Evolution of pipoid frogs:intergeneric relationships of the aquatic frog family Pipidae (Anura)

The 27 species of the aquatic frog family Pipidae are currently arranged in four genera: Xenopus (15 species), Hymenochirus (four species), and the poorly known genus Pseudhymenochirus (one species) occur in Africa; Pipa (seven species) is found in South America and lower Central America. Despite extensive work on the biology of Xenopus from various disciplines, the evolutionary relationships of Xenopus to other pipids have not been resolved. Phylogenetic analyis of morphological features of pipid frogs indicates that, contrary to earlier opinions, Hymenochirus and Pipa are closest relatives (sister-groups); these genera are placed in the subfamily Pipinae. Also, the currently recognized species of Xenopus do not form a natural group; the species tropicalis and epitropicalis are more closely related to Hymenochirus + Pipa than to the remaining species of Xenopus . The two discordant species are transferred to the genus Silurana , which is relegated to the new subfamily Siluraninae; it is the sister-group of the Pipinae. The remaining species of Xenopus constitute a monophyletic group that is placed in the subfamily Xenopodinae as the sister-group of the other genera of pipids.     

Genome duplications within the Xenopodinae do not increase the multiplicity of antimicrobial peptides in Silurana paratropicalis and Xenopus andrei skin secretions

A putative genome duplication event within the Silurana lineage has given rise to the tetraploid frog S. paratropicalis and a second polyploidization within the Xenopus lineage has produced the octoploid frog X. andrei. Peptidomic analysis of norepinephrine-stimulated skin secretions of S. paratropicalis and X. andrei led to identification of multiple peptides with growth-inhibitory activity against Escherichia coli and Staphylococcus aureus. Structural characterization demonstrated that the S. paratropicalis components comprised three peptides belonging to the caerulein-precursor fragment family (CPF-SP1, -SP2 and -SP3), two peptides from the xenopsin-precursor fragment family (XPF-SP1 and -SP2), and one peptide orthologous to peptide glycine-leucine-amide (PGLa-SP1). The CPF peptides showed potent, broad-spectrum antimicrobial activity. The X. andrei components comprised two peptides from the magainin family, (magainin-AN1 and -AN2), two from the XPF family (XPF-AN1 and -AN2), two from the PGLa family(PGLa-AN1 and -AN2), and one caerulein-precursor fragment (CPF-AN1).The primary structures of these peptides indicate a close phylogenetic relationship between X. andrei and the octoploid frog X. amieti. Under the same experimental conditions, seven orthologous antimicrobial peptides were previously isolated from the diploid frog S. tropicalis, nine from the tetraploid frog X. borealis, and five from the tetraploid frog X. clivii. The data indicate, therefore, that nonfunctionalization (gene deletion) has been the most common fate of duplicated antimicrobial peptide genes following polyploidization events in the Silurana and Xenopus lineages.     

Characterization of a Mycobacterium ulcerans-like infection in a colony of African tropical clawed frogs (Xenopus tropicalis)

A nontuberculous Mycobacterium ulcerans-like organism was identified as the causative agent of an epizootic of mycobacteriosis in a colony of African tropical clawed frogs, Xenopus (Silurana) tropicalis, at the University of California, Berkeley. Diverse clinical signs of disease were observed, including lethargy, excess buoyancy, coelomic effusion, cutaneous ulcers, and granulomas. Visceral granulomas, ulcerative and granulomatous dermatitis, coelomitis, and septicemia were common findings at necropsy. Identification of M. ulcerans-like organisms was based on molecular and phenotypical characteristics. The findings of this investigation indicate that this M. ulcerans-like organism is a primary cause of morbidity and mortality in aquatic anurans and should be considered in the differential diagnosis of coelomic effusion in amphibians. Furthermore, if this Mycobacterium species ultimately is identified as M. ulcerans, X. tropicalis should be considered a potential source of this important public health pathogen.     

