An expression vector inhibits gene expression in Xenopus embryos by antisense RNA

Summary An expression vector was constructed containing the entire bovine papilloma virus (BPV-1) genome and part of the a-actin gene of Xenopus laevis cloned in the antisense orientation into the neomycin resistance gene under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter. When this vector is microinjected into X. laevis embryos it replicates extrachromosomally, at least up to the tadpole stage, and a fusion RNA is synthesized after the mid blastula transition (MBT). The expression of the antisense gene results in a morphological abnormality of somites demonstrating that antisense RNA generated by an episomal replicating expression vector can inhibit the expression of a selected gene during early embryogenesis of X. laevis.     

Systematics of the Southeast Asian mongooses (Herpestidae,Carnivora): solving the mystery of the elusive collared mongoose and Palawan mongoose

Although recent molecular studies have clarified the phylogeny of mongooses, the systematics of the Southeast Asian species was incomplete as the collared mongoose Urva semitorquata and some debatable taxa (Hose's mongoose, Palawan mongoose) were missing in the analyses. We sequenced three mitochondrial (cytochrome b, ND2, control region) and one nuclear (beta‐fibrinogen intron 7) fragments of the Southeast Asian mongooses to clarify the systematic position of the different species and populations occurring in this region. Our results showed that the collared mongoose is closely related to the crab‐eating mongoose Urva urva, these two species forming a sister‐group to the short‐tailed mongoose Urva brachyura. Despite Sumatran collared mongooses having a peculiar orange phenotype, we showed that they exhibited very little genetic divergence to individuals from Borneo. In contrast, the populations of the short‐tailed mongoose from Borneo were strongly divergent to those from Peninsular Malaysia and Sumatra, and these might represent separate species. Within the crab‐eating mongoose, we observed little geographical genetic structure. Our study suggests that Hose's mongoose is not a valid species. The Palawan mongooses did not cluster with the other populations of the short‐tailed mongoose; they were closer to the collared mongoose and should be included in this species. © 2014 The Linnean Society of London     

Separation of enantiomers of drugs by capillary electrophoresis,part 7: Gamma-cyclodextrin as chiral solvating agent

Following an extended chiral drug screening program by capillary zone electrophoresis (CZE), the enantioseparation of 86 racemic drugs was tested with γ-cyclodextrin as a chiral solvating agent. Unified conditions were applied to all experiments. In total, 18 drug racemates were separated, 13 entries thereof that had not been separated at the lower CSA concentration applied in an earlier stage of the project. A comparison of the data with the results obtained for α- and β-cyclodextrin points to the significance of partial penetration (“side-on binding”) of aryl groups into the cyclodextrin cavity. Chirality 10:548–554, 1998. © 1998 Wiley-Liss, Inc.     

Timing in the regulation of neural crest cell migration: retarded "maturation" of regional extracellular matrix inhibits pigment cell migration in embryos of the white axolotl mutant

In larvae of the white axolotl mutant (Ambystoma mexicanum), contrary to normal dark ones, trunk pigmentation is restricted because the epidermis is unable to support subepidermal migration of pigment cells from the neural crest (NC). This study examines whether the subepidermal extracellular matrix (ECM) is the defective component which prevents pigment cell migration in the white embryo. We transplanted subepidermal ECM, adsorbed in vivo on membrane microcarriers, from and to white and dark embryos in various combinations. White embryos have demonstrated normal NC cell migration along the medioventral pathway, and in order to test the effects of medial ECM on subepidermal migration, this ECM was similarly transplanted. Carriers with ECM attached were inserted subepidermally in host embryos at a premigratory NC stage. Control carriers without ECM and carriers with subepidermal ECM from white donors did not affect NC cell migration in white or dark embryos. In contrast, subepidermal ECM from dark donors triggered NC cell migration in the subepidermal space of both white and dark hosts. Remarkably, subepidermal ECM from white donors which were older than those normally used also stimulated migration in embryos of both strains. Likewise, medial ECM from white donors elicited migration in white as well as dark hosts. Pigment cells occurred among those NC cells that were stimulated to migrate in response to contact with ECM on carriers. These results indicate that the subepidermal ECM of the white embryo is transiently defective as a substrate for pigment cell migration, implying that "maturation" of the ECM is retarded beyond the times during which pigment cells are able to respond. In contrast, the medial ECM of the white embryo appears to mature normally. These findings suggest that the effect of the d gene is expressed regionally through the subepidermal ECM during a limited period of development. Hence, the action of the d gene seems to retard ECM maturation, bringing it out of phase with the migratory capability of the pigment cells. We propose that such a shift in relative timing of the developmental phenomena involved inhibits pigment cell migration in embryos of the white axolotl mutant and, accordingly, that the restricted pigmentation of the mutant larva is generated through heterochrony.     

The Ectomesenchymal-Endodermal Interaction-System (EEIS) of Triturus alpestris in Tissue Culture

A piece of head neural fold tissue from behind the prospective ear region of a Triturus alpestris neurula was cultivated, together with a piece of ventrolateral pharynx endoderm from the same neurula, in hanging drop cultures. This system, referred to as the Ectomesenchymal-Endodermal Interaction-System (EEIS), offers insight into the visible processes of attachment, migration and differentiation of neural crest cells emigrating from the piece of neural fold.
The neural crest cells were not found to move preferably in the direction of the pharynx endoderm. Therefore, instead of chemotaxis, the concept of contact inhibition¹ provides a more satisfactory explanation for the distribution pattern of emigrating neural crest cells.
During culture, the neural crest cells differentiate into neuroblasts, pigment cells, myoblasts, chondroblasts and, after about 11 days, into perichondrial cells. After 6 days, a large number of neural crest cells, now called mesenchymal cells, persist without any visible differentiation throughout the culture.
Chondroblasts only develop from neural crest cells which have been in contact with the pharynx endoderm, as opposed to all other crest cells differentiating in the EEIS.     

