Biomedical subjects

C Chiang

Publications and source records attributed to C Chiang.

At least 37 records · Page 2Linked to original sources

PubMed1998

Mediolateral patterning of somites: multiple axial signals, including Sonic hedgehog, regulate Nkx-3.1 expression.

The axial structures, the notochord and the neural tube, play an essential role in the dorsoventral patterning of somites and in the differentiation of their many cell lineages. Here, we investigated the role of the axial structures in the mediolateral patterning of the somite by using a newly identified murine homeobox gene, Nkx-3.1, as a medial somitic marker in explant in vitro assays. Nkx-3.1 is dynamically expressed during somitogenesis only in the youngest, most newly-formed somites at the caudal end of the embryo. We found that the expression of Nkx-3.1 in pre-somitic tissue explants is induced by the notochord and maintained in newly-differentiated somites by the notochord and both ventral and dorsal parts of the neural tube. We showed that Sonic hedgehog (Shh) is one of the signaling molecules that can reproduce the effect of the axial structures by exposing explants to either COS cells transfected with a Shh expression construct or to recombinant SHH. Shh could induce and maintain Nkx-3.1 expression in pre-somitic mesoderm and young somites but not in more mature, differentiated ones. The effects of Shh on Nkr-3.1 expression were antagonized by a forskolin-induced increase in the activity of cyclic AMP-dependent protein kinase A. Additionally, we confirmed that the expression of the earliest expressed murine myogenic marker, myf 5, is also regulated by the axial structures but that Shh by itself is not capable of inducing or maintaining it. We suggest that the establishment of somitic medial and lateral compartments and the early events in myogenesis are governed by a combination of positive and inhibitory signals derived from the neighboring structures, as has previously been proposed for the dorsoventral patterning of somites.

Animals↗

PubMed1998

Sonic hedgehog is essential to foregut development.

Congenital malformation of the foregut is common in humans, with an estimated incidence of 1 in 3000 live births, although its aetiology remains largely unknown. Mice with a targeted deletion of Sonic hedgehog (Shh) have foregut defects that are apparent as early as embryonic day 9.5, when the tracheal diverticulum begins to outgrow. Homozygous Shh-null mutant mice show oesophageal atresia/stenosis, tracheo-oesophageal fistula and tracheal and lung anomalies, features similar to those observed in humans with foregut defects. The lung mesenchyme shows enhanced cell death, decreased cell proliferation and downregulation of Shh target genes. These results indicate that Shh is required for the growth and differentiation of the oesophagus, trachea and lung, and suggest that mutations in SHH and its signalling components may be involved in foregut defects in humans.

Animals↗

PubMed1997

Expression of murine Lhx5 suggests a role in specifying the forebrain.

A LIM homeobox gene, Lim5, is known to be expressed in the forebrain of Xenopus and zebrafish (Toyama et al. [1995] Dev. Biol. 170:583-593). Results from developmental and comparative studies of its mouse ortholog, Lhx5, indicate that this gene may play important roles in forebrain development. Lhx5 expression is detected in the most anterior portion of the neural tube at the headfold stage, overlapping partially with Otx2 expression domain. After neural tube closure, Lhx5 is expressed as a transverse stripe, covering most of the diencephalic primordium. This expression recedes to restricted areas as Dlx gene expression occurs. By midgestation, both genes, Lhx5 and Dlx5, are expressed in the diencephalon and ventral telencephalon in an alternating complementary pattern. It may be that Dlx inhibits Lhx5, and this may represent a step of early regionalization of the forebrain. Lhx5 is also expressed in midbrain, hindbrain, and spinal cord, overlapping extensively with Lhx1 starting from day E10.5 of gestation. The early, persistent, and dynamic expression of Lhx5 suggests a regulatory function in forebrain formation.

Amino Acid Sequence↗

PubMed1997

Hoxd-12 differentially affects preaxial and postaxial chondrogenic branches in the limb and regulates Sonic hedgehog in a positive feedback loop.