Speciation of Protopolystoma Bychowsky, 1957 (Monogenea: Polystomatidae) in hosts of the genus shape Xenopus (Anura: Pipidae)

The taxonomy, geographical distribution and hostrange of the polystomatid genus ProtopolystomaBychowsky, 1957 are reviewed. P. xenopodis(Price, 1943) and five new species are recognised,which occur in clawed toads ( Xenopus spp.)throughout subsaharan Africa. Of the two clawed toadsubgenera, Xenopus and Silurana, only theformer is infected. Protopolystoma spp. aredifferentiated by morphological variation of the gut,large hamulus and penis armature. P.xenopodis is found in Xenopus laevis subspeciesin South Africa, Transkei, Zimbabwe, DemocraticRepublic of Congo (D.R.C.), Rwanda, Uganda, Kenya andCameroon ( X. l. poweri and X. l.sudanensis are new host records). It also occurs inintroduced populations of X. l. laevis in theUnited States (southern California) and United Kingdom(South Wales). In subsaharan Africa the speciesdisplays significant, but continuous, geographicalvariation of penis spine size between southernpopulations in X. l. laevis and those in morenortherly host subspecies. Data on the natural hostrange of this parasite were complemented by anexperimental study of host-specificity in the southernform. This can produce patent infections in X.l. victorianus and X. gilli, but not X.wittei nor X. (Silurana) tropicalis. P.simplicis n. sp. is endemic to central and eastAfrican areas, infecting X. laevis subspecies ineastern D.R.C., Rwanda, Uganda and western Kenya, X. wittei-like hosts in eastern D.R.C., westernUganda, Rwanda and Burundi, X. vestitus inwestern Uganda and Xenopus sp. at Nairobi,Kenya. P. ramulosus n. sp. occurs in X.fraseri-like toads in eastern D.R.C. (Gabon andCameroon are also possible literature records), and P. fissilis n. sp. is found in X. fraseri-and X. wittei-like species in Cameroon andeastern D.R.C., and in southern Rwanda, respectively. Two Protopolystoma taxa are found in X.muelleri populations now suspected to representdistinct species: P. occidentalis n. sp. occursin X. muelleri (western form) in Ghana, Togo,Nigeria and Cameroon, while P. orientalis n. sp.is found in X. muelleri (eastern form) in SouthAfrica, Zimbabwe and Tanzania. The allopatricallydistributed species P. ramulosus, P.simplicis, P. occidentalis and P.orientalis form a relatively homogenous grouping withsome interspecific morphological overlap. These taxaare distinguished from P. xenopodis by penisspine morphology and from P. fissilis by hamulusroot form and aspects of gut morphology. Unidentified Protopolystoma sp. have been recorded in X. clivii in Ethiopia, X. fraseri aff. inCameroon and Xenopus sp. in Kenya and Tanzania. At some localities, single host species were infectedby two representatives of Protopolystoma. P. fissilis was recorded in eastern D.R.C. with P. ramulosus, with Protopolystoma sp. inCameroon in X. fraseri-like hosts and with P. simplicis in X. wittei-like hosts in Rwanda. P. xenopodis co-occurred with P. simplicisin X. laevis subspecies through central and eastAfrica.     

Clinical diagnosis and treatment of epidermal chytridiomycosis in African clawed frogs (Xenopus tropicalis)

An investigation was conducted to determine the cause of morbidity and mortality in a collection of 55 adult male Xenopus (Silurana) tropicalis at the University of California, Berkeley. More than 80% of affected frogs died during the epizootic. All frogs were anorectic and lethargic, had dark pigmentation and excess skin sloughing, and lacked a slime layer. Histologic examination revealed severe hyperplastic and spongiotic dermatitis associated with colonization of the stratum corneum by large numbers of zoosporangia diagnostic of Batrachochytrium dendrobatidis. Treatment with a commercial formalin/malachite green solution at a dilution of 0.007 ml/L of tank water for 24 h, repeated every other day for four treatments, eliminated the organism and was curative. These findings are indicative of epidermal chytridiomycosis as a primary cause of death in this collection of X. tropicalis.     

