Keratin alterations during embryonic epidermal differentiation: a presage of adult epidermal maturation

Differentiation of the epidermis during embryonic rabbit development was found to be accompanied by dramatic changes in keratin proteins. Immunofluorescent labeling with keratin antiserum revealed that the undifferentiated epithelium of 12-d embryos was already committed to making keratin proteins. At 18 d of embryogenesis, the epithelium contained keratin proteins in the molecular weight range of 40,000-59,000. The stratification of the epithelium into two cell layers at 20 d of development coincided with the appearance of a 65-kdalton keratin. When a thick stratum corneum developed at 29 d, several additional keratins became prominent, most notably the large keratins (61- and 64-kdalton) and a 54-kdalton keratin. In addition, the 40-kdalton keratin, which had been present in earlier embryonic epidermis, disappeared. Newborn epidermis resembled that of a 29-d embryonic epidermis, with the exception of the appearance or increase in concentration of two more keratin species (46- and 50-kdalton). In vitro culturing of keratinocytes from 12- and 14-d embryonic skin demonstrated that these cells contained essentially the same keratin profiles as the undifferentiated epithelium of 18-d embryos (40-59 kdalton). Keratinocytes grown from older embryos contained increased amounts of keratin, similar to the in vivo situation, but did not synthesize the high molecular weight keratins. The changes observed during embryonic epidermal differentiation appear to be recapitulated during the sequential maturation steps of adult epidermis.     

TIMING OF INCORPORATION OF TRITIATED NUCLEOSIDES INTO DNA AND RNA OF EMBRYONIC CELLS OF RANA PIPIENS

The incorporation of tritiated nucleosides into DNA and RNA has been examined in partially synchronized cells of Rana pipiens embryos at the neurula and tailbud stages. Tritiated thymidine and deoxyguanosine are incorporated into the DNA in two maxima, or waves, during the S phase at both stages. More DNA replicates in the early maximum at the neurula stage than at the tailbud stage. A comparison of the degree of incorporation of labelled deoxyguanosine to labelled thymidine into DNA suggests that earlier replicating DNA at both stages may be GC-rich compared to later replicating DNA. The incorporation of tritiated uridine into RNA during the S phase also differs between the neurula and tailbud stages. Pulse and continuous label experiments indicate that at the neurula stage the highest rate of RNA synthesis occurs late in the S phase whereas at the tailbud stage the higher rate of RNA synthesis has shifted to an interval earlier in the S phase.     

The cDNA sequence of a Type II cytoskeletal keratin reveals constant and variable structural domains among keratins

We present the cDNA and amino acid sequences of a cytoskeletal keratin from human epidermis (Mr = 56K) that belongs to one of the two classes of keratins (Type I and Type II) present in all vertebrates. In these two types of keratins the central approximately 300 residue long regions share approximately 30% homology both with one another and with the sequences of other IF proteins. Within this region, all IF proteins are predicted to contain four helical domains demarcated from one another by three regions of beta-turns. The amino and carboxy termini of the Type II keratin are very different from those of microfibrillar keratins and other nonkeratin IF proteins. However, they contain unusual glycine-rich tandem repeats similar to the amino terminus of the Type I keratin. Thus the size heterogeneity among keratins appears to be a result of differences in the length of the terminal ends rather than the structurally conserved central region.     

Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis

Keratins from the living cell layers of human and neonatal mouse epidermis (prekeratins) have been compared to those from the stratum corneum (SC keratins). Human and mouse epidermis contained four prekeratins, two of each keratin subfamily: type II basic (pI 6.5-8.5; human 68 kDa, 60.5 kDa and mouse 67 kDa, 60 kDa) and type I acidic (pI 4.7-5.7; human 57 kDa, 51 kDa and mouse 58 kDa, 53 kDa,). While all four were present in equal amounts in adult human epidermis, two (67 kDa basic, 58 kDa acidic) were more prominent in neonatal mouse epidermis. Preliminary results with cell fractions (basal, spinous and granular) indicated that quantitative differences were a function of morphology, basal cells containing the smaller member of each subfamily and granular cells the larger. Mouse stratum corneum extracts contained four keratins (three in human): type II neutral-acidic (pI 5.7-6.7; human 65 kDa and mouse 64 kDa, 62 kDa) and type I acidic (pI 4.9-5.4; human 57.5 kDa, 55 kDa and mouse 58.5 kDa, 57.5 kDa). In both species, one-dimensional and two-dimensional peptide mapping (with V8 protease and trypsin respectively) indicated that while all four prekeratins were distinct gene products, similarities existed in the type II basic and the type I acidic keratin subfamilies. A strong homology also existed between type II SC keratins and the larger basic (type II) prekeratin (human 68 kDa and mouse 67 kDa) and between type I SC keratins and the larger acidic (type I) prekeratin (human 57 kDa and mouse 58 kDa). These results indicate a precursor-product relationship within each keratin subfamily, between SC keratins and the prekeratins abundant in the adjacent granular layer. This differentiation-related keratin processing was similar in mouse and human epidermis, and may represent a widespread phenomenon amongst keratinising epithelia.     

