Expression profile of rrbp1 genes during embryonic development and in adult tissues of Xenopus laevis

Recent studies suggest that ribosome-binding protein 1 (RRBP1) is involved in multiple diseases such as tumorigenesis and cardiomyopathies. However, its function during embryonic development remains largely unknown. We searched Xenopus laevis database with human RRBP1 protein sequence and identified two cDNA sequences encoding Xenopus orthologs of RRBP1 including rrbp1a (NM_001089623) and rrbp1b (NM_001092468). Both genes were firstly detected at blastula stage 8 with weak signals in animal hemisphere by whole mount in situ hybridization. Evident expression of rrbp1 was mainly detected in cement gland and notochord at neurula and tailbud stages. Heart expression of rrbp1 was detected at stage 36. RT-PCR results indicated that very weak expression of rrbp1a was firstly detected in oocytes, followed by increasing expression until stage 39. Differently, very weak expression of rrbp1b was firstly observed at stage 2, and then maintained at a lower level to stage 17 followed by an intense expression from stages 19–39. Moreover, both expression profiles were also different in adult tissues. This study reports Xenopus rrbp1 expression during early embryonic development and in adult tissues. Our study will facilitate the functional analysis of Rrbp1 family during embryonic development.     

Expression of muscle LIM protein during early development in Xenopus laevis

We have isolated the Xenopus homologue of Muscle LIM protein (MLP, CRP3) and examined its expression during early embryonic development. MLP is only expressed in the differentiated heart during early development and is expressed in a subset of other striated muscles during later stages. There is no MLP expression during primary myogenesis in the somites, although it is found in adult skeletal muscle.     

Expression of endogenous and microinjected hsp 30 genes in early Xenopus laevis embryos

In the present study, we have examined the regulation of expression of a newly isolated member of the hsp 30 gene family, hsp 30C. Using RT-PCR, we found that this gene was first heat-inducible at the tailbud stage of development. We also examined the expression of two microinjected modified hsp 30C gene constructs in Xenopus embryos. One of the constructs had 404 bp of hsp 30C 5′-flanking region, whereas the other had 3.6 kb. Both gene constructs had 1 kb of 3′-flanking region. RT-PCR assays were employed to detect the expression of these microinjected genes. The presence of extensive 5′- and 3′-flanking regions of the hsp 30C gene did not confer proper developmental regulation, since heat-inducible expression of both of the microinjected constructs was detectable at the midblastula stage. The premature expression of the microinjected hsp 30 gene was not a result of high plasmid copy number or the presence of plasmid DNA sequences. These results suggest that the microinjected genes contain all the cis-acting DNA sequences required for correct heat-inducible regulation but do not contain the elements required for the proper regulation of hsp 30 gene expression during development. It is possible that regulatory elements controlling the developmental expression of the hsp30 genes may reside upstream or downstream of the entire cluster. © 1993Wiley-Liss, Inc.     

Expression patterns of Ephs and ephrins throughout retinotectal development in Xenopus laevis

The Eph family of receptor tyrosine kinases and their ligands the ephrins play an essential role in the targeting of retinal ganglion cell axons to topographically correct locations in the optic tectum during visual system development. The African claw-toed frog Xenopus laevis is a popular animal model for the study of retinotectal development because of its amenability to live imaging and electrophysiology. Its visual system undergoes protracted growth continuing beyond metamorphosis, yet little is known about ephrin and Eph expression patterns beyond stage 39 when retinal axons first arrive in the tectum. We used alkaline phosphatase fusion proteins of EphA3, ephrin-A5, EphB2, and ephrin-B1 as affinity probes to reveal the expression patterns of ephrin-As, EphAs, ephrin-Bs, and EphBs, respectively. Analysis of brains from stage 40 to adult frog revealed that ephrins and Eph receptors are expressed throughout development. As observed in other species, staining for ephrin-As displayed a high caudal to low rostral expression pattern across the tectum, roughly complementary to the expression of EphAs. In contrast with the prevailing model, EphBs were found to be expressed in the tectum in a high dorsal to low ventral gradient in young animals. In animals with induced binocular tectal innervation, ocular dominance bands of alternating input from the two eyes formed in the tectum; however, ephrin-A and EphA expression patterns were unmodulated and similar to those in normal frogs, confirming that the segregation of axons into eye-specific stripes is not the consequence of a respecification of molecular guidance cues in the tectum.     

Reiterated transfer RNA genes of Xenopus laevis

The proportion of the Xenopus laevis genome complementary to “7 S” RNA, unfractionated transfer RNA and some selected aminoacyl-tRNAs, and the sequence complexity of these RNA species, have been determined by following the kinetics of RNA-DNA hybridization on filters under conditions of RNA excess at optimum rate temperature. For hybridization of aminoacyl-labelled tRNAs, conditions for optimum aminoacylation were first determined and, where necessary, aminoacyl-tRNAs were treated with nitrous acid to prevent discharge during annealing. Neither the extent nor rate of hybridization was affected by this treatment.“7 S” RNA, coded for by 580 genes per haploid complement of chromosomes, reacts like a single family of nucleotide sequences, whereas about 43 basic tRNA sequences are coded for by at least 7800 genes. If hybrids are not treated with RNase A, the apparent tDNA redundancy is some 23% greater but no more nucleotide sequences are detectable. Taken together, the results suggest that each tRNA sequence is, on average, 200-fold reiterated.The reiteration varies, however, for different aminoacyl-tRNAs. Thus, hybridization resolves only one valyl-tRNA which is coded for by 240 genes, but at least four leucyl-tRNA sequences can be distinguished by hybridization, each of which is on average 90-fold reiterated. Reiteration also varies for the two methionyl-tRNAs detectable both by hybridization and by reversed phase chromatography: tRNA₁^Met and tRNA₂^Met are 310- and 170-fold reiterated, respectively, and each is kinetically homogeneous. These saturation values are almost exactly additive and are not influenced by the presence of other tRNA species. Thus the results suggest that Xenopus tRNAs are no more heterogeneous than would be predicted by the genetic code, despite the high but variable multiplicity of tRNA cistrons.     