Excision of the Tol2 transposable element of the medaka fish Oryzias latipes in Xenopus laevis and Xenopus tropicalis

The Tol2 transposable element from the medaka fish belong to the hAT family of transposons. In the previous studies, we have identified an autonomous member of this element, which encodes a fully functional transposase, and have shown that it can catalyze transposition in the zebrafish germ lineage. To date, the Tol2 element is the only natural transposon in vertebrates from which an autonomous member has been identified. We report here transposase-dependent excision of the Tol2 element in Xenopus laevis and Xenopus (Silurana) tropicalis embryos. We coinjected a plasmid DNA containing a nonautonomous Tol2 element and the transposase mRNA synthesized in vitro into two-cell-stage embryos, and analyzed DNA extracted from the injected embryos by polymerase chain reaction (PCR). We demonstrated that the Tol2 element could be excised from the plasmid DNA in both X. laevis and X. tropicalis only when it was coinjected with the transposase mRNA. In most cases, a complete loss of the Tol2 sequence was accompanied by addition of a short DNA sequence to the target sequence, indicating that transposase-dependent excision occurred. While these footprints were characteristic to those created upon excision of transposons of the hAT family, the additional bases found in Xenopus were longer and their structures were more complicated than those detected upon excision in zebrafish. This may reflect differences in the activities of host factors involved in either transposition, repair, or both between fish and frog. Our present study suggests that the Tol2 transposon system should be used as a novel genetic tool to develop transgenesis and mutagenesis methods in Xenopus.     

Albumin evolution in polyploid species of the genusXenopus

Electrophoresis of serum from 21 Xenopus species and subspecies reveals variable numbers of albumin bands. The diploid X. tropicalis has one albumin, while the tetraploid species (laevis, borealis, muelleri, clivii, fraseri, epitropicalis) have two. The octoploid species (amieti, boumbaensis, wittei, vestitus, andrei) have two to three bands, and the dodecaploid X. ruwenzoriensis has three. The molecular weight of the Xenopus albumins varies from 68 kd (in the tropicalis group) to 74 kd. The subspecies of X. laevis possess two albumins of different molecular weights (70 and 74 kd), whereas most species have only 70-kd albumins. Peptide maps have been obtained from albumin electromorphs by limited proteolysis in sodium dodecyl sulfate (SDS) gels, using S. aureus V8 protease. The peptide patterns produced by electromorphs from the same tetraploid Xenopus species generally differ from each other, suggesting that the two albumin genes contain a substantial amount of structural differences. In addition, the peptide maps are diagnostic for most tetraploid species and for some subspecies of X. laevis as well. Proteolysis of albumins from most octoploid and dodecaploid species results in patterns which are very similar to the ones produced by the electromorphs from X. fraseri. The albumins of X. vestitus differ from those of the other octoploid species. X. andrei possesses two fraseri-type and one vestitus-type albumin, which indicates that it probably originated by allopolyploidy.     

The amphibian globin gene repertoire as revealed by the Xenopus genome

The draft genome sequence of the Western clawed frog Xenopus (Silurana) tropicalis facilitates the identification, expression analysis and phylogenetic classification of the amphibian globin gene repertoire. Frog and mammalian neuroglobin display about 67% protein sequence identity, with the expected predominant expression in frog brain and eye. Frog and mammalian cytoglobins share about 69% of their amino acids, but the frog protein lacks the mammalian-type extension at the C-terminus. Like in mammals, X. tropicalis cytoglobin is expressed in many organs including neural tissue. Neuroglobin and cytoglobin genomic regions are syntenically conserved in all vertebrate classes. Frog and fish globin X show only 57% amino acid identity, but gene synteny analysis confirms orthology. The expression pattern of X. laevis globin X differs from that in fish, with a prominent expression in the eye and weak expression in most other examined tissues. Globin X is possibly present as two paralogous copies in X. tropicalis, with one copy showing transition stages of non-functionalization. The amphibian genome contains a previously unknown globin type (tentatively named 'globin Y') which is expressed in a broad range of tissues and is distantly related to the cytoglobin lineage. The globin Y gene is linked to a cluster of larval and adult hemoglobin alpha and beta genes which contains substantially more paralogous hemoglobin gene copies than previously published. Database and gene synteny analyses confirm the absence of a myoglobin gene in X. tropicalis.     

