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.     

Distribution of Keratan Sulphate and Chondroitin Sulphate in Wild Type and White Mutant Axolotl Embryos During Neural Crest Cell Migration

In embryos of the white mutant axolotl, prospective pigment cells are unable to migrate from the neural crest (NC) due to a deficiency in the subepidermal extracellular matrix (ECM). This raises the question of the molecular nature of this functional defect. Some PGs can inhibit cell migration on ECM molecules in vitro, and an excess of this class of molecules in the migratory pathways of neural crest cells might cause the restricted migration of prospective pigment cells seen in the white mutant embryo. In the present study, we use several monoclonal antibodies against epitopes on keratan sulphate (KS) and chondroitin sulphate (CS) and LM immunofluorescence to examine the distribution of these glycosaminoglycans at initial (stage 30) and advanced (stage 35) stages of neural crest cell migration. Most KS epitopes are more widely distributed in the white mutant than in the wild type embryo, whereas CS epitopes show very similar distributions in mutant and wild type embryos. This is confirmed quantitatively by immunoblotting: certain KS epitopes are more abundant in the white mutant. TEM immunogold staining reveals that KS as well as CS are present both in the basal lamina and in the interstitial ECM in both types of embryos. It remains to be investigated whether the abundance of certain KS epitopes in the white mutant embryo might contribute to the deficiency in supporting pigment cell migration shown by its ECM.     

Structural and compositional divergencies in the extracellular matrix encountered by neural crest cells in the white mutant axolotl embryo

The skin of the white mutant axolotl larva is pigmented differently from that of the normal dark due to a local inability of the extracellular matrix (ECM) to support subepidermal migration of neural crest-derived pigment cell precursors. In the present study, we have compared the ECM of neural crest migratory pathways of normal dark and white mutant embryos ultrastructurally, immunohistochemically and biochemically to disclose differences in their structure/composition that could be responsible for the restriction of subepidermal neural crest cell migration in the white mutant axolotl. When examined by electron microscopy, in conjunction with computerized image analysis, the structural assembly of interstitial and basement membrane ECMs of the two embryos was found to be largely comparable. At stages of initial neural crest cell migration, however, fixation of the subepidermal ECM in situ with either Karnovsky-ruthenium red or with periodate-lysine-paraformaldehyde followed by ruthenium red-containing fixatives, revealed that fibrils of the dark matrix were significantly more abundant in associated electron-dense granules. This ultrastructural discrepancy of the white axolotl ECM was specific for the subepidermal region and suggested an abnormal proteoglycan distribution. Dark and white matrices of the medioventral migratory route of neural crest cells had a comparable appearance but differed from the corresponding subepidermal ECMs. Immunohistochemistry revealed only minor differences in the distribution of fibronectin, laminin, collagen types I, and IV, whereas collagen type III appeared differentially distributed in the two embryos. Chondroitin- and chondroitin-6-sulfate-rich proteoglycans were more prevalent in the white mutant embryo than in the dark, especially in the subepidermal space. Membrane microcarriers were utilized to explant site-specifically native ECM for biochemical analysis. Two-dimensional gel electrophoresis of these regional matrices revealed a number of differences in their protein content, principally in constituents of apparent molecular masses of 30-90,000. Taken together our observations suggest that local divergences in the concentration/assembly of low and high molecular mass proteins and proteoglycans of the ECM encountered by the moving neural crest cells account for their disparate migratory behavior in the white mutant axolotl.     

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.     

Bone morphogenetic protein-4 and Noggin signaling regulates pigment cell distribution in the axolotl trunk

Abstract Wild-type (dark) and white mutant axolotl ( Ambystoma mexicanum ) embryos were used to investigate the role of the secreted growth factor bone morphogenetic protein-4 (BMP-4) and its antagonist, Noggin, in dorso-lateral trunk neural crest (NC) migration. Implantation of a BMP-4-coated microbead caused a melanophore-free zone around the bead, reduction of the dorsal fin above the bead, and disappearance of myotome tissue. We established a novel method that allows controlled induction of protein synthesis and release. Xenopus animal cap (XAC) cells injected with heat shock-inducible constructs for BMP-4 and Noggin were implanted into axolotl embryos and protein expression was induced at defined time points. With this approach, we could demonstrate for the first time that Noggin can stimulate melanophore migration in the white mutant. We further showed that implantation of BMP-4 expressing XAC cells alters pigment cell distribution without affecting muscle and dorsal fin development.     

