Targeted disruption of the mouse protein phosphatase ppm1l gene leads to structural abnormalities in the brain.

PPM1L, a member of the metal-dependent protein phosphatase (PPM) family, is involved in regulating the stress-activated protein kinase pathway and ceramide trafficking. However, the physiological function of PPM1L in the brain is unclear. In this study, we generated and analyzed ppm1l-deficient mice in order to investigate PPM1L functions in the brain. Our results indicate that ppm1l is highly expressed in the central nervous system during mouse development and that ppm1lΔ/Δ mice display impaired motor performance and morphological abnormalities in the forebrain. Electron microscopic and immunohistochemical analyses suggest that these abnormalities are due to impaired axonal tract formation. Our novel findings suggest an important role for PPM1L in brain development.

Inactivation of Sonic Hedgehog Signaling and Polydactyly in Limbs of Hereditary Multiple Malformation, a Novel Type of Talpid Mutant.

Hereditary Multiple Malformation (HMM) is a naturally occurring, autosomal recessive, homozygous lethal mutation found in Japanese quail. Homozygote embryos (hmm-/-) show polydactyly similar to talpid2 and talpid3 mutants. Here we characterize the molecular profile of the hmm-/- limb bud and identify the cellular mechanisms that cause its polydactyly. The hmm-/- limb bud shows a severe lack of sonic hedgehog (SHH) signaling, and the autopod has 4 to 11 unidentifiable digits with syn-, poly-, and brachydactyly. The Zone of Polarizing Activity (ZPA) of the hmm-/- limb bud does not show polarizing activity regardless of the presence of SHH protein, indicating that either the secretion pathway of SHH is defective or the SHH protein is dysfunctional. Furthermore, mesenchymal cells in the hmm-/- limb bud do not respond to ZPA transplanted from the normal limb bud, suggesting that signal transduction downstream of SHH is also defective. Since primary cilia are present in the hmm-/- limb bud, the causal gene must be different from talpid2 and talpid3. In the hmm-/- limb bud, a high amount of GLI3A protein is expressed and GLI3 protein is localized to the nucleus. Our results suggest that the regulatory mechanism of GLI3 is disorganized in the hmm-/- limb bud.

Role of En2 in the tectal laminar formation of chick embryos.

The chick optic tectum consists of 16 laminae. Here, we report contribution of En2 to laminar formation in chick optic tecta. En2 is specifically expressed in laminae g-j of stratum griseum et fibrosum superficiale (SGFS). Misexpression of En2 resulted in disappearance of En2-expressing cells from the superficial layers (laminae a-f of SGFS), where endogenous En2 is not expressed. Misexpression of En2 before postmitotic cells had left the ventricular layer indicated that En2-misexpressing cells stopped at the laminae of endogenous En2 expression and that they did not migrate into the superficial layers. Induction of En2 misexpression using a tetracycline-inducible system after the postmitotic cells had reached superficial layers also resulted in disappearance of En2-expressing cells from the superficial layers. Time-lapse analysis showed that En2-misexpressing cells migrated back from the superficial layers towards the middle layers, where En2 is strongly expressed endogenously. Our results suggest a potential role of En2 in regulating cell migration and positioning in the tectal laminar formation.

Time-lapse imaging system with shell-less culture chamber.

We have developed a system for imaging whole chick embryos from embryonic day 1.5 (E1.5) to E4.5. Our system consists of a custom-made culture chamber, the top and bottom of which were heated and the inside was humidified. The system also has a fixed stage uplight fluorescent microscope, and long-working distance objective lenses were adopted. The albumen removed-yolk with the embryo in the dish was put in the chamber. It is of importance that we adopted long working distance lenses because the working distance of conventional objective lenses is too short for observation of the embryo in a humidified chamber. The objective lens we adopted has sufficient resolution to detect fluorescent protein expression at the single-cell level. Transparent glass heater set on the top of the chamber helps to reduce dew condensation; the bottom heater keeps the temperature inside the chamber, and the water bath surrounding the egg maintains humidity. This system was used to detect fluorescent protein expressing cells in embryos. We could successfully trace those cells for 17 h in vivo. In conclusion, this system is useful for time-lapse analysis of fluorescent protein expression and distribution for a longer period of time.

Mechanism underlying the block of human Cav3.2 T-type Ca2+ channels by benidipine, a dihydropyridine Ca2+ channel blocker.