Several 5' members of the Hoxd cluster are expressed in nested posterior-distal domains of the limb bud suggesting a role in regulating anteroposterior pattern of skeletal elements. While loss-of-function mutants have demonstrated a regulatory role for these genes in the developing limb, extensive functional overlaps between various different Hox genes has hampered elucidation of the roles played by individual members. In particular, the function of Hoxd-12 in the limb remains obscure. Using a gain-of-function approach, we find that Hoxd-12 misexpression in transgenic mice produces apparent transformations of anterior digits to posterior morphology and digit duplications, while associated tibial hemimelia and other changes indicate that formation/growth of certain skeletal elements is selectively inhibited. If the digital arch represents an anterior bending of the main limb axis, then the results are all reconcilable with a model in which Hoxd-12 promotes formation of postaxial chondrogenic condensations branching from this main axis (including the anteriormost digit) and selectively antagonizes formation of 'true' preaxial condensations that branch from this main axis (such as the tibia). Hoxd-12 misexpression can also induce ectopic Sonic hedgehog (Shh) expression, resulting in mirror-image polydactyly in the limb. Misexpression of Hoxd-12 in other lateral plate derivatives (sternum, pelvis) likewise phenocopies several luxoid/luxate class mouse mutants that all share ectopic Shh signalling. This suggests that feedback activation of Shh expression may be a major function of Hoxd-12. Hoxd-12 can bind to and transactivate the Shh promoter in vitro. Furthermore, expression of either exogenous Hoxd-11 or Hoxd-12 in cultured limb bud cells, together with FGF, induces expression of the endogenous Shh gene. Together these results suggest that certain 5' Hoxd genes directly amplify the posterior Shh polarizing signal in a reinforcing positive feedback loop during limb bud outgrowth.

Animals↗

PubMed1996

Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.

Targeted gene disruption in the mouse shows that the Sonic hedgehog (Shh) gene plays a critical role in patterning of vertebrate embryonic tissues, including the brain and spinal cord, the axial skeleton and the limbs. Early defects are observed in the establishment or maintenance of midline structures, such as the notochord and the floorplate, and later defects include absence of distal limb structures, cyclopia, absence of ventral cell types within the neural tube, and absence of the spinal column and most of the ribs. Defects in all tissues extend beyond the normal sites of Shh transcription, confirming the proposed role of Shh proteins as an extracellular signal required for the tissue-organizing properties of several vertebrate patterning centres.

Animals↗

PubMed1996

Localized pigmented villonodular synovitis of the knee: a report of two cases.

Two cases of localized pigmented villonodular synovitis (PVNS) are reported, both having received surgery. Localized PVNS and diffuse PVNS have not been clearly separated in the literature. Although the clinicopathologic features of the former lesions are similar to diffuse PVNS involving the synovium of the knee joint, the villous component with brownish red color, brownish-stained bloody fluid, and the hemosiderin in the surface-lining cells were lacking in these two cases. Localized PVNS can be treated with simple excision arthroscopically, but it tends to recur as diffuse PVNS if it is excised inadequately. It is suggested that attention must be paid to a soft tissue mass within the knee joint by doing comprehensive pre-operative evaluations which include history taking, computed tomography, and/or magnetic resonance imaging. Intra-operative findings focused on the morphology including the proliferated synovial membrane, gross appearance of the lesion and the pattern of effusion which are all important for distinguishing between localized PVNS and diffuse PVNS. There was no evidence of recurrence in these two cases during the 18 months' follow-up.

Adult↗

PubMed1995

Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage.

