The IkΒ kinase complex (IKK) has a central role in regu-lating the function of NF-kΒ (Ref. 1). IKK is composed

of at least three subunits, two catalytic subunits (IKKa,also known as IKK1, and IKKb) and the regulatory sub-unit IKKg/NEMO. Recently, mice null for the gene encod-ing IKKa have been described. These mice have a lethalphenotype with major effects on differentiation of the epi-dermis and related keratinizing tissues2–4. Interestingly,manipulation of other genes in the NF-kΒ pathway doesnot result in this striking epidermal phenotype, suggestinga novel function for IKKa in epidermal differentiation1.

IKKa2/2 mice are born sheathed by a thickened, un-differentiated epidermis and die shortly after birth. Thedevelopment of hair follicles is also retarded. The limbsand snout of the mice are shortened and blunt, althoughthe axial skeleton is largely intact, as if the thickened epi-dermis restricts outgrowth during development3. Theseskeletal defects in mutant mice are accentuated by theincreased adhesiveness of the undifferentiated epidermis,which results in fusion of fore and hind limbs to the body

wall. The total effect is that of an animal enclosed within athickened epidermis.

The IKKa2/2 phenotype is similar to that described previously for two mutations arising from radiation experi-ments in mice: pupoid fetus (pf ) and repeated epilation(Er). The autosomal recessive mutation, pf, was identifiedoriginally during a search for limb abnormalities byMeredith5. The snout and limbs of pf/pf mice are intact,but shortened, and they appear to be cocooned in a thickened, hyperproliferative epidermis, hence the name(Fig. 1). Animals homozygous for pf are indistinguishableat a gross level from IKKa2/2 mice. Er is a semidominantmutation that results in the same gross phenotype ofstumpy limbs and snout sheathed in a thickened epider-mis6 (Fig. 2). Perinatal lethality of mice homozygous foreither pf or Er is attributed to fusion of oral epithelia andblockage of airways leading to suffocation at birth (Figs 1and 2). This is also a likely factor in IKKa2/2 lethality,although cardiovascular malfunction has been cited ascontributing to perinatal death3.

The similarity among IKKa2/2, pf/pf and Er/Er miceextends to epidermal histology and differentiation. In allthree models it is likely that epidermal hyperplasia and lackof differentiation lead to increased adhesivity of the epider-mis and subsequent gross abnormalities. The histology of

Outlook LETTERS IKKa

TIG November 2000, volume 16, No. 11 0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(00)02121-1

Chris FisherChristopher.Fisher@

am.pnu.com

Cell and MolecularBiology, 7252-267-412,Pharmacia Corporation,

Kalamazoo, MI 49006,USA.

482

IKKa2/2 mice share phenotypewith pupoid fetus (pf/pf ) andrepeated epilation (Er/Er) mutantmice

FIGURE 1. Comparison of pf/pf and normal mice

Gross phenotype of pf/pf mutant pups (left and right) compared to normallittermate (center). The pf/pf phenotype includes stumpy limbs and snout. (b)Scanning electron micrographs of the snouts of normal (left) and pf/pf mutantmice (right) reveal fusion of epithlia of nares (N) and mouth (M), and retardationof hair-follicle formation (revealed by lack of vibrissae follicles; V) in pf/pfmutants. These phenotypic aspects are shared with IKKa2/2 and Er/Er mice.

FIGURE 2. Er/Er mutant phenotype

Intact (right) and mid-sagittal hemisection (left) of newborn Er/Er pups showsblunting of limbs and snout and fusion of oral epithelia (arrow), characteristicsshared with pf/pf and IKKa2/2 mice. Photograph kindly provided by Karen A.Holbrook. Scale bar = 1 cm.