AIMS: Benidipine, a dihydropyridine Ca(2+) channel blocker, has been reported to block T-type Ca(2+) channels; however, the mechanism underlying this effect was unclear. In this study, we characterized the mechanism responsible for this blocking activity. Furthermore, the blocking activity was compared between two enantiomers of benidipine, (S, S)- and (R, R)-benidipine. MAIN METHODS: Human Ca(v)3.2 (hCa(v)3.2) T-type Ca(2+) channels stably expressed in the human embryonic kidney cell line, HEK-293, were studied in whole-cell patch-clamp recordings and Ca(2+) mobilization assay. KEY FINDINGS: In whole-cell patch-clamp recordings, benidipine blocked hCa(v)3.2 T-type Ca(2+) currents elicited by depolarization to a comparable extent as efonidipine. The block was dependent on stimulation frequency and holding potential, but not test potential. Benidipine significantly shifted the steady-state inactivation curve to the hyperpolarizing direction, but had no effect on the activation curve. Benidipine prolonged the recovery from inactivation of hCa(v)3.2 T-type Ca(2+) channels without any effect on the kinetics of activation, inactivation, or deactivation. In the Ca(2+) mobilization assay, benidipine was more potent than efonidipine in blocking Ca(2+) influx through hCa(v)3.2 T-type Ca(2+) channels. (S, S)-Benidipine was more potent than (R, R)-benidipine in blocking hCa(v)3.2 T-type Ca(2+) currents, but there was no difference in blocking the Ca(2+) influx. SIGNIFICANCE: We have characterized the blocking activity of benidipine against hCa(v)3.2 Ca(2+) channels and revealed the difference between the two enantiomers of benidipine. The blocking action of benidipine could be mediated by stabilizing hCa(v)3.2 Ca(2+) channels in an inactivated state.

Epithelial-mesenchymal transition as a possible mechanism of semicircular canal morphogenesis in chick inner ear.

Semicircular canals are sensory organs for balance, consisting of fluid-filled tubules that are arranged perpendicularly to each other in inner ear. The precise mechanism of the morphogenesis of this unique organ is still under investigation. Semicircular canals arise from the flattened pouch of epithelium. The centers of two apposing wall of the pouch approach each other and form a fusion plate. The clearing of the fusion plate makes a hole and leaves the remaining tissue as semicircular canals. Three mechanisms have been proposed for this clearing: programmed cell death, epithelial-mesenchymal transition, and retraction of the cells in the fusion plate to surrounding semicircular canals. Previous studies have revealed programmed cell death in the fusion plate, although other two hypotheses were not disproved. Here we examined the contribution of epithelial-mesenchymal transition and epithelial retraction to the morphogenesis of semicircular canals. We analyzed immunohistochemically the structural change in the epithelium of the developing fusion plate using molecular markers, basal lamina component laminin, cytoskeletal F-actin, and cellular junctional marker beta-catenin. Our observation revealed that fusion plate epithelium lost its apico-basal polarity and intermingled with facing fusion plate cells, associated with the disruption of basal lamina. Moreover, there were several cells with mesenchymal appearance adjacent to the torn basal lamina. We also found the merging of apposing basal laminae at the border between forming canal and breaking fusion plate. These observations suggest that the epithelial-mesenchymal transition, rather than the epithelial retraction, may be responsible for clearing fusion plate cells.

The noncompetitive antagonism of histamine H1 receptors expressed in Chinese hamster ovary cells by olopatadine hydrochloride: its potency and molecular mechanism.

Calcium responses to various concentrations of histamine were monitored in Chinese hamster ovary cells stably expressing the human histamine H(1) receptor. The effects of various histamine H(1) receptor antagonists on the dose-response curve for histamine were evaluated. Olopatadine hydrochloride (olopatadine) inhibited the histamine-induced maximum response (pD(2)': 7.5) but had insignificant effects on histamine EC(50) values. This noncompetitive property exhibited by olopatadine, which was also observed in human umbilical vein endothelial cells, was the most striking among the antihistamines tested in this study. The geometrical isomer of olopatadine (E-isomer), which had a similar binding affinity to the histamine H(1) receptor as olopatadine, showed a mixed antagonistic profile (competitive and noncompetitive). These results indicate that the geometry around the double bond in the dimethylaminopropylidene group is critical for the potent noncompetitive property of olopatadine. Furthermore, binding mode analyses suggest that the protonated amine group in the dimethylaminopropylidene moiety of olopatadine forms an ionic bond with Glu 181 that is present in the second extracellular loop of the histamine H(1) receptor, whereas the amine group of the E-isomer does not. The second extracellular loop in aminergic G-protein-coupled receptors contributes to ligand binding and therefore the noncompetitive property of olopatadine may be explained by the interaction with Glu 181.

