Pigment pattern formation in the larval salamander Ambystoma maculatum

As part of an ongoing comparative study of pigment patterns and their formation in embryos and larvae of ambystomatid salamanders, Ambystoma maculatum from two differnt populations, one in the northern (New York) and one in the central (Tennessee) United States, were investigated. Scanning electron microscopy was used to study early neural crest development. Light microscopy in combination with markers for the two pigment cell types (xanthophores and melanophores) made it possible to follow pigment cell migration before the pigment cells were fully differentiated. A bilateral pigment pattern consisting of two horizontal melanophore stripes surrounding an interstripe area populated by xanthophores formed in the larvae. In both populations, some variation was present in the form of a continuum ranging from clear horizontal stripes to extreme cases with a random pattern. Unlike the other ambystomatids that have been investigated, the neural crest cells in A. maculatum do not form aggregates and no vertical bars are formed. Instead, both the pattern and its formation are very similar to what has been reported for salamandrids. If pattern formation mechanisms can act as developmental constraints we would expect the A. maculatum pattern to be the primitive condition in the Ambystomatidae, using the Salamandridae as the outgroup. There is no strong support for this when aggregate formation is used as a character and mapped onto phylogenies for the group. The aggregate formation mechanism, and the pigment pattern that it leads to, have most likely been secondarily lost in A. maculatum. © 1993 Wiley-Liss, Inc.     

Dorsal ventral polarity and pattern formation in the Drosophila embryo

The establishment of polarity along the dorsal-ventral axis of the Drosophila embryo requires the graded distribution of the dorsal morphogen. Several maternal genes are responsible for the formation of the gradient and their products act in an ordered series of events that begins during oogenesis and involves two different cell types, the oocyte and the follicle cells. The last step in the series results in selective nuclear localization of dorsal proteins, dorsal is thought to regulate the expression of zygotic genes in a concentration dependent way. The zygotic genes determine cell fates in specific regions of the embryo and direct other genes involved in the processes of differentiation.     

Pigment pattern formation in larval ambystomatid salamanders: Ambystoma tigrinum tigrinum

We have begun a comparative study of pigment patterns and their mechanisms of formation in ambystomatid salamanders in an attempt to elucidate the evolution of these traits in this family. In Ambystoma t. tigrinum, the migration of the prospective pigment cells was followed by using scanning electron microscopy and light microscopy combined with markers (dopa incubation for detecting melanophores, ammonia-induced pterin fluorescence for detecting xanthophores). The pigment pattern resulting from the cell migration shares features both with the alternating vertical xanthophore and melanophore bars of A. mexicanum and the horizontal stripes of certain salamandrids and ambystomatids. The pigment pattern of A. t. tigrinum is interpreted here as an intermediate evolutionary step between a primitive horizontal stripe pattern and a derived vertical bar pattern. The initiation of pigment pattern formation resembles the situation in A. mexicanum, probably reflecting the close phylogenetic relationship between the two taxa.     

Pigment cell pattern formation in amphibian embryos: a reexamination of the dopa technique

Neural crest-derived melanophores form species-specific patterns in the dermis of amphibian embryos. Melanophore patterns may be generated by one of two general mechanisms: pigment cell precursors disperse throughout the embryo, with melanophores differentiating in certain regions due to environmental cues, or melanoblasts may localize in different regions as a result of a hierarchy of tissue affinities. Both of these mechanisms have been proposed to be responsible for the dorso-ventral patterning of melanophores in Xenopus laevis. We have reexamined the distribution of melanoblasts in X. laevis and Taricha torosa using the dopa (3,4-dihydroxyphenyl-alanine)-staining technique. We have found that many of the dopa-positive cells identified as melanoblasts by some researchers are actually not derived from the neural crest: dopa-positive cells in T. torosa were identified in the transmission electron microscope to be either leukocytes or erythrocytes, in X. laevis dopa-positive cells are found between the ectoderm and somites where neural crest cells are not found, and X. laevis embryos surgically depleted of neural crest have dopa-staining patterns identical to control embryos. Melanoblasts are apparently not found in the ventralmost regions of early T. torosa and X. laevis embryos, providing additional evidence for the role of differential tissue affinities in directing the formation of embryonic pigment cell patterns.     