Role of the extracellular matrix during neural crest cell migration

Once specified to become neural crest (NC), cells occupying the dorsal portion of the neural tube disrupt their cadherin-mediated cell-cell contacts, acquire motile properties, and embark upon an extensive migration through the embryo to reach their ultimate phenotype-specific sites. The understanding of how this movement is regulated is still rather fragmentary due to the complexity of the cellular and molecular interactions involved. An additional intricate aspect of the regulation of NC cell movement is that the timings, modes and patterns of NC cell migration are intimately associated with the concomitant phenotypic diversification that cells undergo during their migratory phase and the fact that these changes modulate the way that moving cells interact with their microenvironment. To date, two interplaying mechanisms appear central for the guidance of the migrating NC cells through the embryo: one involves secreted signalling molecules acting through their cognate protein kinase/phosphatase-type receptors and the other is contributed by the multivalent interactions of the cells with their surrounding extracellular matrix (ECM). The latter ones seem fundamental in light of the central morphogenetic role played by the intracellular signals transduced through the cytoskeleton upon integrin ligation, and the convergence of these signalling cascades with those triggered by cadherins, survival/growth factor receptors, gap junctional communications, and stretch-activated calcium channels. The elucidation of the importance of the ECM during NC cell movement is presently favoured by the augmenting knowledge about the macromolecular structure of the specific ECM assembled during NC development and the functional assaying of its individual constituents via molecular and genetic manipulations. Collectively, these data propose that NC cell migration may be governed by time- and space-dependent alterations in the expression of inhibitory ECM components; the relative ratio of permissive versus non-permissive ECM components; and the supramolecular assembly of permissive ECM components. Six multidomain ECM constituents encoded by a corresponding number of genes appear to date the master ECM molecules in the control of NC cell movement. These are fibronectin, laminin isoforms 1 and 8, aggrecan, and PG-M/version isoforms V0 and V1. This review revisits a number of original observations in amphibian and avian embryos and discusses them in light of more recent experimental data to explain how the interaction of moving NC cells with these ECM components may be coordinated to guide cells toward their final sites during the process of organogenesis.     

The distribution of fibronectin and tenascin along migratory pathways of the neural crest in the trunk of amphibian embryos

It is generally assumed that in amphibian embryos neural crest cells migrate dorsally, where they form the mesenchyme of the dorsal fin, laterally (between somites and epidermis), where they give rise to pigment cells, and ventromedially (between somites and neural tube), where they form the elements of the peripheral nervous system. While there is agreement about the crest migratory routes in the axolotl (Ambystoma mexicanum), different opinions exist about the lateral pathway in Xenopus. We investigated neural crest cell migration in Xenopus (stages 23, 32, 35/36 and 41) using the X. laevis-X. borealis nuclear marker system and could not find evidence for cells migrating laterally. We have also used immunohistochemistry to study the distribution of the extracellular matrix (ECM) glycoproteins fibronectin (FN) and tenascin (TN), which have been implicated in directing neural crest cells during their migrations in avian and mammalian embryos, in the neural crest migratory pathways of Xenopus and the axolotl. In premigratory stages of the crest, both in Xenopus (stage 22) and the axolotl (stage 25), FN was found subepidermally and in extracellular spaces around the neural tube, notochord and somites. The staining was particularly intense in the dorsal part of the embryo, but it was also present along the visceral and parietal layers of the lateral plate mesoderm. TN, in contrast, was found only in the anterior trunk mesoderm in Xenopus; in the axolotl, it was absent. During neural crest cell migration in Xenopus (stages 25-33) and the axolotl (stages 28-35), anti-FN stained the ECM throughout the embryo, whereas anti-TN staining was limited to dorsal regions. There it was particularly intense medially, i.e. in the dorsal fin, around the neural tube, notochord, dorsal aorta and at the medial surface of the somites (stage 35 in both species). During postmigratory stages in Xenopus (stage 40), anti-FN staining was less intense than anti-TN staining. In culture, axolotl neural crest cells spread differently on FN- and TN-coated substrata. On TN, the onset of cellular outgrowth was delayed for about 1 day, but after 3 days the extent of outgrowth was indistinguishable from cultures grown on FN. However, neural crest cells in 3-day-old cultures were much more flattened on FN than on TN. We conclude that both FN and TN are present in the ECM that lines the neural crest migratory pathways of amphibian embryos at the time when the neural crest cells are actively migrating. FN is present in the embryonic ECM before the onset of neural crest migration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of initial neural crest cell migration in the axolotl embryo by tissue grafts and extracellular matrix transplanted on microcarriers

