Protein Changes in Regressing Tail Tissue of Xenopus laevis during Natural Metamorphosis

Sodium dodecyl sulfate (SDS) gel electrophoresis was used to study the soluble protein fraction of Xenopus laevis tail tissue during in vivo metamorphosis. Prior to morphological signs of tail regression stage 45, a new subunit protein was resolved. At stage 64 three additional subunit proteins were resolved at the end of tail resorption. Results indicate that the altered balance between protein synthesis and degradation has little effect on the protein subunit population prior to morphological signs of tail regression.     

Developmental immunohistology of melanotrophs in Xenopus laevis tadpoles

Summary The adenohypophysial primordium of Xenopus laevis tadpoles at stages 33/34 to 46 (Nieuwkoop and Faber, 1956) were examined immuno-histologically for -MSH, -MSH and ACTH. -MSH was demonstrated from stage 37/38 onwards, and -MSH from stage 39. No signs of ACTH production were detected. -MSH and -MSH occurred in the same cells. No differences were found in the intensity of immunofluorescence between tadpoles which were kept on a black and a white background. The present study lends no support to the hypothesis concerning the derivation of -MSH from ACTH. The observations made suggest that the morphological formation of the pars intermedia is accomplished during stages 37/38 to 39. Acknowledgement. The authors express warm thanks to Dr. M.P. Dubois (Laboratoire de Physiologie de la Reproduction, INRA, Nouzilly, France), who prepared and verified the antibodies. Grants from Swedish Natural Science Research Council and Landshovding Per Westlings minnesfond, Lund, Sweden are gratefully acknowledged     

Developmental expression of Shisa-2 in Xenopus laevis

Shisa is an antagonist of Wnt and FGF signaling, that functions cell autonomously in the endoplasmic reticulum (ER) to inhibit the post-translational maturation of Wnt and FGF receptors. In this paper we report the isolation of a second Xenopus shisa gene (Xshisa-2). Xenopus Shisa-2 shows 30.7% identity to Xshisa. RT-PCR analysis indicated that Xshisa-2 mRNA is present throughout early development and shows an increased expression during neurula and tailbud stages. At neurula stages Xenopus shisa-2 is initially expressed in the presomitic paraxial mesoderm and later in the developing somites. The expression profiles and pattern of Xshisa and Xshisa-2 differ significantly. During gastrulation only Xshisa mRNA is present in the Spemann-Mangold organizer and later on becomes restricted to the neuroectoderm and the prechordal plate.     

Metamorphosis alters the response to spinal cord transection in Xenopus laevis frogs

A series of studies has examined the response of the spinal cord to lesions made at various stages prior to and after metamorphic climax in the clawed frog Xenopus laevis. Complete transections made between Nieuwkoop and Faber (1956) stages 50 and 62 were followed by gradual recovery of righting and coordinated swimming as animals metamorphosed into juveniles (stage 66). Examination of descending axonal projections using horseradish peroxidase (HRP) showed fibers crossing the lesion site and distributing to the caudal lumbar spinal cord. These fibers could be traced from more rostral spinal segments as well as from brainstem injections of HRP. No evidence for rostrally projecting fibers crossing the lesion was obtained. Juvenile frogs of varying ages failed to demonstrate recovery of coordinated swimming or reconstitution of spinal descending pathways. In an additional series of animals, spinal transections were made within 1 or 2 days of tail resorption to assess whether regenerative capacities extended at all into post-metamorphic stages. No evidence for regeneration was found. Studies of metamorphosing frogs after spinal transections showed that fibers crossed the lesion within 5-12 days of transection, well prior to the end of metamorphic climax; however, in some cases in which metamorphosis seemed arrested, little regeneration was observed. Immunocytochemical studies showed that fibers containing serotonin (5-HT) were included in the population of axons that rapidly crossed the lesion after transection at metamorphic stages. These results are compared to those for lesions of the dorsal columns and other systems in developing and juvenile Xenopus. It is suggested that both metamorphosis-related hormonal changes, and axon substrate pathways, may affect the regenerative response in the Xenopus central nervous system (CNS).     