Characterization of bovine keratin genes: similarities of exon patterns in genes coding for different keratins.

Four different genomic clones which contain the genes coding for epidermal keratins Ia (mol. wt. approximately 68 000), Ib (68 000), III (60 000) and VIb (54 500) have been selected using cDNA probes and identified by hybrid-selection translation. The genes vary considerably in length, primarily due to differences in intron sizes: keratin Ia, 9.3 kb (approximately 2.55 kb total exons); keratin Ib, 6.0 kb (2.25 kb exons); keratin III, 6.0 kb (2.2 kb exons); keratin VIb, 4.4 kb (1.85 kb exons). The genes for all three representatives of the basic (type II) cytokeratin subfamily, i.e., keratins Ia, Ib and III, contain eight introns of variable sizes (0.1-1.8 kb) and their exon patterns are very similar. The gene coding for keratin VIb, a representative of the acidic (type I) subfamily, contains seven introns, and the size pattern of its five innermost exons closely resembles that of the genes of the type II keratins. Most of the introns are located in regions coding for the alpha-helical cores of these proteins. Mapping of the intron positions by the S1 nuclease technique and sequencing of some exon-intron boundaries has revealed that some of the introns of all four keratin genes have similar positions to each other and to those of the hamster vimentin gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Targeted deletion of keratins 18 and 19 leads to trophoblast fragility and early embryonic lethality

It has been reported previously that keratin 8 (K8)-deficient mice of one strain die from a liver defect at around E12.5, while those of another strain suffer from colorectal hyperplasia. These findings have generated considerable confusion about the function of K8, K18 and K19 that are co-expressed in the mouse blastocyst and internal epithelia. To resolve this issue, we produced mice doubly deficient for K18 and K19 leading to complete loss of keratin filaments in early mouse development. These embryos died at around day E9.5 with 100% penetrance. The absence of keratins caused cytolysis restricted to trophoblast giant cells, followed by haematomas in the trophoblast layer. Up to that stage, embryonic development proceeded unaffected in the absence of keratin filaments. K18/19-deficient mouse embryos die earlier than any other intermediate filament knockouts reported so far, suggesting that keratins, in analogy to their well established role in epidermis, are essential for the integrity of a specialized embryonic epithelium. Our data also offer a rationale to explore the involvement of keratin mutations in early abortions during human pregnancies.     

Identification of members of the HSP30 small heat shock protein family and characterization of their developmental regulation in heat-shocked xenopus laevis embryos

In the present study we have characterized the synthesis of members of the HSP30 family during Xenopus laevis development using a polyclonal antipeptide antibody derived from the carboxyl end of HSP30C. Two-dimensional PAGE/immunoblot analysis was unable to detect any heat-inducible small HSPs in cleavage, blastula, gastrula, or neurula stage embryos. However, heat-inducible accumulation of a single protein was first detectable in early tailbud embryos with an additional 5 HSPs at the late tailbud stage and a total of 13 small HSPs at the early tadpole stage. In the Xenopus A6 kidney epithelial cell line, a total of eight heat-inducible small HSPs were detected by this antibody. Comparison of the pattern of protein synthesis in embryos and somatic cells revealed a number of common and unique heat inducible proteins in Xenopus embryos and cultured kidney epithelial cells. To specifically identify the protein product of the HSP30C gene, we made a chimeric gene construct with the Xenopus HSP30C coding sequence under the control of a constitutive promoter. This construct was microinjected into fertilized eggs and resulted in the premature and constitutive synthesis of the HSP30C protein in gastrula stage embryos. Through a series of mixing experiments, we were able to specifically identify the protein encoded by the HSP30C gene in embryos and somatic cells and to conclude that HSP30C synthesis was first heat-inducible at the early tailbud stage of development. The differential pattern of heat-inducible accumulation of members of the HSP30 family during Xenopus development suggests that these proteins may have distinct functions at specific embryonic stages during a stress response.     