Expression of the N-myc proto-oncogene during the early development of Xenopus laevis

The N-myc proto-oncogene is expressed in a wide range of tissues during mammalian embryogenesis. This observation, along with the oncogenic capacity of this gene, has led to the suggestion that N-myc plays an important role in early development. However, due to the complexity of the expression pattern and the difficulty of manipulating mammalian embryos, little progress has been made towards understanding the developmental function of this gene. To enable a more detailed analysis of the role of this gene in early development, a study of the Xenopus homologue of N-myc was undertaken. Xenopus N-myc cDNA clones were isolated from a neurula library using a murine N-myc probe. Analysis of the timing of expression of N-myc mRNA and of the distribution of N-myc protein during Xenopus development indicate that this gene may be playing an important role in the formation of a number of embryonic structures, including the nervous system. N-myc is initially expressed as a maternal RNA, but this mRNA is degraded by the gastrula stage of development. Zygotic expression does not commence until late neurula. Examination of the distribution of the N-myc protein by whole-mount immunohistochemistry indicates that the early embryonic expression occurs in the central nervous system, the neural crest, the somites and the epidermis. Later expression is mostly within the head and somites. Specific structures within the head that express the protein include the eye, otic vesicle, fore and hindbrain and a number of cranial nerves. The results demonstrate that while N-myc is expressed in the developing nervous system of Xenopus, the timing of expression indicates that it is unlikely to be involved in regulation of the very first stages of neurogenesis.     

Expression analysis of the peroxiredoxin gene family during early development in Xenopus laevis

Development in the frog, Xenopus laevis, requires the utilization of yolk glyco-lipo-proteins in a temporally- and spatially-dependent manner. The metabolism of the yolk produces hydrogen peroxide (H₂O₂), a potent reactive oxygen species (ROS). Peroxiredoxins (prdxs) are a family of six anti-oxidant enzymes that, amongst other roles, reduce H₂O₂. Prdxs reduce H₂O₂ through a thiol-redox reaction at conserved cysteine residues which results in the creation of disulfide bonds. Recently the thiol-redox reaction of Prdxs has also been implicated in several cell signaling systems. Here we report the cloning and expression patterns during development of six peroxiredoxin homologs from the frog X. laevis. Sequence analysis confirmed their identity as well as their evolutionary relationship with peroxiredoxins from several other species. Using RT-PCR and in situ hybridization analysis we have shown that there is early and robust expression of all six homologs during development. All six X. laevis peroxiredoxins are expressed in neural regions including the brain, eyes, as well as the somites. Different expression patterns for each peroxiredoxin are also observed in the pronephric region, including the proximal and distal tubules. Expression of several peroxiredoxins was also observed in the blood precursors and the olfactory placode. These results suggest important roles for all six peroxiredoxins during early development. These roles may be restricted to their functions as anti-oxidant enzymes, but may also be related to their emerging roles in redox signaling.     

Expression of adult and tadpole specific globin genes from Xenopus laevis in transgenic mice.

Transgenic mice were generated which carried the adult alpha and beta-globin genes and the major tadpole specific beta-globin gene of Xenopus laevis. The adult specific alpha and beta genes were found to express in erythroid tissues in adult mice, while the major tadpole specific beta gene (beta T1) was expressed in blood from 12.5 day embryos. The pattern of expression of the beta T1 gene during mouse development was consistent with its being regulated as an embryonic globin gene in the mouse. This observation suggests that some of the factors mediating globin switching have been conserved during the evolution of modern amphibia and mammals and raises interesting questions concerning the evolution of vertebrate globin gene switching.     

Emerin expression in early development of Xenopus laevis

Emerin is an integral protein of the inner nuclear membrane in the majority of differentiated vertebrate cells. In humans, deficiency of emerin causes a progressive muscular dystrophy of the Emery-Dreifuss type. The physiological role of emerin is poorly understood. By screening and sequencing of EST clones we have identified two emerin homologues in Xenopus laevis, Xemerin1 and Xemerin2. Xemerins share with mammalian emerins the N-terminal LEM domain and a single transmembrane domain at the C-terminus. As shown by immunoblot analysis with Xemerin-specific antibodies, both proteins have an apparent molecular mass of 24 kDa but differ in their isoelectric points. Xemerin1 and Xemerin2 proteins are not detectable in oocytes nor during early embryogenesis. Protein expression is first found at stage 43 and persists in somatic cells. However, RT-PCR and Northern blot analysis show Xemerin mRNAs of approximately 4.0 kb to be present in oocytes and throughout embryogenesis. During embryogenesis the level of Xemerin mRNAs increases at stage 22 and is particularly abundant in mesodermal and neuro-ectodermal regions of the embryo. These data provide the necessary background to further investigate the role of emerin in nuclear envelope assembly, gene expression and organ development of X. laevis as a model organism.