Differential expression of creatine kinase isozymes during development of Xenopus laevis: An unusual heterodimeric isozyme appears at metamorphosis

The creatine kinase (CK) repertoire of Xenopus laevis, which is more complex than that of most other vertebrates, involves at least four genomic loci, all showing developmental and tissue-specific expression. The differential expression of this multilocus CK isozyme system was investigated by immunohistology. Specific monoclonal antibodies (mAb) against the three cytoplasmic CK isozymes of Xenopus laevis were isolated and characterized. Two of these mAbs, anti-CK-IV (DM16) and anti-CK-III (JRM4), were specific for CK-IV and CK-III subunits respectively, as well as for the corresponding homodimeric isozymes, CK-IV/IV and CK-III/III. Anti-CK-II (MRX7) mAb recognizes CK-II subunits and CK-II/III heterodimers; the homodimeric CK-II/III does not occur. Immunohistological localization on larval and adult tissue sections reveals that CK-IV epitopes, beside a generalized tissue distribution, are especially concentrated in the cytoplasm of some particular cells such as the photoreceptors in the outer segment of the retina, certain nerve cells of the spinal cord and spinal ganglia, and in larval hepatocytes. The CK-III III isozyme is specifically expressed in skeletal muscle, its appearance and accumulation occurring in parallel with myoblast differentiation. The CK-II antigen is detected first at the time of metamorphosis is skeletal muscles, as well as in the heart, eyes and brain. In striated musculature the expression of CK-II subunits during metamorphosis results in almost complete replacement of CK-III/III homodimers by CK-II/III heterodimers, as indicated by the progressive masking of CK-III epitope and the corresponding appearance of CK-II antigen. In the adult eyes, CK-II antigens localize at the same particular site of photoreceptors as do CK-IV antigens. Since that antigen represents a heterodimeric CK-II/III isozyme, this implies the activation of both CK-II and CK-III genes, none of which is expressed in larval retina.     

Purification and characterization of cytoplasmic creatine kinase isozymes ofXenopus laevis

The soluble creatine kinase isozymes CK-II, CK-III, and CK-IV fromXenopus laevis have been purified to apparent homogeneity and their subunits characterized by means of molecular weight, peptide pattern, and dissociation-reassociation experiments. CK-III and CK-IV are homodimeric isozymes whose subunits are distinct in both molecular weight (42,000 and 41,000, respectively) andStaphylococcus aureus V₈ peptide pattern. In dissociation-reassociation experiments, those two subunits do form active heterodimeric isozymes with one another or with rabbit M-CK subunits. Hybrid CK-III/IV isozymes occur also during embryonic differentiation and in adult heart muscle, whereas most other adult tissues contain only homodimeric CK-III or CK-IV isozymes. The CK-II isozyme is a heterodimer composed of one CK-III subunit and another subunit specific to CK-II (M _r =41,000). Neitherin vivo norin vitro does this subunit seem able to form homodimers or heterodimers with CK-IV and rabbit M-CK subunits. If we take into account the apparent association of CK-I isozyme with cellular organelles, these results corroborate earlier statements and suggest that the CK isozyme system ofX. laevis is encoded by at least four differentially regulated genomic loci.     

Two transitions of haemoglobin expression in Xenopus: from embryonic to larval and from larval to adult

Electrophoretic analyses of haemoglobin and globin phenotypes in families of Xenopus borealis and Xenopus l. laevis revealed two developmental haemoglobin transitions during ontogeny. The first transition occurs at the developmental stage when tadpoles begin to feed. It is characterized by the decline of embryonic-specific globins in favour of novel, tadpole-specific globins (X. borealis) correlated to changes in the haemoglobin pattern. We suppose that this switch results from the replacement of a primitive, ventral blood island-dependent erythrocyte population by tadpole erythrocytes from other erythropoietic sites. Several other globin chains and haemoglobins are present in both young tadpoles and throughout larval life. The second, well-known transition occurs during metamorphosis, where all tadpole haemoglobins are replaced by adult haemoglobins composed of entirely different globin chains.     

Two Transitions of Haemoglobin Expression in Xenopus: from Embryonic to Larval and from Larval to Adult

Abstract. Electrophoretic analyses of haemoglobin and globin phenotypes in families of Xenopus borealis and Xenopus l. laevis revealed two developmental haemoglobin transitions during ontogeny. The first transition occurs at the developmental stage when tadpoles begin to feed. It is characterized by the decline of embryonic-specific globins in favour of novel, tadpole-specific globins ( X. borealis ) correlated to changes in the haemoglobin pattern. We suppose that this switch results from the replacement of a primitive, ventral blood island-dependent erythrocyte population by tadpole erythrocytes from other erythropoietic sites. Several other globin chains and haemoglobins are present in both young tadpoles and throughout larval life. The second, well-known transition occurs during metamorphosis, where all tadpole haemoglobins are replaced by adult haemoglobins composed of entirely different globin chains.     