Developmental Fates of Blastomeres of Eight-Cell-Stage Xenopus laevis Embryos

The developmental fates of animal, vegetal, dorsal, and ventral egg regions of Xenopus laevis embryos were examined. For this purpose, a tracer enzyme (horseradish peroxidase) was injected bilaterally into pairs of eight-cell-stage blastomeres and the clonal organization of marked cells in the early tail-bud embryos was examined. The epidermis over most of the body originated from animal-ventral micromeres, but that in the head originated from animal-dorsal blastomeres and that in the area surrounding the anus originated from vegetal-ventral blastomeres. The neural tube originated from animal-dorsal, vegetal-dorsal, and animal-ventral blastomeres. These results were consistent with those of previous studies. But in contrast to previous findings, results showed that the entire notochord is derived from animal-dorsal micromeres and that the somites originate from all four bilateral pairs of blastomeres in the eight-cell stage. These results are discussed in relation to the current maps of prospective fates based on results of vital-dye staining. Morphogenetic movements are also discussed on the basis of the clonal organization demonstrated in the present study.     

Developmental pattern and molecular identification of globin chains in Xenopus laevis

Summary High-resolution electrophoresis of larval and adult hemoglobins of Xenopus laevis reveals stage-specific differences in the number and mobility of the globin chains. To establish the relationship between the globin chains and the previously described globin genes, the corresponding mRNAs were hybrid-selected from total erythroblast RNA by representative cDNA clones, and translated in vitro. Electrophoretic separation of the translation products allowed identification of a major and a minor -globin chain in the larval and adult stages. This also holds for the adult -chains, however in the larval stage a difference in abundance is only detectable in the -mRNAs, but not in the translation products, because they comigrate. The fact that major and minor globin chains can be assigned to genes, which are located in two clusters, suggests that the related genes are expressed coordinately, but at different levels. Analysis of the globin patterns during development reveals that transition from the larval to the adult globin chains coincides with metamorphosis. Moreover, there is evidence of two globin chains that are only expressed in early larval stages and hence might be related to additional larval -globin genes of as yet unknown genomic location.     

Neurogenesis during optic tectum regeneration in Xenopus laevis

The regenerative neurogenesis of the optic tectum of larval Xenopus laevis has been studied analyzing the proliferative and morphogenetic phases of the regeneration process after removal of one optic lobe. To this end, short‐term and long‐term pulses were carried out using the thymidine analog BrdU, selectively incorporated into cells during the S phase of the cell cycle. Results indicate that while in early larvae (stage 49/50, according to Nieuwkoop & Faber 1967 ) regeneration occurs mainly at the expense of the stem cells present in extensive proliferation zones (“matrix areas”) of the midbrain, in late larvae (stage 55/56) regeneration occurs at the expense of stem cells present in very limited matrix areas of the brain and of quiescent cells, which re‐enter the cell cycle following trauma. Moreover, in early larvae, morphogenesis of the optic tectum is carried out according to a precise spatio‐temporal order from rostro‐caudal to latero‐medial. By contrast, in late larvae, the topographical order of the regenerative morphogenesis of the optic lobe is completely altered. As a consequence, the regenerated optic tectum in early larvae has an apparently normal structure, while the regenerated optic tectum in late larvae lacks stratification.     

Cell intercalation during notochord development in Xenopus laevis

Morphometric data from scanning electron micrographs (SEM) of cells in intact embryos and high-resolution time-lapse recordings of cell behavior in cultured explants were used to analyze the cellular events underlying the morphogenesis of the notochord during gastrulation and neurulation of Xenopus laevis. The notochord becomes longer, narrower, and thicker as it changes its shape and arrangement and as more cells are added at the posterior end. The events of notochord development fall into three phases. In the first phase, occurring in the late gastrula, the cells of the notochord become distinct from those of the somitic mesoderm on either side. Boundaries form between the two tissues, as motile activity at the boundary is replaced by stabilizing lamelliform protrusions in the plane of the boundary. In the second phase, spanning the late gastrula and early neurula, cell intercalation causes the notochord to narrow, thicken, and lengthen. Its cells elongate and align mediolaterally as they rearrange. Both protrusive activity and its effectiveness are biased: the anterioposterior (AP) margins of the cells advance and retract but produce much less translocation than the more active left and right ends. The cell surfaces composing the lateral boundaries of the notochord remain inactive. In the last phase, lasting from the mid- to late neurula stage, the increasingly flattened cells spread at all their interior margins, transforming the notochord into a cylindrical structure resembling a stack of pizza slices. The notochord is also lengthened by the addition of cells to its posterior end from the circumblastoporal ring of mesoderm. Our results show that directional cell movements underlie cell intercalation and raise specific questions about the cell polarity, contact behavior, and mechanics underlying these movements. They also demonstrate that the notochord is built by several distinct but carefully coordinated processes, each working within a well-defined geometric and mechanical environment.     