BACKGROUND: Sonic hedgehog (Shh), a vertebrate homolog of the Drosophila segment polarity gene hedgehog (hh), has been implicated in patterning of the developing chick limb. Such a role is suggested by the restricted expression of Shh along the posterior limb bud margin, and by the observation that heterologous cells expressing Shh have limb-polarizing activity resembling that of cells from the polarizing region of the posterior limb bud margin. It has not been demonstrated, however, that the Sonic hedgehog protein (SHH) alone is sufficient for limb patterning. SHH has been shown to undergo autoproteolytic cleavage in vitro, yielding two smaller products. It is of interest, therefore, to determine whether processing of SHH occurs in the developing limb and how such processing influences the function of SHH. RESULTS: We demonstrate that SHH is proteolytically processed in developing chick limbs. Grafts of cells expressing SHH protein variants that correspond to individual cleavage products demonstrate that the ability to induce patterned gene expression and to impose morphological pattern upon the limb bud is limited to the amino-terminal product (SHH-N) of SHH proteolytic cleavage. We also demonstrate that bacterially synthesized and purified SHH-N, released from implanted beads, is sufficient for limb-patterning activity. Finally, we show that the endogenous amino-terminal cleavage product is tightly localized to the posterior margin of the limb bud. CONCLUSIONS: Our data show that, of the two cleavage products resulting from SHH autoproteolysis, SHH-N expressed in grafted heterologous cells or supplied in purified form is sufficient to impose pattern upon the developing limb. Moreover, the restricted localization of the endogenous amino-terminal SHH cleavage product to the posterior border of the chick limb bud makes it unlikely that its patterning activity results from it being distributed in a broad gradient across the antero-posterior axis. More consistent with the observed localization is a model in which the amino-terminal SHH cleavage product exerts its patterning effects by local induction in or near the polarizing region, initiating a cascade of gene expression that ultimately extends across the developing limb.

Animals↗

PubMed1995

Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis.

The differentiation of floor plate cells and motor neurons can be induced by Sonic hedgehog (SHH), a secreted signaling protein that undergoes autoproteolytic cleavage to generate amino- and carboxy-terminal products. We have found that both floor plate cells and motor neurons are induced by the amino-terminal cleavage product of SHH (SHH-N). The threshold concentration of SHH-N required for motor neuron induction is about 5-fold lower than that required for floor plate induction. Higher concentrations of SHH-N can induce floor plate cells at the expense of motor neuron differentiation. Our results suggest that the induction of floor plate cells and motor neurons by the notochord in vivo is mediated by exposure of neural plate cells to different concentrations of the amino-terminal product of SHH autoproteolytic cleavage.

Animals↗

PubMed1995

Long-range sclerotome induction by sonic hedgehog: direct role of the amino-terminal cleavage product and modulation by the cyclic AMP signaling pathway.

A long-range signal encoded by the Sonic hedgehog (Shh) gene has been implicated as the ventral patterning influence from the notochord that induces sclerotome and represses dermomyotome in somite differentiation. Long-range effects of hedgehog (hh) signaling have been suggested to result either from local induction of a secondary diffusible signal or from the direct action of the highly diffusible carboxy-terminal product of HH autoproteolytic cleavage. Here we provide evidence that the long-range somite patterning effects of SHH are instead mediated by a direct action of the amino-terminal cleavage product. We also show that pharmacological manipulations to increase the activity of cyclic AMP-dependent protein kinase A can selectively antagonize the effects of the amino-terminal cleavage product. Our results support the operation of a single evolutionarily conserved signaling pathway for both local and direct long-range inductive actions of HH family members.

Animals↗

PubMed1995

Induction of midbrain dopaminergic neurons by Sonic hedgehog.

Midbrain dopaminergic neurons, whose loss in adults results in Parkinson's disease, can be specified during embryonic development by a contact-dependent signal from floor plate cells. Here we show that the amino-terminal product of Sonic hedgehog autoproteolysis (SHH-N), an inductive signal expressed by floor plate cells, can induce dopaminergic neurons in vitro. We show further that manipulations to increase the activity of cyclic AMP-dependent protein kinase A, which is known to antagonize hedgehog signaling, can block dopaminergic neuron induction by floor plate cells. Our results and those of other studies indicate that SHH-N can function in a dose-dependent manner to induce different cell types within the neural tube. Our results also provide the basis for a potential cell transplantation therapy for Parkinson's disease.