Expression of Xenopus tropicalis noggin1 and noggin2 in early development: two noggin genes in a tetrapod

We report the identification of two distinct noggin genes in the tetrapod Xenopus tropicalis. Noggin functions to antagonize BMP signaling in many developmental contexts, and much work has explored its role in early vertebrate development. We have identified two noggin genes in the tropical clawed frog, X. tropicalis, a diploid anuran which is being explored for its potential as a genetic model system for early vertebrate development. Here we report the cloning and characterization of the Xenopus tropicalis noggin1 and noggin2 genes, which have distinct expression domains in the early embryo with one overlapping domain in the anterior neural tissue. X. tropicalis noggin1 expression is very similar to that of noggin in Xenopus laevis, with expression beginning in the blastula organizer region and continuing through gastrulation and neurulation in the organizer and notochord. Later, it is also expressed in the anterior neural ridge and subsequent forebrain; noggin1 is also expressed in the pharyngeal arches after neural tube closure. At the tadpole stage expression is maintained in the dorsal neural tube and is present in the otic vesicle. However, the expression of noggin2 is much more similar to the expression of noggin2 in D. rerio with expression in the forebrain, hindbrain, and somites, but unlike D. rerio, X. tropicalis noggin2 is expressed in the heart by stage 28. This work presents the first example of a tetrapod with at least two noggin genes.     

Experimental analysis of lens-forming capacity in Xenopus borealis larvae

Previously, the only anuran amphibians known to have the capacity to regenerate a lens after lentectomy were Xenopus laevis and Xenopus tropicalis. This regeneration process occurs during the larval life through transdifferentiation of the outer cornea promoted by inductive factors produced by the retina and accumulated inside the vitreous chamber. However, the capacity of X. tropicalis to regenerate a lens is much lower than that of X. laevis. This study demonstrates that Xenopus borealis, a species more closely related to X. laevis than to X. tropicalis, is not able to regenerate a lens after lentectomy. Nevertheless, some morphological modifications corresponding to the first stages of lens regeneration in X. laevis were observed in the outer cornea of X. borealis. This suggested that in X borealis the regeneration process was blocked at early stages. Results from histological analysis of X. borealis and X. laevis lentectomized eyes and from implantation of outer cornea fragments into the vitreous and anterior chambers demonstrated that: (i) in X. borealis eye, the lens-forming competence in the outer cornea and inductive factors in the vitreous chamber are both present, (ii) no inhibiting factors are present in the anterior chamber, the environment where lens regeneration begins, (iii) the inability of X. borealis to regenerate a lens after lentectomy is due to an inhibiting action exerted by the inner cornea on the spreading of the retinal factor from the vitreous chamber towards the outer cornea. This mechanical inhibition is assured by two distinctive features of X. borealis eye in comparison with X. laevis eye: (i) a weaker and slower response to the retinal inducer by the outer cornea; (ii) a stronger and faster healing of the inner cornea. Unlike X. tropicalis and similar to X. laevis, in X. borealis the competence to respond to the retinal factor is not restricted to the corneal epithelium but also extends to the pericorneal epidermis.     

Are there major developmentally regulated H4 gene classes in Xenopus?

Primer extension analysis has been used to study the principal H4 mRNAs present at different developmental stages and in several adult tissues of Xenopus borealis and X. laevis. In X. borealis a single sequence class predominates in oocytes, tadpoles and cultured fibroblasts. There is also a polymorphic minor type which shows no developmental regulation. The primer extension bands obtained from adult liver and kidney RNA appear to be the same as ovary and therefore these tissues almost certainly contain the same major H4 mRNA species. This is confirmed by S1 mapping of the 3' end of the mRNA. Thus for H4 genes in X. borealis there is no evidence of the kind of switches in histone gene expression seen in sea urchins or certain protostomes. The situation in X. laevis is complicated by considerably higher gene variability both within and between individuals. Nevertheless, in this species, as in X. borealis, there seems to be no major developmental switch in the regulation of H4 gene expression, a conclusion that also holds for an H1C and an H3 gene.     

Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.

The accessibility of DNA in chromatin to psoralen was assayed to compare the chromatin structure of the rRNA coding and spacer regions of the two related frog species Xenopus laevis and Xenopus borealis. Isolated nuclei from tissue culture cells were photoreacted with psoralen, and the extent of cross-linking in the different rDNA regions was analyzed by using a gel retardation assay. In both species, restriction fragments from the coding regions showed two distinct extents of cross-linking, indicating the presence of two types of chromatin, one that contains nucleosomes and represents the inactive gene copies, and the other one which is more cross-linked and corresponds to the transcribed genes. A similar cross-linking pattern was obtained with restriction fragments from the enhancer region. Analysis of fragments including these sequences and the upstream portions of the genes suggests that active genes are preceded by nonnucleosomal enhancer regions. The spacer regions flanking the 3' end of the genes gave different results in the two frog species. In X. borealis, all these sequences are packaged in nucleosomes, whereas in X. laevis a distinct fraction, presumably those flanking the active genes, show a heterogeneous chromatin structure. This disturbed nucleosomal organization correlates with the presence of a weaker terminator at the 3' end of the X. laevis genes compared with those of X. borealis, which allows polymerases to transcribe into the downstream spacer.     