The present experiments were designed to test whether the onset of neural crest cell migration in the embryonic axolotl trunk is stimulated by surrounding tissues and their associated extracellular matrix (ECM). Tissue grafts, or embryonic ECM adsorbed in vivo onto inert "microcarriers" prepared from Nuclepore filters, were placed close to the premigratory neural crest cells, and the embryos were then incubated to a specific stage. The experiments were evaluated with light microscopy, SEM, and TEM. It was found that grafts from the dorsal epidermis were especially effective in locally stimulating initial neural crest cell migration in the region under the graft. The microcarrier experiments showed that the subepidermal ECM alone could initiate neural crest cell migration, implying that the ECM of the epidermal grafts was the stimulating factor. These results indicate that the premigratory neural crest cells along the trunk have migratory capability but that they need to be triggered from the environment, probably from the surrounding ECM, to start migration. It is proposed that ECM, as substrate for cell locomotion, initiates and regulates the onset of neural crest cell migration.     

Axolotls with an under- or oversupply of neural crest can regulate the sizes of their dorsal root ganglia to normal levels

How animals adjust the size of their organs is a fundamental, enduring question in biology. Here we manipulate the amount of neural crest (NC) precursors for the dorsal root ganglia (DRG) in axolotl. We produce embryos with an under- or over-supply of pre-migratory NC in order to find out if DRG can regulate their sizes during development. Axolotl embryos are perfectly suitable for this research. Firstly, they are optimal for microsurgical manipulations and tissue repair. Secondly, they possess, unlike most other vertebrates, only one neural crest string located on top of the neural tube. This condition and position enables NC cells to migrate to either side of the embryo and participate in the regulation of NC cell distribution. We show that size compensation of DRG in axolotl occurs in 2 cm juveniles after undersupply of NC (up-regulation) and in 5 cm juveniles after oversupply of NC (down-regulation). The size of DRG is likely to be regulated locally within the DRG and not via adaptations of the pre-migratory NC or during NC cell migration. Ipsi- and contralateral NC cell migration occurs both in embryos with one and two neural folds, and contralateral migration of NC is the only source for contralateral DRG formation in embryos with only one neural fold. Compensatory size increase is accompanied by an increase in cell division of a DRG precursor pool (PCNA+/SOX2−), rather than by DRG neurons or glial cells. During compensatory size decrease, increased apoptosis and reduced proliferation of DRG cells are observed.     

Genetic analysis of steel and the PG-M/versican-encoding gene AxPG as candidates for the white (d) pigmentation mutant in the salamander Ambystoma mexicanum