Pigment formation in the dermatophytes

Avstract The moist mycelium ofT. tonsurans was extracted with 3 % w/v sodium hydroxide to yield a purple to violet solution which was passed through a cation-exchange resin column, evaporated partially, dialyzed and lyophilized. The lyophilized powder was extracted using absolute ethanol, acetone and chloroform (Process A) and separately by defatting with petroleum ether and n-hexane followed by chloroform-methanol 2:1 (Process B).Separation was achieved for the extract of Process B on thin layer chromatography using Silica Gel G and butanol-ethanol-water as the solvent system. Ultra-violet and visible spectrophotometric absorption data are reported for the lyophilized extract ofT. tonsurans, hydrolyzates of the lyophilized extract, and for sodium hydroxide extracts ofT. megnini, T. mentagrophytes, T. rubrum, T. tonsurans andT. violaceum. The spectra were similar in all cases.A review of pigment substance in dermatophytes is made, which together with the experimental data secured withT. tonsurans and previous experimentation, suggest the possible effects of drying procedures, contusion, solvents, acidic additives and method of extraction on the removal of pigment from the mycelium of dermatophytes. It is suggested that all the above conditions influence the substances ultimately extracted and that some alteration of a basic pigment association in the organism occurs to release pigment fragments or fractions of varying complexity. Oxidation and polymerization may influence the end result. It is possible that the mechanism presented can account for the number of pigment substances reportedly occurring in the dermatophytes and that a similar pigment structure or complex exists in other Trichophyton species.Grateful thanks are extended to the Medical Research Council of Canada for financial support.     

Absence of global genomic cytosine methylation pattern erasure during medaka (Oryzias latipes) early embryo development

Two techniques were used to analyze global genomic 5-methyl cytosine methylation at CCGG sites of medaka embryo DNA. DNA was labeled by incorporation of microinjected radiolabeled deoxynucleotide into one-cell embryos. After Hpa II or Msp I digestion the radiolabeled DNA was fractionated in agarose gels and the distribution of label quantified throughout each sample lane to detect differences in fragment distribution. Alternately isolated DNA was digested with Hpa II or Msp I and the resulting generated termini end-labeled. The end-labeled digestion products were then analyzed for fragment distribution after gel fractionation. These techniques proved to be extremely sensitive, allowing comparison of genomic DNA methylation values from as few as 640 fish cells. The data suggest that in medaka embryos the vast majority (>90%) of genomic DNA is methylated at CCGG sites. Furthermore, these data support the conclusion that the extent of methylation at these sites does not change or changes very little during embryogenesis (from 16 cells to the hatchling). These data argue against active demethylation, or loss of methylation patterns by dilution, during the developmental stages between the one cell zygote and gastrulation. From a comparative viewpoint, these data may indicate that mammals and fishes methylate and demethylate their genomes in very different manners during development.     

A pattern formation mechanism to control spatial organization in the embryo of Drosophila melanogaster

It is known that cells are already committed to a particular segment at the cellular blastoderm stage during embryogenesis of Drosophila melanogaster. Recently, several segmentation genes have been observed to be expressed in a sequence of banded spatial patterns in the syncytial blastoderm, prior to the formation of the cellular blastoderm. It is demonstrated in this paper that a two component reaction-diffusion (RD) system with net production functions which are antisymmetric with respect to the uniform steady-state values, is capable of producing a sequence of seven spatial patterns in the syncytial blastoderm. The sequence of patterns obtained exhibit a strong preference for banded or striped patterns. The first pattern is a simple anteroposterior gradient while the second is a gradient in the dorsoventral direction. The next five patterns are a sequence of banded patterns which exhibit frequency doubling, i.e. the number of bands in each pattern tend to be double the number in the previous pattern. The predicted pattern sequence is comparable to that observed in the expression of some segmentation genes. It is suggested that a pattern formation mechanism based on such an RD system may exist in the embryo where it produces a sequence of prepatterns to regulate the expression of various segmentation genes leading ultimately to a segmented embryo. There is sufficient spatial information in the sequence of banded prepatterns for the segments to be unique.     

Pigment cell mechanism of postembryonic stripe pattern formation in the Japanese four‐lined snake

Postembryonic changes in the dermal and epidermal pigment cell architecture of the striped and nonstriped morph of the Japanese four‐lined snake Elaphe quadrivirgata were examined to reveal stripe pattern formation after hatching. The striped and nonstriped morphs were distinguishable at the hatching, suggesting that the basis of stripe pattern was formed during embryonic development. In the striped morph, the color of stripes changed from red‐brown in juveniles to vivid dark‐brown in adults, and density of dermal melanophore increased much more in the stripe than background dorsal scales with growth. This increase in density of dermal melanophore was accompanied not only by the increased epidermal melanophore density but also by the change in vertical structures of dermal melanophore. By contrast, the density of epidermal and dermal melanophore evenly increased over the dorsal scales in the nonstriped morph. Thus, the increased vividness of the stripe pattern after hatching is achieved through localized increase of melanophore density particularly in the stripe region but not over the whole dorsal scales. J. Morphol. 277:196–203, 2016. © 2015 Wiley Periodicals, Inc.     

Pigment epithelium of the eye in the human embryo in vitro

A model is developed and described for studying cytological differentiation of pigment epithelium cells of the human eye in vitro. It is shown that the differentiation pattern depends on the stage of embryogenesis.     