Formation of pigment cell patterns in Triturus alpestris embryos

The distribution of melanophores and xanthopores in developing tailbud stages of Triturus alpestris was investigated. In stage 27 embryos (curved tailbuds), melanophores are distributed evenly but sparsely over the entire dorsolateral trunk. With progressive development melanophores arrange themselves into compact dorsal and lateral bands present in stage 34 embryos (9 to 10-mm-long larvae). On the inner surface of detached pieces of skin from early tailbuds which were investigated in the scanning electron microscope xanthophores were discovered in addition to and mixed with melanophores. Unlike melanophores they are invisible from outside. Later in development they occupy the zone between the melanophore bands and also the dorsal fin. Thus, formation of pigment cell patterns in Triturus embryos is a process which seems to depend on the differential sorting out of two populations of neural crest-derived chromatophore cell types.     

Developmental origins and evolution of jaws: new interpretation of "maxillary" and "mandibular"

Cartilage of the vertebrate jaw is derived from cranial neural crest cells that migrate to the first pharyngeal arch and form a dorsal "maxillary" and a ventral "mandibular" condensation. It has been assumed that the former gives rise to palatoquadrate and the latter to Meckel's (mandibular) cartilage. In anamniotes, these condensations were thought to form the framework for the bones of the adult jaw and, in amniotes, appear to prefigure the maxillary and mandibular facial prominences. Here, we directly test the contributions of these neural crest condensations in axolotl and chick embryos, as representatives of anamniote and amniote vertebrate groups, using molecular and morphological markers in combination with vital dye labeling of late-migrating cranial neural crest cells. Surprisingly, we find that both palatoquadrate and Meckel's cartilage derive solely from the ventral "mandibular" condensation. In contrast, the dorsal "maxillary" condensation contributes to trabecular cartilage of the neurocranium and forms part of the frontonasal process but does not contribute to jaw joints as previously assumed. These studies reveal the morphogenetic processes by which cranial neural crest cells within the first arch build the primordia for jaw cartilages and anterior cranium.     

Changes in the distribution of melanophores and xanthophores inTriturus alpestris embryos during their transition from the uniform to banded pattern

Summary The change in distribution of melanophores from stage 28+ (uniform melanophore pattern) to stage 34 (banded melanophore pattern) and the participation of xanthophores in these changes has been investigated inTriturus alpestris embryos by studying the social behaviour of single cells. While melanophores are clearly visible from outside the embryo at stage 28+, xanthophores cannot be recognized from the outside until after stage 34. In ultrathin sections of stage 34 embryos, xanthophores are observed alternating with melanophores in a zonal distribution (Epperlein 1982). Using detached pieces of dorsolateral trunk skin, which retain their chromatophores after detachment, the entire distribution of melanophores and xanthophores can be visualized in a scanning electron microscope (SEM). In ambiguous cases (early stages), cells were reprocessed for transmission electron microscopy (TEM) and the presence of the characteristic pigment organelles was assessed. In addition, xanthophores were specifically identified by pteridine fluorescence with dilute ammonia. Pteridines were also identified chromatographically in skin homogenates. The combination of these methods allowed precise identification and quantitative determination of melanophores and xanthophores. Both cell types were present as codistributed, biochemically differentiated cells at stage 28+. Changes in the pattern up to stage 34 were due to the rearrangement at the epidermal-mesodermal interface of a relatively fixed number of melanophores which became preferentially localised at the dorsal somite edge and at the lateral plate mesoderm, and to the distribution of an increasing number of xanthophores to subepidermal locations in the dorsal fin and between the melanophore bands. Concomitant was an increase in the thickness of the epidermal basement membrane and a change in shape of chromatophores from elongate via stellate to rosette shaped, which may be correlated with a shift from migratory to sessile phases.     

Immunohistochemical demonstration of hyaluronan and its possible involvement in axolotl neural crest cell migration

Hyaluronan (HA), an extracellular matrix component, is involved mainly in the control of cell proliferation, neural crest and tumor cell migration, and wound repair. We investigated the effect of hyaluronan on neural crest (NC) cell migration and its ultrastructural localization in dark (wild-type) and white mutant embryos of the Mexican axolotl (Ambystoma mexicanum, Amphibia). The axolotl system is an accepted model for studying mechanisms of NC cell migration. Using a biotinylated hyaluronan binding protein (HABP), major extracellular matrix (ECM) spaces, including those of NC cell migration, reacted equally positive on cryosections through dark and white embryos. Since neural crest-derived pigment cells migrate only in subepidermal spaces of dark embryos, HA does not seem to influence crest cell migration in vivo. However, when tested on different alternating substrates in vitro, migrating NC cells in dark and white embryos prefer HA to fibronectin. In vivo, such an HA migration stimulating effect might exist as well, but be counteracted to differing degrees in dark and white embryos. The ultrastructural localization of HA was studied by means of transmission electron microscopic immunohistochemistry using HABP and different protocols of standard chemical fixation, cryofixation, embedding, and immunolabeling. The binding reaction of HA to HABP was strong and showed an equal distribution throughout ECM spaces after both standard chemical fixation/freeze substitution and cryofixation. A preference for the somite or subepidermal side was not observed. Following standard fixation/freeze substitution HABP-labeled "honeycomb"-like networks reminiscent of fixation artifacts were more prominent than labeled fibrillar or irregular net-like structures. The latter predominated in adequately frozen specimens following high-pressure freezing/freeze substitution. For this reason fibrillar or irregular net-like structures very likely represent hyaluronan in the complex subepidermal matrix of the axolotl embryo in its native arrangement.