Morphogenesis and regulated gene activity are independent of DNA replication in Xenopus embryos.

Xenopus embryos were transferred into media containing aphidicolin at late blastula, mid-gastrula, and early neurula stages. In each case, embryos continued to differentiate in the absence of DNA replication. When the inhibitor was added at late blastula, embryos continued to develop for about 8 h. However, when aphidicolin was added at the early neurula stage, development could be seen for up to 40 h after addition. The influence of replication on embryonic gene activity was studied by RNA blot analysis. Of the genes we examined only histone gene expression was down regulated by the addition of aphidicolin. The expression of various embryo-specific genes was unaffected by the lack of DNA synthesis. Even after several hours of treatment with aphidicolin, replication-inhibited tailbud and tadpole stages showed the same levels of specific mRNAs as control embryos containing 4-5 times more DNA. We conclude that morphogenesis and embryo-specific gene activity are independent of both DNA replication and a precise amount of DNA per embryo.     

Cytology, embryology, and evolution of the developmental arrest syndrome in newts of the genus Triturus (Caudata: Salamandridae)

We have examined embryonic development in three species (T. carnifex, T. cristatus, and T. marmoratus) of European newts of the genus Triturus (subgenus Neotriton) in which developmental arrest occurs in embryos that are homomorphic for a chromosomal heteromorphism involving chromosome 1 (Horner and Macgregor: J. Herpetol., 19:261-270, 1985). Embryonic arrest occurred during tailbud stages in all three species, but at a slightly earlier stage in T. marmoratus. Two phenotypes were identified among the arrested embryos. One of these is indistinguishable in embryonic morphology from normal embryos at all stages up to the time of arrest, but the other is characterized by a protruding yolk plug, which persists from the late gastrula/early neurula stage to the tailbud arrest stage and apparently interferes with normal morphogenesis. Evidence is presented that the two arrested phenotypes, which occur in approximately equal numbers, represent embryos that carry the two alternative homomorphic chromosome pairs of chromosome 1 heteromorphism. We conclude that developmental arrest reflects a balanced lethal heterozygosity probably resulting from an unequal exchange of genic material between the homologues of chromosome 1 which occurred in a common ancestor of the Neotriton species.     

Keratin polypeptide expression in mouse epidermis and cultured epidermal cells

Adult mouse epidermis contains up to 11 distinct keratin polypeptides, as resolved by two-dimensional gel electrophoresis. These include both basic (Type II; 67-, 65-, 63-, 62-, and 60-kDa) and acidic (Type I; 61- to 59-, 54-, 52-, 49-, and 48-kDa) keratins that exhibit multiple isoelectric forms. Several, but not all, of these keratins, identified by immunoblotting, were found to be actively synthesized in the skin when assayed in short-term pulse-labeling experiments. When compared to the adult, newborn mouse epidermis expresses fewer keratin subunits. However, greater amounts of keratins associated with differentiated suprabasal cells and stratum corneum, which is more pronounced morphologically in the newborn, were identified. We also observed strain-specific differences in the expression of a Type I acidic keratin. This 61-kDa (pI, approx. 5.3) keratin was produced exclusively by the CF-1 mouse and, based on peptide mapping, appeared to be related to the acidic 59-kDa keratin that was identified in this strain as well as all other mouse strains. The 61-kDa keratin was not expressed in vitamin A-deficient animals, suggesting that its appearance may be related to a retinoid-dependent posttranslational modification. In comparison to keratin expression in vivo, primary mouse keratinocyte monolayer cultures maintained in low Ca2+ (less than 0.08 mM) did not express the terminal differentiation keratins of 67-kDa (basic) or 59-kDa (acidic), although enhanced synthesis of the 60-kDa (basic) and the 52-kDa and 59-kDa (acidic) keratins associated with proliferation were observed. In addition, a subpopulation of nonadherent cells was continuously produced by the primary keratinocyte cultures that expressed the 67-kDa (basic) keratin specific for terminal differentiation. When the keratinocyte cultures were induced to terminally differentiate with Ca2+, the overall pattern of keratin expression was not changed significantly. Taken together, these results provide further evidence for the variable nature of keratin expression in mouse epidermal keratinocytes under different growth conditions.     