Animals↗

PubMed1995

Control of Drosophila tracheal branching by the novel homeodomain gene unplugged, a regulatory target for genes of the bithorax complex.

We have identified a novel Drosophila homeodomain gene, unplugged (unp), whose function is required for formation of the tracheal branches that penetrate the CNS. In unp mutant embryos the segmentally repeated ganglionic branches stall and fail to penetrate the CNS and the segment-specific cerebral branch and associated cerebral anastomosis fail to form. Expression of unp in the founder cells for the cerebral branch within the first tracheal metamere is repressed in posterior segments by Ubx and other bithorax complex genes. This pattern of expression and homeotic gene regulation is reproduced by an unusual 2.6 kb cis-regulatory sequence located downstream of the unp transcription unit. Since the unp protein is localized to the nucleus of tracheal precursor cells as they migrate and extend, unp protein appears to play a regulatory role in neural branching of the tracheae, and the segment-specific aspects of these neural branching patterns appear to be generated by homeotic regulation of unp expression.

Amino Acid Sequence↗

PubMed1994

Expression of a novel Toll-like gene spans the parasegment boundary and contributes to hedgehog function in the adult eye of Drosophila.

Many proteins involved in signal transduction and cell adhesion are characterized by the presence of an extracellular domain with repeated copies of a leucine-rich motif (LRR). Here we report the isolation and characterization of a novel gene, tlr (for Toll-like receptor), which encodes a protein containing multiple LRRs in its presumed extracellular domain, a single transmembrane segment and homology to the cytoplasmic domain of the interleukin 1 receptor in its presumed intracellular domain. The pattern of tlr expression at the extended germ band stage is characterized by 15 transverse stripes in the gnathal and trunk segments, with four patches of expression corresponding to head segments and an additional patch of expression in the presumptive hindgut. The segmentally repeated tlr stripes in the trunk overlap both the wingless and engrailed stripes and thus span the parasegment boundary. The tlr stripes require pair rule gene function for their establishment and later become dependent upon segment-polarity gene function for their maintenance. Segmental modulation of tlr expression later in the tracheal system is dependent upon the function of the homeotic genes of the bithorax complex. The tlr gene also is prominently expressed in the imaginal discs. In the eye disc, this expression occurs in two stripes at the anterior and posterior margins of the morphogenetic furrow; this expression is consistent with a genetic interaction between a tlr mutation and an eye-specific allele of hedgehog. All of these data combine to suggest a role for tlr in interactions between cells at critical boundaries during development.

Aging↗

PubMed1994

Products, genetic linkage and limb patterning activity of a murine hedgehog gene.

The hedgehog (hh) segmentation gene of Drosophila melanogaster encodes a secreted signaling protein that functions in the patterning of larval and adult structures. Using low stringency hybridization and degenerate PCR primers, we have isolated complete or partial hh-like sequences from a range of invertebrate species including other insects, leech and sea urchin. We have also isolated three mouse and two human DNA fragments encoding distinct hh-like sequences. Our studies have focused upon Hhg-1, a mouse gene encoding a protein with 46% amino acid identity to hh. The Hhg-1 gene, which corresponds to the previously described vhh-1 or sonic class, is expressed in the notochord, ventral neural tube, lung bud, hindgut and posterior margin of the limb bud in developing mouse embryos. By segregation analysis the Hhg-1 gene has been localized to a region in proximal chromosome 5, where two mutations affecting mouse limb development previously have been mapped. In Drosophila embryos, ubiquitous expression of the Hhg-1 gene yields effects upon gene expression and cuticle pattern similar to those observed for the Drosophila hh gene. We also find that cultured quail cells transfected with a Hhg-1 expression construct can induce digit duplications when grafted to anterior or mid-distal but not posterior borders within the developing chick limb; more proximal limb element duplications are induced exclusively by mid-distal grafts. Both in transgenic Drosophila embryos and in transfected quail cells, the Hhg-1 protein product is cleaved to yield two stable fragments from a single larger precursor. The significance of Hhg-1 genetic linkage, patterning activity and proteolytic processing in Drosophila and chick embryos is discussed.