Conformational significance of EH21A1-A4, phenolic derivatives of geldanamycin, for Hsp90 inhibitory activity.

Hsp90 is an attractive chemotherapeutic target because it is essential to maturation of multiple oncogenes. We describe the conformational significance of EH21A1-A4, phenolic derivatives of geldanamycin isolated from Streptomyces sp. Their native free structures are similar to the active form of geldanamycin bound to Hsp90 protein. Their conformational character is a probable reason for their high-affinity binding. Lack of toxic benzoquinone in EH21A1-A4 also adds to their potential as lead compounds for anti-tumor drugs.

Role of Bone morphogenetic protein 4 in zebrafish semicircular canal development.

Bone morphogenetic proteins (BMPs) are known to play roles in inner ear development of higher vertebrates. In zebrafish, there are several reports showing that members of the BMP family are expressed in the otic vesicle. We have isolated a novel zebrafish mutant gallery, which affects the development of the semicircular canal. Gallery merely forms the lateral and the immature anterior protrusion, and does not form posterior and ventral protrusions. We found that the expression of bmp2b and bmp4, both expressed in the normal optic vesicle at the protrusion stage, are extremely upregulated in the otic vesicle of gallery. To elucidate the role of BMPs in the development of the inner ear of zebrafish, we have applied excess BMP to the wild-type otic vesicle. The formation of protrusions was severely affected, and in some cases, they were completely lost in BMP4-treated embryos. Furthermore, the protrusions in gallery treated with Noggin were partially rescued. These data indicate that BMP4 plays an important role in the development of protrusions to form semicircular canals.

Dan is required for normal morphogenesis and patterning in the developing chick inner ear.

During vertebrate inner ear development, compartmentalization of the auditory and vestibular apparatuses along two axes depends on the patterning of transcription factors expressed in a region-specific manner. Although most of the patterning is regulated by extrinsic signals, it is not known how Nkx5.1 and Msx1 are patterned. We focus on Dan, the founding member of the Cerberus/Dan gene family that encodes BMP antagonists, and describe its function in morphogenesis and patterning. First, we confirmed that Dan is expressed in the dorso-medial region of the otic vesicle that corresponds to the presumptive endolymphatic duct and sac (ed/es). Second, we used siRNA knockdown to demonstrate that depletion of Dan induced both a severe reduction in the size of the ed/es and moderate deformities of the semicircular canals and cochlear duct. Depletion of Dan also caused suppression of Nkx5.1 in the dorso-lateral region, suppression of Msx1 in the dorso-medial region, and ectopic induction of Nkx5.1 and Msx1 in the ventro-medial region. Most of these phenotypes also appeared following misexpression of the constitutively active form of BMP receptor type Ib. Thus, Dan is required for the normal morphogenesis of the inner ear and, by inhibiting BMP signaling, for the patterning of the transcription factors Nkx5.1 and Msx1.

Cloning and developmental expression of a chick G-protein-coupled receptor SCGPR1.

To identify spinal motor neuron subtype-specific transcripts, we employed a single cell subtractive screen of mRNAs in chick embryos. We cloned a differentially expressed gene that termed spinal cord G-protein-coupled receptor 1 (SCGPR1) from its expression pattern that change dynamically in the developing spinal cord. The vertebrate orthologue of SCGPR1 is termed Gpr37 (GPCR/CNS1, ET(B)R-LP-1, Pael-R), however the specific ligand of this receptor has not been identified. Recent studies indicate that Pael-R can associate with parkin, a ubiquitin ligase which accumulates in Lewy bodies in dopaminergic neurons and is associated with Parkinson's disease. Although SCGPR1 (Gpr37) expression has been examined in adult tissues, the embryonic expression has not reported. Here, we have defined the expression pattern of SCGPR1 by in situ hybridization during chick development. SCGPR1 was first detected at HH stage 7 in the neural tube and notochord. As development progressed, SCGPR1 expression became restricted to the ventral neural tube. SCGPR1 expression was also present in the developing telencephalon, mesencephalon, retina, visceral-class motor neurons, myotome and thyroid invagination.

Role of Gbx2 and Otx2 in the formation of cochlear ganglion and endolymphatic duct.