skin of Er/Er and pf/pf mice is similar, in that both have athickened, hyperproliferative epidermis and the completeabsence of a stratum corneum7–9 (Fig. 3). Failure of granu-lar-layer formation is nearly complete in pf/pf mutants,while Er/Er mice have a variable granular layer that isreduced or missing in some areas and hyperplastic in others7,10 (Fig. 3). This difference between pf/pf and Er/Ermice is reflected in the pattern of expression of markers ofdifferentiation including the keratohyalin proteins profi-laggrin and filaggrin7,10. Mice homozygous for pf expresslittle or no profilaggrin, which correlates with the absenceof epidermal granular layer formation. By contrast, Er/Ermice express profilaggrin, correlating with the variablegranular layer, but do not proteolytically process it tofilaggrin. This lack of processing is, apparently, due to thefailure of the mutant epidermis to form a stratumcorneum7. Therefore, a delay in the program of epidermaldifferentiation results in clear abnormalities in epidermalhistology and protein expression in both pf/pf and Er/Ermice.

Until the IKKa2/2 mice were described, these abnormal-ities of epidermal differentiation were unique to pf/pf andEr/Er mice. IKKa2/2 mice also have a failure of epidermaldifferentiation that is remarkably similar to Er/Er andpf/pf mice2–4. The similarities include absence of stratumcorneum and a block in filaggrin production. The lack ofprofilaggrin expression and an almost complete absence ofa granular cell layer in the IKKa2/2 mice make them moresimilar to pf/pf, rather than Er/Er mice. However, express-ion of keratin 14 (K14) is similar in pf/pf, Er/Er andIKKa2/2 animals. In normal epidermis, K14 is detectedonly in the basal cells, while all layers of the epidermis ofpf/pf, Er/Er and IKKa2/2 mice stain positive for K14 byimmunohistochemistry2,10. These results, again, suggestthat all three mutants share a similar phenotype.

Epidermal histology does reveal some interesting distinctions between the mice, however. Blood vessels thatenter during development and extend high into the epider-mis are often seen in pf/pf mice8 (Fig. 3). The Er/Er mutantdoes not have this characteristic histology, but histologyof IKKa2/2 mice shows blood vessels high within the epidermis2 suggesting a similar histogenesis for pf/pf andIKKa2/2 mice. However, it is important to note that all three mice have been examined on different geneticbackgrounds, which may contribute to differences in phenotype.

Despite the close similarities among phenotypes, allindications suggest that they are caused by differentgenes. Both pf and Er mutations map to chromosome 4(Refs 11, 12) but complementation tests indicate thatthey are not alleles8. IKKa, which was previously clonedas the mouse gene encoding CHUK (Ref. 13), maps tomouse chromosome 19 (Ref. 14). The unique nature ofthe phenotype of the IKKa knockout mouse, compared toknockouts of other members of the NF-kΒ pathway,points to a branchpoint of IKKa function that regulatesepidermal differentiation. The similar phenotypes of theEr/Er, pf/pf and IKKa2/2 mutant mice suggest that thesegenes all act in the same pathway.

Experiments demonstrate that grafting pf/pf and Er/Erskin to a normal host corrects the abnormal epidermalphenotype, determined by morphological and biochemicalcriteria9,10,15,16. If the proteins encoded by pf and Er actsystemically, as these experiments suggest, they mightencode factors activating IKKa in the epidermis. Cloning

and characterization of the pf and Er genes would facili-tate our understanding of the relationship between theseand the epidermal IKKa pathway and how they interact toregulate epidermal differentiation.

The vertebrate integument has been a popular modelsystem for studying basic processes regulating organo-genesis for over 60 years. Classical studies elucidated theimportance of epithelial–mesenchymal interactions in regu-lating the formation and differentiation of formation during development and into adulthood. Recent studieshave focused on the role of a growing list of regulatory el-ements implicated in controlling embryonic development,including bone morphogenic proteins, fibroblast growthfactors, sonic hedgehog, Notch signaling, and related ele-ments, in appendage morphogenesis and pattern forma-tion17. However, the earliest determinants specifyingappendage pattern remain unknown, despite the substan-tial progress in this area. The IKKa2/2 mice represent thefirst demonstration in vivo of a signaling moleculeinvolved in establishing an important, early stage of differ-entiation of interfollicular epidermis. It is too early toknow whether IKKa participates directly in the signalingpathway that specifies the initial appearance of the