Lateral line placodes are induced during neurulation in the axolotl

In order to determine the time window for induction of lateral line placodes in the axolotl, we performed two series of heterotopic and isochronic transplantations from pigmented to albino embryos at different stages of embryogenesis and assessed the distribution of pigmented neuromasts in the hosts at later stages. First, ectoderm from the prospective placodal region was transplanted to the belly between early neurula and mid tailbud stages (stages 13-27). Whereas grafts from early neurulae typically differentiated only into epidermis, grafts from late neural fold stages on reliably resulted in differentiation of ectopic pigmented neuromasts. Second, belly ectoderm was transplanted to the prospective placodal region between early neurula and tailbud stages (stages 13-35). Normal lateral lines containing pigmented neuromasts formed in most embryos when grafts were performed prior to early tailbud stages (stage 24) but not when they were performed later. Our findings indicate that lateral line placodes, from which neuromasts originate, are already determined at late neural fold stages (first series of grafts) but are inducible until early tailbud stages (second series of grafts). A further series of heterochronic transplantations demonstrated that the decline of inducibility at mid tailbud stages is mainly due to the loss of ectodermal competence.     

Xenopus neurula left-right asymmetry is respeficied by microinjecting TGF-beta5 protein

A variety of TGF-beta-related ligands regulate the left-right asymmetry of vertebrates but the involvement of TGF-betas in left-right specification has not been reported. We assessed whether TGF-beta signaling is involved in the left-right specification of Xenopus post-gastrula embryos by microinjecting Xenopus TGF-beta5 protein into the left or right flank of neurula-tailbud embryos. Injection on the right side of neurulae caused left-right reversal of the internal organs in 93% of the embryos, while injection on the left side caused less than 5% left-right reversal. Expression of Xenopus nodal related-1 (Xnr-1 ), Xenopus antivin and Xenopus Pitx2, which are normally expressed on the left, was unaltered by the left-side injection. In contrast, right-side injection into neurulae induced the expression of these genes predominantly on the right side. Right-side injection into tailbud embryos caused bilateral expression of these handed genes. Time course analysis of asymmetric gene expression revealed that Xnr-1 could be induced by TGF-beta5 at late neurula stage, while antivin and Pitx2 could be induced by TGF-beta5 at the latertail bud stage. Injection of the antisense morpholino oligonucleotide against Xenopus TGF-beta5 into the left dorsal blastomere inhibited the normal left-handed expression of Xnr-1 and Pitx2, and caused the organ reversal in the injected embryos. These results suggest that normal left-right balance of endogenous TGF-beta5 signaling in the neurula embryo may be needed to determine the laterality of the asymmetric genes and to generate the correct left-right axis.     

The human keratins: biology and pathology

The keratins are the typical intermediate filament proteins of epithelia, showing an outstanding degree of molecular diversity. Heteropolymeric filaments are formed by pairing of type I and type II molecules. In humans 54 functional keratin genes exist. They are expressed in highly specific patterns related to the epithelial type and stage of cellular differentiation. About half of all keratins--including numerous keratins characterized only recently--are restricted to the various compartments of hair follicles. As part of the epithelial cytoskeleton, keratins are important for the mechanical stability and integrity of epithelial cells and tissues. Moreover, some keratins also have regulatory functions and are involved in intracellular signaling pathways, e.g. protection from stress, wound healing, and apoptosis. Applying the new consensus nomenclature, this article summarizes, for all human keratins, their cell type and tissue distribution and their functional significance in relation to transgenic mouse models and human hereditary keratin diseases. Furthermore, since keratins also exhibit characteristic expression patterns in human tumors, several of them (notably K5, K7, K8/K18, K19, and K20) have great importance in immunohistochemical tumor diagnosis of carcinomas, in particular of unclear metastases and in precise classification and subtyping. Future research might open further fields of clinical application for this remarkable protein family.     

Expression pattern of BXR suggests a role for benzoate ligand-mediated signalling in hatching gland function

The Xenopus laevis nuclear receptor BXR has recently been shown to be activated by a class of endogenous benzoate metabolites, indicating the presence of a novel and unsuspected benzoate ligand-dependent signalling pathway. The receptor is expressed ubiquitously in blastula and gastrula stage embryos, and its expression declines during neurula stages. In order to examine further this novel vertebrate signalling system, we have examined the expression of the BXR gene in tailbud stage embryos and adults. We show here that in Xenopus tailbud stage embryos expression is restricted to the hatching gland, suggesting a role in hatching gland function. Neither BXR nor a BXR-VP16 fusion is sufficient to specify hatching gland in neurally-induced tissue. In adults, BXR expression is abundant in the brain and gonads. This expression pattern in adults is distinct from any of the putative mammalian homologues. A nuclear receptor that mediates benzoate signalling has yet to be found in mammals.     