Tunicamycin-inducible polypeptide synthesis during Xenopus laevis embryogenesis

Tunicamycin treatment of Xenopus laevis embryos enhanced the synthesis of a specific set of polypeptides with molecular masses of 98, 78, 59 and 58 kDa. The 78-kDa polypeptide was tentatively identified as glucose-regulated protein (GRP) 78 on the basis of molecular mass, pl (5.2), and tunicamycin inducibility, which took place upon treating embryos after the midblastula transition (MBT). The synthesis of a polypeptide with this electrophoretic mobility was detected but was not tunicamycin-inducible at stages prior to the MBT. GRP78 mRNA was detectable before the MBT but was not inducible by tunicamycin until the tailbud stage. A comparison of tunicamycin-induced polypeptide synthesis in Xenopus embryos, A6 cell line, and white blood cells by 2D-PAGE and fluorography revealed three spots in the GRP78 region of the gel. One was observed in both embryos and adult cells; another was adult-specific; and the third one was possibly an embryo-specific form. These results suggest that GRP78 synthesis might undergo a switch from an embryonic to an adult pattern during Xenopus development.     

Crystallins during Xenopus laevis free lens formation

Summary The ontogeny and localization of crystallins during free lens development (i.e. lens development without the optic vesicle) were investigated in Xenopus laevis using the indirect immunofluorescence staining method with an antiserum raised against homologous total lens soluble proteins. Since the developing free lenses pass through stages similar to those of the lenses regenerated from the inner cell layer of the outer cornea following lentectomy in the same species Freeman's classification was used to identify the stages of free lens development. The first appearance of a positive reaction occurred at early stage IV in a number of cells in an area where future lens fibre cells would develop. With further differentiation of the free lens more and more cells in the fibre area started to show a positive reaction and the first positive reaction in the epithelium was observed late in stage V. Histological examination revealed that a fully differentiated free lens and a normally developed lens are similar but that the free lens is smaller.     

Protamine polymorphism in Xenopus laevis laevis

Protamines from individual frogs of the subspecies Xenopus laevis laevis were compared by electrophoresis on polyacrylamide gels containing acetic acid, urea, and Triton X-100 to determine if the expression of protamine genes differs among individuals. Two electrophoretic bands, SP2a and SP2b, appeared to be expressed as allelic variants. Of 33 frogs, 19 expressed only SP2a, 11 expressed both SP2a and SP2b, and three expressed only SP2b. Electrophoretic analysis of partial V8 protease digests could not distinguish the peptides released from SP2a and SP2b. Differences in sperm development between individuals were not detected by light or electron microscopy. The results suggest that protamine polymorphism can exist among individuals of a species without an apparent effect on sperm development or sperm function.     

Testosterone 5α-Reductase in Spinal Cord of Xenopus laevis

Abstract: Testosterone 5α-reductase, the enzyme that converts testosterone to 5α-dihydrotestosterone, is present in the spinal cord of Xenopus laevis. In adult males the enzymatic activity is optimal at pH 7.4 and 27°C; the apparent K_m is 2.0 × 10⁻⁵ m and the V _max is 10.0 pmol/mg protein/h. Enzymatic activity was assayed in segments of the spinal cord in each of four groups: control untreated males, females, castrated males, and sexually active clasping males. Striking differences in both the amount of dihydrotestosterone produced with time and in the pattern of its distribution were seen in spinal cords of clasping males compared with those of the other groups. The differences are greatest in the basal medulla and rostral segments of the spinal cord. Neurons in these segments innervate the muscles primarily involved in clasping.