Cell lineage analysis of pattern formation in the Tubifex embryo. II. Segmentation in the ectoderm

Ectodermal segmentation in the oligochaete annelid Tubifex is a process of separation of 50-microm-wide blocks of cells from the initially continuous ectodermal germ band (GB), a cell sheet consisting of four bandlets of blast cells derived from ectoteloblasts (N, O, P and Q). In this study, using intracellular lineage tracers, we characterized the morphogenetic processes that give rise to formation of these ectodermal segments. The formation of ectodermal segments began with formation of fissures, first on the ventral side and then on the dorsal side of the GB; the unification of these fissures gave rise to separation of a 50-microm-wide block of approximately 30 cells from the ectodermal GB. A set of experiments in which individual ectoteloblasts were labeled showed that as development proceeded, an initially linear array of blast cells in each ectodermal bandlet gradually changed its shape and that its contour became indented in a lineage-specific manner. These morphogenetic changes resulted in the formation of distinct cell clumps, which were separated from the bandlet to serve as segmental elements (SEs). SEs in the N and Q lineages were each comprised of clones of two consecutive primary blast cells. In contrast, in the O and P lineages, individual blast cell clones were distributed across SE boundaries; each SE was a mixture of a part of a more anterior clone and a part of the next more posterior clone. Morphogenetic events, including segmentation, in an ectodermal bandlet proceeded normally in the absence of neighboring ectodermal bandlets. Without the underlying mesoderm, separated SEs failed to space themselves at regular intervals along the anteroposterior axis. We suggest that ectodermal segmentation in Tubifex consists of two stages, autonomous morphogenesis of each bandlet leading to generation of SEs and the ensuing mesoderm-dependent alignment of separated SEs.     

The role of tolloid/mini fin in dorsoventral pattern formation of the zebrafish embryo.

A highly conserved TGF-&bgr; signaling pathway is involved in the establishment of the dorsoventral axis of the vertebrate embryo. Specifically, Bone Morphogenetic Proteins (Bmps) pattern ventral tissues of the embryo while inhibitors of Bmps, such as Chordin, Noggin and Follistatin, are implicated in dorsal mesodermal and neural development. We investigated the role of Tolloid, a metalloprotease that can cleave Chordin and increase Bmp activity, in patterning the dorsoventral axis of the zebrafish embryo. Injection of tolloid mRNA into six dorsalized mutants rescued only one of these mutants, mini fin. Through chromosomal mapping, linkage and cDNA sequence analysis of several mini fin alleles, we demonstrate that mini fin encodes the tolloid gene. Characterization of the mini fin mutant phenotype reveals that Mini fin/Tolloid activity is required for patterning ventral tissues of the tail: the ventral fin, and the ventroposterior somites and vasculature. Gene expression studies show that mfn mutants exhibit reduced expression of ventrally restricted markers at the end of gastrulation, suggesting that the loss of ventral tail tissues is caused by a dorsalization occurring at the end of gastrulation. Based on the mini fin mutant phenotype and the expression of tolloid, we propose that Mini fin/Tolloid modifes the Bmp activity gradient at the end of gastrulation, when the ventralmost marginal cells of the embryo are in close proximity to the dorsal Chordin-expressing cells. At this time, unimpeded Chordin may diffuse to the most ventral marginal regions and inhibit high Bmp activity levels. In the presence of Mini fin/Tolloid, however, Chordin activity would be negatively modulated through proteolytic cleavage, thereby increasing Bmp signaling activity. This extracellular mechanism is amplified by an autoregulatory loop for bmp gene expression.     

The maternal gene nanos has a central role in posterior pattern formation of the Drosophila embryo

A group of maternal genes, the posterior group, is required for the development of the abdominal region in the Drosophila embryo. We have used genetic as well as cytoplasmic transfer experiments to order seven of the posterior group genes (nanos, pumilio, oskar, valois, vasa, staufen and tudor) into a functional pathway. An activity present in the posterior pole plasm of wild-type embryos can restore normal abdominal development in posterior group mutants. This activity is synthesized during oogenesis and the gene nanos most likely encodes this activity. The other posterior group genes have distinct accessory functions: pumilio acts downstream of nanos and is required for the distribution or stability of the nanos-dependent activity in the embryo. Staufen, oskar, vasa, valois and tudor act upstream of nanos. Embryos from females mutant for these genes lack the specialized posterior pole plasm and consequently fail to form germ-cell precursors. We suggest that the products of these genes provide the physical structure necessary for the localization of nanos-dependent activity and of germ line determinants.     