Host-defense peptides in skin secretions of the tetraploid frog Silurana epitropicalis with potent activity against methicillin-resistant Staphylococcus aureus (MRSA)

A putative genome duplication event within the Silurana lineage has given rise to the tetraploid Cameroon clawed frog Silurana epitropicalis (Fischberg, Colombelli, and Picard, 1982). Peptidomic analysis of norepinephrine-stimulated skin secretions of S. epitropicalis led to identification of 10 peptides with varying degrees of growth-inhibitory activity against Escherichia coli and Staphylococcus aureus. Structural characterization identified the peptides as belonging to the magainin family (magainin-SE1), the caerulein-precursor fragment family (CPF-SE1, -SE2 and -SE3), the xenopsin-precursor fragment family (XPF-SE1, SE-2, SE-3 and -SE4), and the peptide glycine-leucine-amide family (PGLa-SE1 and -SE2). In addition, peptide phenylalanine-glutamine-amide (FLGALLGPLMNLLQ·NH(2)) was isolated from the secretions that lacked antimicrobial activity. Comparison of the multiplicity of orthologous peptides in S. epitropicalis and the diploid Silurana tropicalis indicates that extensive nonfunctionalization (deletion or silencing) of antimicrobial peptide genes has occurred after polyploidization in the Silurana lineage, as in the Xenopus lineage. CPF-SE2 (GFLGPLLKLGLKGAAKLLPQLLPSRQQ; MIC=2.5μM) and CPF-SE3 (GFLGSLLKTGLKVGSNLL·NH(2); MIC=5μM) showed potent growth-inhibitory activity against a range of clinical isolates of methicillin-resistant S. aureus (MRSA). Their utility as systemic anti-infective drugs is limited by significant hemolytic activity against human erythrocytes (LC(50)=50μM for CPF-SE2 and 220μM for CPF-SE3) but the peptides may find application as topical agents in treatment of MRSA skin infections and decolonization of MRSA carriers.     

Paramphistome digeneans from shape Xenopus species (Pipidae) in Africa: taxonomy,host-specificity and biogeography

The taxonomy, host range and geographical distribution of paramphistome digeneans from Xenopus spp. in sub-Saharan Africa are reviewed. Two representatives of Progonimodiscus Vercammen-Grandjean, 1960 are recognised, both of which are narrowly or primarily specific to Xenopus. An analysis of morphometric and meristic characters indicated geographical variation in Progonimodiscus doyeri (Ortlepp, 1926), with two allopatric forms showing significant, but continuous, variation in testis size and vitelline follicle number. P. colubrifer n. sp. is distinguished from P. doyeri by the form of muscular elevations on the acetabulum accessory peduncle. It infects Xenopus ( Silurana) tropicalis-like toads from lowland tropical rain forest zones in Nigeria, Togo and the Ivory Coast, while P. doyeri occurs in hosts of the subgenus Xenopus from a wide variety of biotypes. Previous literature records indicate the presence of the southern P. doyeri morphological variant in X. laevis laevis in South Africa and Zimbabwe and the northern variant in X. l. victorianus, X. fraseri aff. and X. muelleri in the Democratic Republic of Congo, X. wittei in Uganda and the Democratic Republic of Congo, and the ranid Conraua crassipes in Cameroon (the only record of Progonimodiscus from a non-pipid host). New host and/or geographical records for this species are of the northern form in X. l. victorianus, X. l. bunyoniensis and X. vestitus in Uganda, X. l. sudanensis in Cameroon, X. borealis in Kenya, X. pygmaeus in the Democratic Republic of Congo, X. fraseri aff. in Cameroon, X. wittei aff. in the Democratic Republic of Congo and X. muelleri in Cameroon and Nigeria. While the geographical limits of the two P. doyeri variants are not known with precision, existing data are consistent with a turnover in the region of 15°S, where a notable discontinuity occurs in the distributions of other Xenopus parasites. Species of Diplodiscus Diesing, 1836 in Xenopus hosts are rare. Diplodiscus peregrinator n. sp. was recovered from X. tropicalis at a single locality in the Ivory Coast and distinguished by a combination of body size, egg size, genital pore position and acetabulum morphology. D. fischthalicus Meskal, 1970 was not found during the present study.     