 Vertebrate non-retinal pigment cells are derived from neural crest (NC) cells, and several mutations have been identified in the Mexican axolotl Ambystoma mexicanum (Ambystomatidae) that affect the development of these cell lineages. In ”white” (d) mutant axolotls, premigratory NC cells differentiate as pigment cells, yet fail to disperse, survive, or both, and this leads to a nearly complete absence of pigment cells in the skin. Previous studies revealed that d affects pigment cell development non-autonomously, and have reported differences between white and wild-type axolotls in the structure and composition of the extracellular matrix through which NC and pigment cells migrate. Here we test the correspondence of d and two candidate genes: steel and AxPG. In amniotes, Steel encodes the cytokine Steel factor (mast cell growth factor; stem cell factor; kit ligand), which is expressed along the migratory pathways of melanocyte precursors and is required by these cells for their migration and survival; mammalian Steel mutants resemble white mutant axolotls in having a deficit or complete absence of pigment cells. In contrast, AxPG encodes aPG-M/versican-like proteoglycan that may promote the migration of A. mexicanum pigment cells, and AxPGexpression is reduced in white mutant axolotls. We cloned a salamander orthologue of steel and used a partial genetic linkage map of Ambystoma to determine the genomic locations of steel, AxPG, and d. We show that the three genes map to different linkage groups, excluding steel and AxPG as candidates for d. Received: 11 November 1998 / Accepted: 30 January 1999     

Mitogen-induced B-cell differentiation in Xenopus laevis

Abstract. Four genes are known to affect pigmentation in the Mexican axolotl. The purpose of this article is to review previous information pertinent to these genes and to reevaluate such information in light of new evidence that demonstrates (in a preliminary way) how pigments, and subsequently phenotypes, are affected by the various pigment genes. Each of the mutant phenotypes – m (melanoid), ax (axanthic), a (albino), and d (white) - is compared to the wild type (D). All of these genes are recessives, all of them affect phenotypic changes during development, and three of the four ( m, a , and d ) also affect specific biochemical (i.e., pigment) changes during development. In the axolotl, color patterns can be directly correlated to the presence(or absence) of a variety of pigments that are normally found in discrete pigment cells. Qualitative and quantitative analyses of the bright-colored pigments (pteridines and flavins in this case) present in axolotl skin demonstrate that these pigments vary significantly among the various phenotypes under consideration. These analyses raise some interesting questions with regard to how each of the pigment genes is believed to act, and numerous possibilities for continued experimentation are suggested.     

Myocardial cell relationships during morphogenesis in normal and cardiac lethal mutant axolotls, Ambystoma mexicanum

Sarcomere formation has been shown to be deficient in the myocardium of axolotl embryos homozygous for the recessive cardiac lethal gene c. We examined the developing hearts of normal and cardiac mutant embryos from tailbud stage 33 to posthatching stage 43 by scanning electron microscopy in order to determine whether that deficiency has any effect on heart morphogenesis. Specifically, we investigated the relationships of myocardial cells during the formation of the heart tube (stage 33), the initiation of dextral looping (stages 34-36), and the subsequent flexure of the elongating heart (stages 38-43). In addition, we compared the morphogenetic events in the axolotl to the published accounts of comparable stages in the chick embryo. In the axolotl (stage 33), changes in cell shape and orientation accompany the closure of the myocardial trough to form the tubular heart. The ventral mesocardium persists longer in the axolotl embryo than in the chick and appears to contribute to the asymmetry of dextral looping (stages 34-36) in two ways. First, as a persisting structure it places constraints on the simple elongation of the heart tube and the ability of the heart to bend. Second, after it is resorbed, the ventral myocardial cells that contributed to it are identifiable by their orientation, which is orthogonal to adjacent cells: a potential source of shearing effects. Cardiac lethal mutant embryos behave identically during these events, indicating that functional sarcomeres are not necessary to these processes. The absence of dynamic apical myocardial membrane changes, characteristic of the chick embryo (Hamburger and Hamilton stages 9-11), suggests that sudden hydration of the cardiac jelly is less likely to be a major factor in axolotl cardiac morphogenesis. Subsequent flexure (stages 38-43) of the axolotl heart is the same in normal and cardiac lethal mutant embryos as the myocardial tube lengthens within the confines of a pericardial cavity of fixed length. However, the cardiac mutant begins to exhibit abnormalities at this time. The lack of trabeculation (normally beginning at stage 37) in the mutant ventricle is evident at the same time as an increase in myocardial surface area, manifest in extra bends of the heart tube at stage 39. Nonbeating mutant hearts (stage 41) have an abnormally large diameter in the atrioventricular region, possibly the result of the accumulation of ascites fluid. In addition, mutant myocardial cells have a larger apical surface area compared to normals.     