The boundary of gene expression of transcription factors often plays a role in making a signaling center in development. In the otic vesicle, Gbx2 is expressed in the dorso-medial region including the endolymphatic duct, and Otx2 in the ventral region. Fgf10 is expressed between their expression boundaries, and the cochleovestibular ganglion develops close to the medial side of the Fgf10 expressing domain. Similar expression patterns are observed in the central nervous system, where Otx2 and Gbx2 expression abut at the mid-hindbrain boundary, and the repressive interaction between Otx2 and Gbx2 defines the mid-hindbrain boundary. These analogous expression patterns raise a question about the role of the interaction between Gbx2 and Otx2 in the otic vesicle. To address this, we misexpressed Gbx2 and Otx2 to the otic epithelium. Ectopic Gbx2 expression could repress Otx2 expression and vice versa. In addition, Fgf10 expression was repressed and cochlear ganglion formation was interfered with. Moreover, endolymphatic duct was severely hypomorphic in the Otx2 misexpressing embryos. These results suggest that the interaction between Gbx2 and Otx2 in developing inner ear defines Fgf10 expression domain to induce the cochlear ganglion. It is also suggested that Gbx2 expression is important for the formation of the endolymphatic duct.

Gain- and loss-of-function in chick embryos by electroporation.

It remained very difficult to manipulate gene expression in chick embryos until the advent of in ovo electroporation which enabled the induction of both gain-of-function, and recently loss-of-function, of a gene of interest at a specific developmental stage. Gain-of-function by electroporation is so effective that it has become widely adopted in developmental studies in the chick. Recently, it became possible to induce loss-of-function by introducing an siRNA expression vector by electroporation. In this review, the methods of electroporation for gain-of-function and for loss-of-function by siRNA are discussed.

Six1 controls patterning of the mouse otic vesicle.

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.

How can free energy component analysis explain the difference in protein stability caused by amino acid substitutions? Effect of three hydrophobic mutations at the 56th residue on the stability of human lysozyme.

To elucidate the molecular mechanism of thermal stability, it is essential to determine what are the major free energy components that contribute significantly to the total free energy difference caused by amino acid mutations. In this work, we carried out free energy calculations based on all-atom molecular dynamics simulations to investigate the effect of three hydrophobic mutations at the same position, I56A, I56V and I56F of human lysozyme. The calculated free energy differences are in good agreement with the experimental values in all cases. From free energy component analysis, we found that small changes in stability in the I56A and I56V mutants originate from the short-range Lennard-Jones interactions, whereas the I56F mutant is largely destabilized owing to the changes in the long-range electrostatic interactions. The calculated results are also compared with the free energy components determined by an empirical relationship based on the native-state structure and thermodynamic data. Although this relationship has been shown to be very successful in reproducing the stability changes caused by various amino acid substitutions in several proteins, the changes of stability in I56V and I56F mutants are not reproduced very well. By comparing the free energy components calculated by these two approaches, we showed that the effect of the long-range interaction on the stability changes may be underestimated in the empirical relationships when the structural change caused by mutation is relatively small, as in I56F. It is also suggested that estimation of the change in accessible surface area, deltadeltaASA, may be overestimated if the structure around the mutation site in the denatured state is native-like, which would cause overestimation of the free energy change as in the case of I56V. Our results clearly show that the combined approach of the free energy calculation based on the all-atom molecular dynamics simulation and the empirical relationships is very useful for understanding the detailed mechanism of protein stability.

Positive contribution of hydration structure on the surface of human lysozyme to the conformational stability.

Water molecules make a hydration structure with the network of hydrogen bonds, covering on the surface of proteins. To quantitatively estimate the contribution of the hydration structure to protein stability, a series of hydrophilic mutant human lysozymes (Val to Ser, Tyr, Asp, Asn, and Arg) modified at three different positions on the surface, which are located in the alpha-helix (Val-110), the beta-sheet (Val-2), and the loop (Val-74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by x-ray crystallography at 100 K, respectively. The introduced polar residues made hydrogen bonds with protein atoms and/or water molecules, sometimes changing the hydration structure around the mutation site. Changes in the stability of the mutant proteins can be evaluated by a unique equation that considers the conformational changes resulting from the substitutions. Using this analysis, the relationship between the changes in the stabilities and the hydration structures for mutant human lysozymes substituted on the surface could be quantitatively estimated. The analysis indicated that the hydration structure on protein surface plays an important role in determining the conformational stability of the protein.