Systematic relationships of Nematomorpha based on molecular and morphological data

Abstract. We sequenced the 18S rRNA gene from 11 nematomorph species from 9 genera and derived hypotheses concerning the sister group of Nematomorpha and relationships within this taxon. The molecular and morphological data are consistent with the monophyly of Nematomorpha, a sister-group relationship between Nematomorpha and Nematoda, and a sister-group relationship between the marine genus Nectonema and all of the freshwater taxa, Gordiida. Hypotheses of relationships within Gordiida support the traditional taxa Gordiidae, Chordodidae, and Chordodinae but reject Parachordodinae and Spinochordodidae. The molecular results differ from those of previous morphological studies by suggesting a reduction of the two tail lobes at the posterior end of males in Chordodinae, monophyly of the genus Paragordionus , and paraphyly of the genus Chordodes .     

Xenopus tropicalis nodal-related gene 3 regulates BMP signaling: an essential role for the pro-region

In vertebrates, nodal-related genes are crucial for specifying mesendodermal cell fates. Six nodal-related genes have been identified in Xenopus, but only one, nodal, has been identified in the mouse. The Xenopus nodal-related gene 3 (Xnr3), however, lacks the mesoderm-inducing activity of the other five nodal-related genes in Xenopus, and can directly induce neural tissue in animal caps by antagonizing BMP signals. In this study, we isolated three clones of the Xenopus (Silurana) tropicalis nodal-related gene 3 (Xtnr3) and analyzed their function. The Xtnr3 genes show high homology to Xnr3 and have the same activity. Southern blot and genomic PCR analyses indicate that the X. tropicalis genome has duplications in the Xtnr3 gene sequences and our three clones represent separate gene loci. We also found a partial clone of Xtnr3 that coded for the N-terminal part of its pro-region. Surprisingly, this sequence also induced neural tissue by antagonizing BMP signals, and its coded protein physically associated with BMP4 mature protein. Furthermore, we showed that the pro-region of Xnr5 has the same activity. Together, these findings indicate that the pro-region of nodal-related genes acts antagonistically towards BMP signals, which identifies a novel mechanism for the inhibition of BMP signaling.     

High-throughput transgenesis in Xenopus using I-SceI meganuclease

In this report we describe an easy, highly efficient transgenesis method for Xenopus. The method is very simple; a commercially available meganuclease, I-SceI, is incubated with a transgene construct carrying its recognition sites, and is subsequently microinjected into fertilized eggs. Approximately 30% (in Xenopus tropicalis) or 20% (in Xenopus laevis) of injected embryos exhibit non-mosaic, promoter-dependent transgene expression, and transgenes from the founder animals are transmitted to offspring. The method is compatible with mRNA or antisense morpholino oligonucleotide injection, and these secondary reagents can be introduced simultaneously or sequentially with a transgene to test their interaction. This high-throughput transgenic technique will be a powerful tool for studying the complex wiring of regulatory networks at the genome-wide level, as well as for facilitating genetic studies in the rapidly breeding diploid frog, X. tropicalis.     

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.     