Animals↗

PubMed1994

The novel homeodomain gene buttonless specifies differentiation and axonal guidance functions of Drosophila dorsal median cells.

We have identified a novel homeodomain gene, buttonless (btn), that is specifically expressed in 20 cells of a single type during Drosophila embryonic development. These cells, the dorsal median (DM) cells, are arranged as a single pair within each segment along the dorsal midline of the CNS. Distinctive features of the DM cells include a large cell body and a long thick process extending laterally to the muscle attachment site. In the absence of btn gene function the initial commitment to the DM cell fate is made but differentiation fails to occur and the DM cells are lost. The btn mutation thus specifically eliminates the DM cells, and this genetic ablation in turn reveals a requirement for DM cells as cellular cues for axonal guidance during transverse nerve outgrowth and bifurcation of the median nerve.

Amino Acid Sequence↗

PubMed1993

A 5-hydroxytryptamine2 agonist augments gamma-aminobutyric acid and excitatory amino acid inputs to noradrenergic locus coeruleus neurons.

We examined the effects of the 5-hydroxytryptamine2 receptor agonist, (+-)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, on spontaneous and evoked discharge of locus coeruleus neurons in the rat. Extracellular recordings were obtained from single locus coeruleus neurons while (+-)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane was injected systemically or locally into the locus coeruleus. Systemic, but not local, administration of (+-)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane decreased spontaneous discharge of locus coeruleus neurons in a dose-dependent manner while simultaneously increasing responses evoked by somatosensory stimulation, consistent with previous studies using 5-hydroxytryptamine2 agonists. Increased responsiveness was observed after both low- and high-intensity stimulation and, in the latter, resulted from the addition of a second, longer latency response after (+-)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane administration, when cells typically responded to each stimulation with two driven spikes instead of one. Both of these effects could be completely reversed by systemic administration of the 5-hydroxytryptamine2 receptor antagonist, ketanserin. Furthermore, we report that: (i) the (+/-)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane-induced decrease in spontaneous firing was blocked by local infusion of the GABA antagonists bicuculline or picrotoxin into the locus coeruleus, but not by local infusion of the alpha-2 adrenoceptor antagonist, idazoxan; and (ii) the enhancement of locus coeruleus sensory responses after high-intensity stimulation was blocked by local application of the selective antagonist of N-methyl-D-aspartate receptors, 2-amino-5-phosphonopentanoic acid, but not by local infusion of the preferential antagonist of non-N-methyl-D-aspartate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione. Together, these results lead us to propose that systemic 5-hydroxytryptamine2 agonists influence locus coeruleus indirectly, causing tonic activation of a GABAergic input to the locus coeruleus, and facilitating sensory inputs that act via excitatory amino acid receptors within locus coeruleus.

2-Amino-5-phosphonovalerate↗

PubMed1993

Response of locus coeruleus neurons to footshock stimulation is mediated by neurons in the rostral ventral medulla.