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TIG November 2000, volume 16, No. 11 483

FIGURE 3. Comparison of the skin of normal, Er/Erand pf/pf mice

Histology of skin from (a) normal newborn, (b) newborn Er/Er and (c) newbornpf/pf mice. Normal epidermis has stratum corneum, but this fails to form ineither Er/Er or pf/pf mice. The granular layer of Er/Er mutants is partially formedand contains keratohyalin granules (KG). This layer is missing from pf/pf mice.Blood vessels (arrow) in pf/pf epidermis extend high into epidermis.

King-Chuen Chowbckchow@ust.hk

Department ofBiochemistry, The Hong

Kong University ofScience and Technology,

Clear Water Bay,Kowloon, Hong Kong.

terminally differentiating-keratinocyte lineage. An alterna-tive possibility is that loss of IKKa (or pf or Er) promotesa hyperproliferative phenotype that delays the develop-mental program leading to epidermal differentiation.

Nevertheless, IKKa, pf and Er are an intriguing set of elements that might act jointly to specify the initialappearance of a differentiation program that persiststhroughout the life of the mouse.

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TIG November 2000, volume 16, No. 11484

References1 Zandi, E. and Karin, M. (1999) Bridging the gap: Composition,

regulation, and physiological function of the lkΒ kinasecomplex. Mol. Cell. Biol. 19, 4547–4551

2 Takeda, K. et al. (1999) Limb and skin abnormalities in micelacking IKKa. Science 284, 313–316

3 Hu, Y. et al. (1999) Abnormal morphogenesis but intact IKKactivation in mice lacking the IKKa subunit of lkΒ kinase.Science 284, 316–320

4 Li, Q. et al. (1999) IKK1-deficient mice exhibit abnormaldevelopment of skin and skeleton. Genes Develop. 13,1322–1328

5 Meredith, R. (1964) Communication. Mouse News Lett. 31, 256 Guenet, J.L. et al. (1979) Repeated epilation: a genetic

epidermal syndrome in mice. J. Hered. 70, 90–947 Holbrook, K.A. et al. (1982) Abnormal epidermal keratinization

in the repeated epilation mutant mouse. J. Cell Biol. 92,387–397

8 Fisher, C. (1987) Abnormal development in the skin of pupoidfetus mutant mouse: abnormal keratinization, recovery of anormal phenotype, and relationship to the repeated epilation(Er/Er) mutant mouse. Curr. Top. Dev. Biol. 22, 209–234

9 Fisher C. (1994) The Cellular Basis of Development andDifferentiation in Mammalian Keratinizing Epithelia. In: TheKeratinocyte Handbook (Lane, B. et al., eds), CambridgeUniversity Press

10 Fisher, C. et al. (1987) Abnormal expression and processing ofkeratins in pupoid fetus (pf/pf) and repeated epilation (Er/Er)mutant mice. J. Cell Biol. 1807–1819

11 Green, M.C. (1982) Genetic Strains and Variants of theLaboratory Mouse. Fischer, Stuttgart

12 Mouse Genome Database (MGD), Mouse Genome Informatics

Site, The Jackson Laboratory (http://www.informatics.jax.org/).13 Connelly, M.A. and Marcu, K.B. (1995) CHUK, a new member of

the helix-loop-helix and leucine zipper families of interactingproteins, contains a serine-theonine kinase domain. Cell. Mol.Biol. 41, 537–549

14 Mock, B.A. et al. (1995) CHUK, a conserved helix-loop-helixubiquitous kinase, maps to human chromosome 10 andmouse chromosome 19. Genomics 27, 348–351