Host-defense peptides from skin secretions of the tetraploid frogs Xenopus petersii and Xenopus pygmaeus, and the octoploid frog Xenopus lenduensis (Pipidae)

Peptidomic analysis of norepinephrine-stimulated skin secretions led to the identification of host-defense peptides belonging to the magainin, peptide glycine-leucine-amide (PGLa), and caerulein precursor fragment (CPF) families from the tetraploid frogs, Xenopus petersii (Peters' clawed frog) and Xenopus pygmaeus (Bouchia clawed frog), and the octoploid frog Xenopus lenduensis (Lendu Plateau clawed frog). Xenopsin-precursor fragment (XPF) peptides were not detected. The primary structures of the antimicrobial peptides from X. petersii demonstrate a close, but not conspecific relationship, with Xenopus laevis whereas the X. pygmaeus peptides show appreciable variation from previously characterized orthologs from other Xenopus species. Polyploidization events within the Xenopodinae (Silurana+Xenopus) are associated with extensive gene silencing (nonfunctionization) but unexpectedly the full complement of four PGLa paralogs were isolated from X. lenduendis secretions. Consistent with previous data, the CPF peptides showed the highest growth-inhibitory activity against bacteria with CPF-PG1 (GFGSLLGKALKIGTNLL.NH(2)) from X. pygmaeus combining high antimicrobial potency against Staphylococcus aureus (MIC=6 μM) with relatively low hemolytic activity (LC(50)=145 μM).     

Cross-fertilization and structural comparison of egg extracellular matrix glycoproteins from Xenopus laevis and Xenopus tropicalis

While the anuran amphibian Xenopus laevis is a widely used vertebrate model system, it is not optimal for genetic manipulations due to its tetraploid genome and long generation time. A current alternative amphibian model system, Xenopus tropicalis, has the advantages of a diploid genome and a much shorter generation time. We undertook a comparative investigation of X. tropicalis egg extracellular matrix glycoproteins in relation to those already characterized in X. laevis. Fertilization methods and isolation of egg extracellular molecules were directly transferable from X. laevis to X. tropicalis. Cross-fertilizations were successful in both directions, indicating similar molecules involved in sperm-egg interactions. Egg envelopes analyzed by SDS-PAGE were found to have almost identical gel patterns, whereas jelly component profiles were similar only for the larger macromolecules (>90 kDa). The cDNA sequences for egg envelope glycoproteins ZPA, ZPB, ZPC, ZPD and ZPAX, and also egg cortical granule lectin involved in the block to polyspermy, were cloned for X. tropicalis and showed a consistent approximately 85% amino acid identity to the X. laevis sequences. Thus, homologous egg extracellular matrix molecules perform the same functions, and the molecular and cellular mechanisms of fertilization in these two species are probably equivalent.     

Characterization of histone genes isolated from Xenopus laevis and Xenopus tropicalis genomic libraries

Using a cDNA clone for the histone H3 we have isolated, from two genomic libraries of Xenopus laevis and Xenopus tropicalis, clones containing four different histone gene clusters. The structural organization of X. laevis histone genes has been determined by restriction mapping, Southern blot hybridization and translation of the mRNAs which hybridize to the various restriction fragments. The arrangement of the histone genes in X. tropicalis has been determined by Southern analysis using X. laevis genomic fragments, containing individual genes, as probes. Histone genes are clustered in the genome of X. laevis and X. tropicalis and, compared to invertebrates, show a higher organization heterogeneity as demonstrated by structural analysis of the four genomic clones. In fact, the order of the genes within individual clusters is not conserved.     

Genetic and genomic prospects for Xenopus tropicalis research

Research using Xenopus laevis has made enormous contributions to our understanding of vertebrate development, control of the eukaryotic cell cycle and the cytoskeleton. One limitation, however, has been the lack of systematic genetic studies in Xenopus to complement molecular and cell biological investigations. Work with the closely related diploid frog Xenopus tropicalis is beginning to address this limitation. Here, we review the resources that will make genetic studies using X. tropicalis a reality.     

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.