Lung specific developmental expression of the Xenopus laevis surfactant protein C and B genes

Efforts to characterize the mechanisms underlying early lung development have been confounded by the absence of a model that permits study of lung development prior to the onset of endodermal differentiation. Since Xenopus laevis development occurs in an extrauterine environment, we sought to determine whether the classical molecular markers of lung development and function, surfactant protein genes, are expressed in X. laevis. Surfactant protein C (SP-C) is a specific marker for lung development, expressed early in development and exclusively in the lung. Surfactant protein B (SP-B) expression is essential for life, as its absence results in neonatal death in mice and gene mutations have been associated with neonatal respiratory failure in humans. Here, we report the cloning of the first non-mammalian SP-C and SP-B genes (termed xSP-C and xSP-B) using the Xenopus model. The processed mature translated regions of both xSP-C and xSP-B have high homology with both human and mouse genes. xSP-C and xSP-B are both expressed throughout the lung of the X. laevis swimming tadpoles soon after the initiation of lung development as assessed by RT-PCR and whole mount in situ hybridization. The temporal expression patterns of xSP-C and xSP-B are consistent with the expression patterns in mammalian models of lung development. In both the tadpole and the adult X. laevis, xSP-C and xSP-B are expressed only in lung. Knowledge of the sequence and expression pattern of these two surfactant proteins in Xenopus might allow for use of this organism to study early lung development.     

Chromosomal mapping of Xenopus 5S genes: somatic-type versus oocyte-type.

Xenopus 5S RNA genes exhibit a pattern of differential expression during development in which some members (oocyte-type) are transcribed only in oocytes, while others (somatic-type) are expressed in both oocytes and somatic cells. Using cloned DNA probes specific for each gene type, we determined the positions of these genes on Xenopus metaphase chromosomes by in situ hybridization. Somatic-type 5S genes in both X. laevis and X. borealis are located at the distal end of the long arm of only one chromosome (number 9). The oocyte-type 5S RNA genes are found at the distal ends of the long arms of most Xenopus chromosomes, including chromosome 9. Thus, large scale differences in chromosomal location cannot explain the selective expression of these genes, as suggested previously.     

Toward an unbiased evolutionary platform for unraveling Xenopus developmental gene networks

The availability of both the Xenopus tropicalis genome and the soon to be released Xenopus laevis genome provides a solid foundation for Xenopus developmental biologists. The Xenopus community has presently amassed expression data for ～2,300 genes in the form of published images collected in the Xenbase, the principal Xenopus research database. A few of these genes have been examined in both X. tropicalis and X. laevis and the cross-species comparison has been proven invaluable for studying gene function. A recently published work has yielded developmental expression profiles for the majority of Xenopus genes across fourteen developmental stages spanning the blastula, gastrula, neurula, and the tail-bud. While this data was originally queried for global evolutionary and developmental principles, here we demonstrate its general use for gene-level analyses. In particular, we present the accessibility of this dataset through Xenbase and describe biases in the characterized genes in terms of sequence and expression conservation across the two species. We further indicate the advantage of examining coexpression for gene function discovery relating to developmental processes conserved across species. We suggest that the integration of additional large-scale datasets--comprising diverse functional data--into Xenbase promises to provide a strong foundation for researchers in elucidating biological processes including the gene regulatory programs encoding development.     

A new method to remove hybridization bias for interspecies comparison of global gene expression profiles uncovers an association between mRNA sequence divergence and differential gene expression in Xenopus

The recent sequencing of a large number of Xenopus tropicalis expressed sequences has allowed development of a high-throughput approach to study Xenopus global RNA gene expression. We examined the global gene expression similarities and differences between the historically significant Xenopus laevis model system and the increasingly used X.tropicalis model system and assessed whether an X.tropicalis microarray platform can be used for X.laevis. These closely related species were also used to investigate a more general question: is there an association between mRNA sequence divergence and differences in gene expression levels? We carried out a comprehensive comparison of global gene expression profiles using microarrays of different tissues and developmental stages of X.laevis and X.tropicalis. We (i) show that the X.tropicalis probes provide an efficacious microarray platform for X.laevis, (ii) describe methods to compare interspecies mRNA profiles that correct differences in hybridization efficiency and (iii) show independently of hybridization bias that as mRNA sequence divergence increases between X.laevis and X.tropicalis differences in mRNA expression levels also increase.     

A reliable new cell marker in Xenopus

A new reliable and durable method for marking cells in Xenopus is described. It is based on the differential staining of the nuclei of different Xenopus species, e.g., X. laevis and X. borealis, with the fluorescent dye quinacrine. This method permits us to recognize with certainty each cell in mitosis and interphase of X. borealis origin in any tissue combination with most of the other Xenopus species tested so far. This holds for all stages of development following grafting experiments, including adult tissues. The method is applicable in smears and squash preparations as well as in microtome sections. The method is particularly useful for marking migrating cells which are difficult to track, for instance, in embryos and in the circulatory system.