15 Fisher, C. et al. (1984) Abnormal keratinization in pupoidfetus (pf/pf) mutant mice. Dev. Biol. 102, 290–299

16 Salaün, J. et al. (1986) The differentiation of repeatedepilation (Er/Er) mouse mutant skin in organ culture and ingrafts. Anat. Embryol. 174, 195–205

17 Oro, A.E. and Scott, M.P. (1998) Splitting hairs: Dissectingroles of signaling systems in epidermal development. Cell 95,575–578

It is generally believed that modifications of nucleotidesor DNA segments are the results of errors that occur

during DNA replication and when DNA-repair systemsfail. Such errors eventually lead to genetic variation and,in turn, provide the basis for natural selection. In otherwords, biological evolution derives from errors inbiological functions. Werner Arber argues that, on thecontrary, there are actually several biological functionsthat generate genetic diversity. Therefore, Arber proposesthat biological evolution, rather than being the result of apassive accumulation of errors, is driven by a multitude ofspecific biological functions1 that are conferred by certaingenetic determinants. Arber advocates the term ‘evolutiongenes’ to differentiate these genes from those involved inhousekeeping or accessory functions2–4.

The concept of an ‘evolution gene’ is certainly appeal-ing. However, Arber confines his discussion to the evolu-tionary contribution of genetic determinants whoseencoded products directly affect DNA stability. He hasnot touched on an important issue, namely the activitymodulation of ‘evolution gene’ products, without whichbiological evolution is not much different from chemicalevolution (except that the former is more complex). In thisregard, several lines of evidence point to the fact that theheat-shock protein Hsp70 (also known as DnaK inEscherichia coli) can facilitate many activities that lead tothe generation of genetic diversity. The hsp70 gene has avery special role in the course of biological evolution.

The gene hsp70 should be classified, prima facie, as ahousekeeping gene because, in addition to its well-knownhigh-temperature-induced response for thermal stress pro-tection, this gene is always expressed constitutively undernormal growth conditions, and the gene product isinvolved in protein maturation, translocation and proteo-lysis (see Ref. 5 for a review). However, Hsp70 is also involved in a number of DNA sequence-alteration

activities. Ogata et al. 6 showed that the recombination fre-quency of a dnaK-defective E. coli mutant was impaired.We found that overexpression of dnaK could improve therepairing of mismatched nucleotides7. It seems that in thesetwo cases the presence of Hsp70 can stimulate the activitiesof the RecF-recombination system. DnaK had been shownto participate in DNA repair by enhancing the proper fold-ing of the DNA-repair protein UmuC (Ref. 8). Zou et al.9

were able to demonstrate that, in vitro, the presence ofDnaK could improve the activity of UvrA, which, in turn,increased the loading cycles of UvrB and eventually led to ahigher efficiency of nucleotide excision repair. We had alsoshown that overexpression of dnaK in E. coli could stimu-late the activity of the transposable element Tn5 (Ref. 10).Such stimulation is believed to be mediated by the effect ofDnaK on DNA gyrase. In a very recent bacterial conjuga-tion study, we found that the conjugation efficiency of adnaK-defective E. coli strain was only 1024 of that of thewild type at the permissive temperature of 30°C (K-C.Chow, unpublished). Moreover, we cannot neglect the factthat DnaK was originally shown to be indispensable for λ-phage DNA replication (see Ref. 11 for a review), and theability of the λ phage to carry out transduction and illegitimate recombination has been well documented (seeRef. 12 for a review).

As DNA repair, recombination, transposition, conju-gation and transduction are the very general means adoptedby bacterial systems for the generation or modulation ofgenetic variation, and as all these activities (from pointmutation to horizontal genetic transfer) are facilitated bythe presence of Hsp70, this heat-shock protein can be justifiably referred to, at least in bacterial systems, as an‘evolution facilitator’.

It is interesting to note that the hsp70 gene is present inalmost all organisms, except for a few archaeal species13

(all of which occupy the lowest positions on the universal

Hsp70 (DnaK) – an evolutionfacilitator?

0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(00)020105-3