) nerve is also formed poorly. Furthermore, the dorsal root ganglia are fused with the spinal cord, and the dorsal and ventral roots of the spinal nerves are lacking in many segments. These results indicate that Hes1 and Hes5 play an important role in the formation of the cranial and spinal nerve systems.

Roles of bHLH genes in neural stem cell differentiation.

Neural stem cells change their characteristics over time during development: they initially proliferate only and then give rise to neurons first and glial cells later. In the absence of the repressor-type basic helix-loop-helix (bHLH) genes Hes1, Hes3 and Hes5, neural stem cells do not proliferate sufficiently but prematurely differentiate into neurons and become depleted without making the later born cell types such as astrocytes and ependymal cells. Thus, Hes genes are essential for maintenance of neural stem cells to make cells not only in correct numbers but also in full diversity. Hes genes antagonize the activator-type bHLH genes, which include Mash1, Math and Neurogenin. The activator-type bHLH genes promote the neuronal fate determination and induce expression of Notch ligands such as Delta. These ligands activate Notch signaling and upregulate Hes1 and Hes5 expression in neighboring cells, thereby maintaining these cells undifferentiated. Thus, the activator-type and repressor-type bHLH genes regulate each other, allowing only subsets of cells to undergo differentiation while keeping others to stay neural stem cells. This regulation is essential for generation of complex brain structures of appropriate size, shape and cell arrangement.

Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation.

Radial glial cells derive from neuroepithelial cells, and both cell types are identified as neural stem cells. Neural stem cells are known to change their competency over time during development: they initially undergo self-renewal only and then give rise to neurons first and glial cells later. Maintenance of neural stem cells until late stages is thus believed to be essential for generation of cells in correct numbers and diverse types, but little is known about how the timing of cell differentiation is regulated and how its deregulation influences brain organogenesis. Here, we report that inactivation of Hes1 and Hes5, known Notch effectors, and additional inactivation of Hes3 extensively accelerate cell differentiation and cause a wide range of defects in brain formation. In Hes-deficient embryos, initially formed neuroepithelial cells are not properly maintained, and radial glial cells are prematurely differentiated into neurons and depleted without generation of late-born cells. Furthermore, loss of radial glia disrupts the inner and outer barriers of the neural tube, disorganizing the histogenesis. In addition, the forebrain lacks the optic vesicles and the ganglionic eminences. Thus, Hes genes are essential for generation of brain structures of appropriate size, shape and cell arrangement by controlling the timing of cell differentiation. Our data also indicate that embryonic neural stem cells change their characters over time in the following order: Hes-independent neuroepithelial cells, transitory Hes-dependent neuroepithelial cells and Hes-dependent radial glial cells.

Roles of the bHLH gene Hes1 in retinal morphogenesis.

During retinal development, common precursors give rise to various types of cells in a time course specific to each cell type. Previously, we demonstrated that the bHLH gene Hes1 inhibits neuronal differentiation whereas, in Hes1-null retina, precursors prematurely differentiate into neurons and form abnormal rosette-like structures. Thus, Hes1 is essential for maintenance of precursors and morphogenesis of the neural retina. However, the precise causal link between premature differentiation and abnormal structures remains to be determined. Here, we found that misexpression of Hes1 in the developing retina promotes formation of undifferentiated precursor-like cells, whereas in Hes1-null retina, precursors are not properly maintained and prematurely differentiate into ganglion cells. Strikingly, those prematurely differentiated ganglion cells erupt into the subretinal space through the regions where precursors and the outer limiting membrane are lost. These results indicate that Hes1 maintains precursors and the outer limiting membrane and thereby regulates retinal morphogenesis.