Global cell sorting in the C. elegans embryo defines a new mechanism for pattern formation

4D microscopic observations of Caenorhabditis elegans development show that the nematode uses an unprecedented strategy for development. The embryo achieves pattern formation by sorting cells, through far-ranging movements, into coherent regions before morphogenesis is initiated. This sorting of cells is coupled to their particular fate. If cell identity is altered by experiment, cells are rerouted to positions appropriate to their new fates even across the whole embryo. This cell behavior defines a new mechanism of pattern formation, a mechanism that is also found in other animals. We call this new mechanism "cell focusing". When the fate of cells is changed, they move to new positions which also affect the shape of the body. Thus, this process is also important for morphogenesis.     

Pattern formation in the Drosophila embryo

Three plausible hypotheses about developmental commitments in the Drosophila embryo propose that: (1) a micromosaic of localized determinants in the egg trigger somatic commitments; (2) monotonic anterior-posterior and dorsal-ventral gradients in the egg specify positions by a series of threshold values; (3) sequential subdivision of the early embryo into 'anterior' or 'posterior' 'middle' or 'end', 'dorsal' or 'ventral', 'odd' or 'even' compartmental domains encodes the somatic commitment in each region in a combinatorial epigenetic code. Evidence in favour of such a combinatorial code includes its capacity to account for major features of transdetermination and for many single and coordinated homoeotic transformations. In particular, both these metaplasias often cause transformations between ectodermal tissues such as antenna and genitalia, whose anlagen lie far apart on the blastoderm fate map. This phenomenon is not naturally explained by monotonic gradient models. In contrast, not only transformation between distant regions of the fate map, but also the observed geometries of compartmental boundaries on the wing, and probable ones in the early embryo, are naturally explained by reaction-diffusion models. These systems form a discrete succession of differently shaped monotonic and nonmonotonic eigenfunction gradient patterns of the same morphogens, as the tissue containing the chemical system changes in size and shape, or in other parameters. The successive mirror symmetries in non-monotonic gradients predict that distant regions of the embryo make similar developmental commitments, and also predict specific classes of pattern mutants forming mirror symmetric structures along the embryo on a variety of length scales. Finally, reaction diffusion systems spontaneously generate transverse gradients of the underlying chemicals when more than one eigenfunction is amplified at once, and therefore specify two-dimensional positional information within domains. Although it is attractive, no feature of the combinatorial code hypothesis is verified. Current data relating to whether the sequential formation of compartmental boundaries actually reflects the commitment of the two isolated 'polyclones' to alternative fates, whether any genes act continuously to maintain disc commitments, and whether homoeotic mutants actually 'switch' disc determined states, are assessed.     

Cell lineage analysis of pattern formation in the Tubifex embryo. I. Segmentation in the mesoderm.

Annelids are strongly segmented animals that display a high degree of metamerism in their body plan. The embryonic origin of metameric segmentation was examined in an oligochaete annelid Tubifex using lineage tracers. Segmental organization arises sequentially in the anterior-to-posterior direction along the longitudinal axis of the mesodermal germ band, a coherent column of primary blast cells that are produced from the mesodermal teloblast. Shortly after its birth, each primary blast cell undergoes a spatiotemporally stereotyped sequence of cell divisions to generate three classes of cells (in terms of cell size), which together give rise to a distinct cell cluster. Each cluster is composed of descendants of a single primary blast cell; there is no intermingling of cells between adjacent clusters. Relatively small-sized cells in each cluster become localized at its periphery, and they form coelomic walls including an intersegmental septum to establish individuality of segments. A set of cell ablation experiments showed that these features of mesodermal segmentation are not affected by the absence of the overlying ectodermal germ band. These results suggest that each primary blast cell serves as a founder cell of each mesodermal segment and that the boundary between segments is determined autonomously. It is concluded that the metameric body plan of Tubifex arises from an initially simple organization (i.e., a linear series) of segmental founder cells.     

Development of the pancreas in medaka,Oryzias latipes,from embryo to adult

To address conserved and unique features of fish pancreas development, we performed extensive analyses of pancreatic development in medaka embryos and adults using pdx1‐ and ptf1a‐transgenic medaka, in situ hybridization and immunohistochemistry. The markers used in these analyses included pdx1, nkx6.1, nkx6.2, nkx2.2, Islet1, insulin, Somatostatin, glucagon, ptf1a, ela3l, trypsin, and amylase. The double transgenic (Tg) fish produced in the present study visualizes the development of endocrine (pdx1+) and exocrine (ptf1a+) parts simultaneously in living fishes. Like other vertebrates, the medaka pancreas develops as two (dorsal and ventral) buds in the anterior gut tube, which soon fuse into a single anlagen. The double Tg fish demonstrates that the differential property between the two buds is already established at the initial phase of bud development as indicated by strong pdx1 expression in the dorsal one. This Tg fish also allowed us to examine the gross morphology and the structure of adult pancreas and revealed unique characters of medaka pancreas such as broad and multiple connections with the gut tube along the anterior–posterior axis.