ULTRASTRUCTURE OF THE 'GERMINAL PLASM' IN XENOPUS EMBRYOS AFTER CLEAVAGE

The endodermal location of 'germinal plasm'-bearing cells (GPBCs) and the ultrastructure of the 'germinal plasm' were studied in Xenopus laevis embryos at gastrula, neurula, tailbud and younger tadpole stages. Primordial germ cells (PGCs) of feeding tadpoles were also observed ultrastructurally.
GPBCs were found in the inner endoderm and in the yolk plug region at the late gastrula stage, in the middle and in the dorsal part of the endoderm cell mass at the late neurula and late tailbud stages, respectively. At the younger tadpole stage they were observed in the uppermost dorsal part of the endoderm. Germinal granules were always present in GPBCs at all stages examined but were not found in PGCs of feeding tadpoles. Irregularly shaped-stringlike bodies (ISBs) which seemed to have changed from germinal granules were first noticed in GPBCs at the late neurula stage, and were still present in PGCs of tadpoles, while 'granular materials' were not seen in GPBCs until the feeding tadpole stages. These facts and ultrastructural similarities shared by these organelles lead us to conclude that the change of the germinal granule through ISB, to the 'granular material' takes place during the differentiation of GPBCs into PGCs.     

The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments

The four major keratins of normal human epidermis (molecular mass 50, 56.5, 58, and 65-67 kD) can be subdivided on the basis of charge into two subfamilies (acidic 50-kD and 56.5-kD keratins vs. relatively basic 58-kD and 65-67-kD keratins) or subdivided on the basis of co-expression into two "pairs" (50-kD/58-kD keratin pair synthesized by basal cells vs. 56.5-kD/65-67-kD keratin pair expressed in suprabasal cells). Acidic and basic subfamilies were separated by ion exchange chromatography in 8.5 M urea and tested for their ability to reassemble into 10-nm filaments in vitro. The two keratins in either subfamily did not reassemble into 10-nm filaments unless combined with members of the other subfamily. While electron microscopy of acidic and basic keratins equilibrated in 4.5 M urea showed that keratins within each subfamily formed distinct oligomeric structures, possibly representing precursors in filament assembly, chemical cross-linking followed by gel analysis revealed dimers and larger oligomers only when subfamilies were combined. In addition, among the four major keratins, the acidic 50-kD and basic 58-kD keratins showed preferential association even in 8.5 M urea, enabling us to isolate a 50-kD/58-kD keratin complex by gel filtration. This isolated 50-kD/58-kD keratin pair readily formed 10-nm filaments in vitro. These results demonstrate that in tissues containing multiple keratins, two keratins are sufficient for filament assembly, but one keratin from each subfamily is required. More importantly, these data provide the first evidence for the structural significance of specific co-expressed acidic/basic keratin pairs in the formation of epithelial 10-nm filaments.     

Monoclonal antibody analysis of bovine epithelial keratins. Specific pairs as defined by coexpression

We have characterized the keratin proteins of various bovine epithelial tissues by one- and two-dimensional gel electrophoresis, coupled with the immunoblot technique using AE1, AE2, AE3, AE5, CA20, BE14, and 6.11 monoclonal antikeratin antibodies. The results indicate that all known bovine keratins can be divided into two subfamilies. The "acidic" (Type I) subfamily consists of 41-, 43-, 45-, 46-, 50-, 54-, 56-, and 56.5-kDa keratins, all of which have a pI of less than 5.6, and most of them are recognized by our AE1 antibody, whereas the "neutral-to-basic" (Type II) subfamily consists of 55-, 57-, 58-, 62-65-, 66-, and 67-kDa keratins, all of which have a pI of greater than 6.0 and are recognized by our AE3 antibody. Tissue distribution data and cell culture studies show that, within the two subfamilies, keratins with similar "size ranks" form a "pair" as defined by frequent co-expression. Furthermore, within most "keratin pairs," the basic keratin is larger than the acidic one by 8-10 kDa. These results provide further support for the concepts of "keratin subfamilies" and keratin pairs and are consistent with the possibility that the acidic and basic members of at least some keratin pairs may interact specifically during in vivo tonofilament assembly and/or function. Immunoblotting data derived from the use of several monospecific antibodies show that although the size, charge, and pattern of expression of most bovine keratins are similar to those of the human counterparts, there are important exceptions to this rule.