Physical influences on neural crest cell migration in avian embryos: contact guidance and spatial restriction

Several ideas on how neural crest (NC) cell migration in bird embryos might be dependent on the physical qualities of the internal embryonic environment were studied. Contact guidance has been suggested to direct NC cells ventrally in the trunk, but this has been subject to doubt (see Newgreen and Erickson, 1986, Int. Rev. Cytol. 103, 118-119). On reexamination, in situ extracellular matrix (ECM) and cell processes on the medial face of the somites were found appropriately oriented for this function. In addition, tissue culture models of oriented ECM could induce orientation of NC cells which mimicked that observed in the embryo. It is concluded that in this situation, oriented structures contribute to directed migration of NC cells in vivo, but the mechanism of contact guidance (i.e., steric or adhesive guidance) could not be ascertained. Contact guidance, in the form of steric guidance, has also been suggested as limiting ventrad NC cell movement at the midbrain level due to an insurmountable ridge on the side of the midbrain. The presence of this ridge was confirmed but it is unlikely to be responsible for prevention of ventrad migration, because, although it subsides very rapidly, the cells still refuse to move ventrad, and because models of this ridge in vitro proved to be no obstacle to NC cells. NC cell migration is also described as being limited by gross space between other organs or tissues. In vitro, NC cells could penetrate Nucleopore filters with pore diameters of 0.86 micron or greater. Observation of cell-free spaces in embryos showed that these were almost all much larger than the minimum pore size established experimentally. It is therefore concluded that in general the dimensions of gross tissue spaces probably do not set important limits for NC cell migration, but that the dimensions of transiently distensible microspaces between ECM fibrils may be a critical physical parameter.     

Promotion of chromatophore differentiation in isolated premigratory neural crest cells by extracellular matrix material explanted on microcarriers

This study was undertaken to determine whether premigratory neural crest cells of the axolotl embryo differentiate autonomously into chromatophores, or whether stimuli from the environment, particularly from the extracellular matrix, are required for this process. Neural crest cells were excised from the dorsal part of the premigratory crest cord and cultured alone, either in a serum-free salt solution or in the presence of fetal calf serum (FCS), and together with explants of the neural tube or dorsal epidermis. A "microcarrier" technique was developed to assay the possible effects of subepidermal extracellular matrix (ECM) on chromatophore differentiation. ECM was adsorbed in vivo onto microcarriers prepared from Nuclepore filters, by inserting such carriers under the dorsolateral epidermis in the embryonic trunk. Neural crest cells were then cultured on the substrate of ECM deposited on the carriers. Melanophores were detected by DOPA incubation, revealing phenol oxidase activity, or by externally visible accumulation of melanin. Prospective xanthophores were visualized before they became overtly differentiated by alkali-induced pteridine fluorescence. Isolated premigratory neural crest cells did not transform autonomously into any of these phenotypes. Conversely, coculture with the neural tube or the dorsal epidermis, and also the initial presence or later addition of FCS during incubation, resulted in differentiation of neural crest cells into chromatophores. Both chromatophore phenotypes were also expressed on the ECM substrate deposited on the microcarriers. The results indicate that neural crest cells do not differentiate autonomously into melanophores and xanthophores, but that interactions with components of, or factors associated with the extra cellular matrix surrounding the premigratory neural crest and present along the dorsolateral migratory pathway are crucial for the expression of these chromatophore phenotypes in the embryo.     