While it is well documented that locus coeruleus neurons are potently activated by foot-pinch or sciatic nerve stimulation, little is known about the circuit producing this sensory response. Previous work in our laboratory has identified the medullary nucleus paragigantocellularis as a major excitatory afferent to the locus coeruleus. Here, we use local microinjections into the paragigantocellularis to test whether this nucleus is a link in the pathway mediating the activation of locus coeruleus neurons by subcutaneous footpad stimulation, or footshock, in anesthetized rats. Lidocaine HCl microinjected into the paragigantocellularis reversibly attenuated footshock-evoked activation of 50 out of 56 locus coeruleus cells, with responses in 20 cells completely blocked. Microinjections of GABA into the paragigantocellularis reduced the footshock-evoked responses of 17 out of 27 locus coeruleus cells (seven complete blocks); microinjections of the GABAB agonist baclofen had no effect (0 out of 11 cells blocked). Microinjections of a synaptic decoupling cocktail of manganese and cadmium also attenuated locus coeruleus activation in eight out of nine cells with two complete blocks. With each agent, the most effective injection placement for complete blockade of responses was the ventromedial paragigantocellularis; injections bordering this region attenuated responses, while those outside of the paragigantocellularis (dorsal medullary reticular formation, nucleus tractus solitarius, or facial nucleus), or vehicle injections, were ineffective. These results are consistent with previous findings that pharmacologic blockade of paragigantocellularis-evoked locus coeruleus activity also blocks footshock-evoked responses of locus coeruleus neurons [Ennis and Aston-Jones (1988) J. Neurosci. 8, 3644-3657], and support the view that this somatosensory response, and perhaps other sensory-evoked responses of locus coeruleus neurons, involve the nucleus paragigantocellularis.

Animals↗

PubMed1992

Thermal denaturation of plastocyanin: the effect of oxidation state, reductants, and anaerobicity.

The thermal stability of plastocyanin (PC) was determined as a function of oxidation state of the copper center and the presence of oxidants, reductants, oxygen, and EDTA. It was found that the copper center and its ligands play a crucial role in maintaining the stability of PC. Thermal denaturation was monitored by using far-uv circular dichroism (CD) spectra to monitor changes in secondary structure, the near-uv CD ellipticity at 280 nm to monitor changes in tertiary structure, and the absorbance at 597 nm and the 255-nm CD transition to monitor changes in the copper center. Reduced PC (Tm = 71 degrees C) was found to be more stable than the oxidized form (Tm = 61 degrees C). The Tm was increased by addition of reductants, removal of oxygen, or addition of EDTA. Two distinct denatured forms (designated D1 and D2) were separated by anion exchange fast protein liquid chromatography. Neither form contained a native copper center. Form D2 retained the characteristic 280-nm CD band but showed an altered far-uv CD spectrum. Its formation was inhibited by the addition of reductants or the removal of oxygen. It could be refolded to form native, Cu-PC upon incubation with copper plus a reductant such as dithionite. These results suggest that its formation involves the reversible oxidation of a group on the PC molecule, possibly a ligand to the copper such as Cys 84 or Met 92. Form D1 occurred in the presence of ferricyanide or at high temperatures in the presence of oxygen. EDTA inhibited its formation. Form D1 lost the 280-nm CD transition and its far-uv CD spectrum was altered. No renaturation was observed suggesting that Form D1 is the product of an irreversible oxidation step possibly involving a histidine ligand to the copper. Forms D1 and D2 are not interconvertible and represent the endpoints of two different denaturation pathways.

Anaerobiosis↗

PubMed1992

Detection of blood group surface antigens of urinary bladder tumours using monoclonal antibodies with the avidin-biotin complex technique.

We examined 8 normal bladder transitional epithelia and 65 transitional cell carcinomas of the urinary bladder of various stages and grades for the presence of ABO(H) blood group surface isoantigens using the immunoperoxidase staining via the Avidin-Biotin Complex (ABC) methods. It was found that 27% of patients with grade I tumours, 50% with grade II tumours and 82% with grade III tumours had loss of cell surface isoantigens. When correlated with the clinical stage the tumours showed no surface isoantigens in stage D, while 65% of patients with stage A tumours were positive for surface antigens. From among 37 patients (57%), 28 (43%) survived for more than five years. Our results suggest that surface antigens of transitional cell carcinoma of the urinary bladder tended to disappear as the histologic changes of the tumour progressed. It also was noted that a loss of ABH(O) surface isoantigens was a bad prognostic sign.

ABO Blood-Group System↗