Phycobilisome composition and possible relationship to reaction centers

The photosynthetic apparatus was studied in Anacystis nidulans wild type and in a spontaneous pigment mutant 85Y which had improved growth in far-red light (greater than 650 nm). Two phycobiliproteins, C-phycocyanin (lambda max 625) and allophycocyanin (lambda max 650), were present in a molar ratio of approximately 3:1 in the wild type and approximately 0.4:1 in the mutant. Phycobilisomes of wild type cells were larger (57 X 30 nm) than those of the mutant 85Y (28 X 15 nm). In the mutant they seemed to consist primarily of the allophycocyanin core. Fluorescence emission maxima of wild type and mutant 85Y phycobilisomes were at 680 nm (23 degrees C) and 685 nm (-196 degrees C). Excitation maxima of phycobilisomes were at 630 and 650 nm for the wild type and the mutant 85Y, respectively. The phycobilisomes of wild type cells whether grown in white or far-red light had the same size and pigment composition. A typical wild type cell in white light had a thylakoid area of 22.8 microns 2, but in far-red light the area was reduced to 13.5 microns 2, which was close to that of 85Y at 13.6 microns 2. Chlorophyll molecules per cell decreased in far-red light from 1.1 X 10(7) in wild type (white light) to 4.5 X 10(6) in mutant 85Y (far-red). The number of phycobilisomes per cell (approx 2 X 10(4)), calculated from the phycobiliprotein content and phycobilisome size, was about the same in wild type (white light) and mutant 85Y (far-red light), but the number of phycobilisomes per unit area of thylakoid was significantly greater in mutant 85Y than in wild type. The present results suggest that the phycobilisomes are linked with reaction centers and that the PSII complement (photo-system II and phycobilisome) was fully maintained in far-red light.     

Basal lamina is not a barrier to neural crest cell emigration: documentation by TEM and by immunofluorescent and immunogold labelling

One of the factors proposed to control initiation of migration of neural crest (NC) cells is disruption of the basal lamina (BL) that is presumed to exist over the dorsal portion of the neural tube. Previously, we discovered that, in the mouse embryo, a continuous BL is not deposited over the dorsal portion of the neural tube until emigration of the NC cells is terminated. Here, we show that the pattern of BL deposition in chick embryos is similar, but not identical, to that in the mouse. In particular, (i) patches of BL are deposited on the premigratory NC cells in the chick but not in the mouse and (ii) BL is thicker and more interstitial matrix is deposited at the same stage of development in the chick. In addition, immunofluorescent and immunogold labelling of collagen IV, laminin and fibronectin show that (i) patches of young BL contain all three molecules; (ii) collagen IV and laminin are present in BL throughout neurulation but fibronectin either disappears or becomes masked in more mature BL and (iii) collagen IV and especially fibronectin are present in the interstitial matrix, but the relative abundance of fibronectin changes with time. The simultaneous use of immunolabelling for both light and TEM sections has allowed us to determine unambiguously that presence of a basement membrane (light microscopy) does not necessarily imply presence of basal lamina. We conclude that, as in mouse, the BL cannot be involved in the timing of the initiation of migration of NC cells. Our evidence in both the mouse and the chick, together with work in the axolotl, suggests that the basic pattern of BL deposition during neurulation may be a general phenomenon in embryonic development. Moreover, these results, in conjunction with the work of others, suggest that the critical step for initiation of migration of NC cells may be the loss of adhesions between cells.     

Melanotropin as a Potential Regulator of Pigment Pattern Formation in Embryonic Skin

Frozen tissue sections of developing axolotl embryos were labeled by indirect immunofluorescence with anti-alpha-MSH. Anti-MSH immunoreactivity is first detectable in embryos when neural crest cells are migrating from the neural tube. Antibody labeling is visible around the lateral and ventral edges of the neural tube and in the embryonic ectoderm. As development progresses, the amount of labeling increases greatly, particularly in developing ectoderm. Western blots of soluble proteins extracted from various developmental stages of axolotl embryo ectoderm reveal that MSH activity is associated directly with several high molecular weight components that may be part of the embryonic extracellular matrix. Thus, we suggest that melanotropin activity is present in embryonic axolotl skin, is associated with the extracellular matrix, and is thereby in a position to play a supportive and/or directive role in the establishment of embryonic pigment patterns.     

Retarded nuclear migration in Drosophila embryos with aberrant F-actin reorganization caused by maternal mutations and by cytochalasin treatment

Three X-linked mutations of Drosophila melanogaster, gs(1)N26, gs(1)N441 and paralog, had a common maternal-effect phenotype. Mutant embryos show reduced egg contraction that normally occurs at an early cleavage stage in wild-type embryos. In addition, the mutants exhibited retarded nuclear migration while synchronous nuclear divisions were unaffected. The retarded migration causes nuclei to remain in the anterior part of the embryo retaining their spherical distribution even in a late cleavage stage. This consequently results in an extreme delay in nuclear arrival in the posterior periplasm. A mutant phenocopy was induced in wild-type embryos that were treated with cytochalasin B or D at a very early cleavage stage. Remarkable differences were noticed in the organization of cortical F-actin between the mutants and the wild type throughout the cleavage stage: obvious F-actin aggregates were dispersed in the cortex of mutant embryos, in contrast to the wild type where the cortical F-actin layer was smooth and underlying F-actin aggregates were smaller than those in the mutants; the transition of the distribution pattern of F-actin in the yolk mass, from the centralized to the fragmented type, occurred later in the mutants than in wild type. The results suggest that these mutations affect the mechanism underlying establishment and transition of F-actin organization required for normal egg contraction and nuclear migration in the cleavage embryos.     

Structure and interactions of aggrecans: statistical thermodynamic approach

Weak polyelectrolytes tethered to cylindrical surfaces are investigated using a molecular theory. These polymers form a model system to describe the properties of aggrecan molecules, which is one of the main components of cartilage. We have studied the structural and thermodynamical properties of two interacting aggrecans with a molecular density functional theory that incorporates the acid-base equilibrium as well as the molecular properties: including conformations, size, shape, and charge distribution of all molecular species. The effect of acidity and salt concentration on the behavior is explored in detail. The repulsive interactions between two cylindrical-shaped aggrecans are strongly influenced by both the salt concentration and the pH. With increasing acidity, the polyelectrolytes of the aggrecan acquire charge and with decreasing salt concentration those charges become less screened. Consequently the interactions increase in size and range with increasing acidity and decreasing salt concentration. The size and range of the forces offers a possible explanation to the aggregation behavior of aggrecans and for their ability to resist compressive forces in cartilage. Likewise, the interdigitation of two aggrecan molecules is strongly affected by the salt concentration as well as the pH. With increasing pH, the number of charges increases, causing the repulsions between the polymers to increase, leading to a lower interdigitation of the two cylindrical polymer layers of the aggrecan molecules. The low interdigitation in charged polyelectrolytes layers provides an explanation for the good lubrication properties of polyelectrolyte layers in general and cartilage in particular.     

原位杂交检测pal-1 mRNA在秀丽小杆线虫野生型和突变体早期胚胎中的分布

pal-1是秀丽小杆线虫(Caenorhabditis elegans)早期胚胎发育中决定体细胞命运的重要基因,也是转录因子,调控后续基因的表达,凡含有该基因表达的细胞发育成体细胞。本文通过整体原位杂交技术检测pal-1mRNA在C·elegans野生型和par-1、par-2、par-3、par-4突变体、spn-4突变体、mex-5/mex-6突变体早期胚胎中的分布,探讨这些基因在胚胎发育早期对pal-1mRNA的影响。实验结果表明:par-1、par-3、par-4突变使4细胞胚胎pal-1mRNA完全丧失了野生型不对称分布模式,pal-1mRNA分布在所有卵裂球中;par-2对pal-1mRNA的分布影响较小,在par-2突变体4细胞胚胎中pal-1mRNA分布与野生型相同。spn-4、mex-5、mex-6也能影响pal-1mRNA的分布,使其分布丧失不对称性。在par-1、par-4突变的情况下,pal-1mRNA广泛存在,但PAL-1蛋白也不表达,显示对pal-1mRNA的翻译调控是PAL-1蛋白空间和时序不对称分布的主要原因[动物学报51(5):884-891,2005]。