Each cell lineage specified in the preimplantation mammalian embryo depends on intrinsic factors for its development, but there is also mutual interdependence between them. OCT4 is required for the ICM/epiblast lineage, and at transient high levels for extraembryonic endoderm, but also indirectly through its role in regulating Fgf4 expression, for the establishment and proliferation of extraembryonic ectoderm from polar trophectoderm. The transcription factor SOX2 has also been implicated in the regulation of Fgf4 expression. We have used gene targeting to inactivate Sox2, examining the phenotypic consequences in mutant embryos and in chimeras in which the epiblast is rescued with wild-type ES cells. We find a cell-autonomous requirement for the gene in both epiblast and extraembryonic ectoderm, the multipotent precursors of all embryonic and trophoblast cell types, respectively. However, an earlier role within the ICM may be masked by the persistence of maternal protein, whereas the lack of SOX2 only becomes critical in the chorion after 7.5 days postcoitum. Our data suggest that maternal components could be involved in establishing early cell fate decisions and that a combinatorial code, requiring SOX2 and OCT4, specifies the first three lineages present at implantation. Early embryonic development in mammals is characterized by a series of cell fate decisions that restrict developmental potential in an asymmetric fashion. There is, however, no evidence that this is caused by differential allocation of maternal cytoplasmic determinants as in many other animals. Although the fertilized egg may have a polarity that can predict the definitive axes of the later postimplantation embryo, this at best confers a bias to what is a very regulative system. Cell position seems more important. Thus, the first restriction of developmental potential to the trophectoderm lineage occurs in blastomeres located on the outside of the morula, whereas inside cells become inner cell mass (ICM). Subsequently, the ICM is specified into two lineages, embryonic ectoderm (epiblast), which gives rise to all cell types of the embryo as well as to extraembryonic mesoderm, and extraembryonic (primitive) endoderm, which is found on the surface of the ICM adjacent to the blastocoel cavity and contributes to the yolk sac (Lu et al. 2001).There is a dependence on each of these three early distinct lineages for the survival, patterning, and differentiation of each of the others during subsequent development postimplantation. The polar trophectoderm receives signals from the underlying ICM, triggering its proliferation and differentiation into extraembryonic ectoderm (ExE). This continues to proliferate and gives rise to the various trophoblast cell types of the placenta and to structures such as the chorion. Conversely, the mural trophectoderm, which is not in contact with the ICM, ceases to divide and terminally differentiates into primary trophoblast giant cells (Rossant and Cross 2001). The primitive endoderm also forms two distinct cell types...

Eukaryotic chromosomes are capped with repetitive telomere sequences that protect the ends from damage and rearrangements. Telomere repeats are synthesized by telomerase, a ribonucleic acid (RNA)-protein complex. Here, the cloning of the RNA component of human telomerase, termed hTR, is described. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (TTAGGG)n. Germline tissues and tumor cell lines expressed more hTR than normal somatic cells and tissues, which have no detectable telomerase activity. Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity. HeLa cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings. Thus, human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells.

Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.

The transcription factor SOX2 is expressed most notably in the developing CNS and placodes, where it plays critical roles in embryogenesis. Heterozygous de novo mutations in SOX2 have previously been associated with bilateral anophthalmia/microphthalmia, developmental delay, short stature, and male genital tract abnormalities. Here we investigated the role of Sox2 in murine pituitary development. Mice heterozygous for a targeted disruption of Sox2 did not manifest eye defects, but showed abnormal anterior pituitary development with reduced levels of growth hormone, luteinizing hormone, and thyroid-stimulating hormone. Consequently, we identified 8 individuals (from a cohort of 235 patients) with heterozygous sequence variations in SOX2. Six of these were de novo mutations, predicted to result in truncated protein products, that exhibited partial or complete loss of function (DNA binding, nuclear translocation, or transactivation). Clinical evaluation revealed that, in addition to bilateral eye defects, SOX2 mutations were associated with anterior pituitary hypoplasia and hypogonadotropic hypogonadism, variable defects affecting the corpus callosum and mesial temporal structures, hypothalamic hamartoma, sensorineural hearing loss, and esophageal atresia. Our data show that SOX2 is necessary for the normal development and function of the hypothalamo-pituitary and reproductive axes in both humans and mice. Introduction SOX2 is a member of the sex-determining region of the Y chromosome-related (SRY-related) high-mobility group (HMG) box (SOX) family of transcription factors, encoded by 20 genes in humans and mice, each of which carries a 79-amino acid HMG box DNA-binding domain similar to that of SRY as well as domains implicated in transcriptional regulation (1, 2). Based on HMG box homology, they are grouped into different subfamilies. SOX1, SOX2, and SOX3 belong to the B1 subfamily and are expressed in various phases of embryonic development and cell differentiation, where they play critical roles in embryogenesis (3, 4). All 3 mark neuroepithelial progenitors and stem cells from the earliest stages of development, and there is a strong, but not absolute, tendency for them to be downregulated as cells differentiate.In the mouse, Sox2 RNA is first detected in cells at the morula stage (2.5 dpc) and then in the inner cell mass of the blastocyst (3.5 dpc).

The late cornified envelope (LCE) gene cluster within the epidermal differentiation complex on human chromosome one (mouse chromosome three) contains multiple conserved genes encoding stratum-corneum proteins. Within the LCE cluster, genes form "groups" based on chromosomal position and protein homology. We link a recently accepted nomenclature for the LCE cluster (formerly XP5, small proline-rich-like, late-envelope protein genes) to gene structure, groupings, and chromosomal organization, and carry out a pan-cluster quantitative expression analysis in a variety of tissues and environmental conditions. This analysis shows that (i) the cluster organizes into two "skin" expressing groups and a third group with low-level, tissue-specific expression patterns in all barrier-forming epithelia tested, including internal epithelia; (ii) LCE genes respond "group-wise" to environmental stimuli such as calcium levels and ultraviolet (UV) light, highlighting the functional significance of groups; (iii) in response to UV stimulation there is massive upregulation of a single, normally quiescent, non-skin LCE gene; and (iv) heterogeneity occurs between individuals with one individual lacking expression of an LCE skin gene without overt skin disease, suggesting LCE genes affect subtle attributes of skin function. This quantitative and pan-cluster expression analysis suggests that LCE groups have distinct functions and that within groups regulatory diversification permits specific responsiveness to environmental challenge.

AKT activity has been reported in the epidermis associated with keratinocyte survival and differentiation. We show in developing skin that Akt activity associates first with post-proliferative, para-basal keratinocytes and later with terminally differentiated keratinocytes that are forming the fetal stratum corneum. In adult epidermis the dominant Akt activity is in these highly differentiated granular keratinocytes, involved in stratum corneum assembly. Stratum corneum is crucial for protective barrier activity, and its formation involves complex and poorly understood processes such as nuclear dissolution, keratin filament aggregation, and assembly of a multiprotein cell cornified envelope. A key protein in these processes is filaggrin. We show that one target of Akt in granular keratinocytes is HspB1 (heat shock protein 27). Loss of epidermal HspB1 caused hyperkeratinization and misprocessing of filaggrin. Akt-mediated HspB1 phosphorylation promotes a transient interaction with filaggrin and intracellular redistribution of HspB1. This is the first demonstration of a specific interaction between HspB1 and a stratum corneum protein and indicates that HspB1 has chaperone activity during stratum corneum formation. This work demonstrates a new role for Akt in epidermis.The epidermis is the primary environmental barrier, protecting from infection, allergens, and damage from UV radiation. The major constituent of this epidermal barrier is the terminally differentiated, anuclear keratinocyte. This structure is bounded by a cornified envelope, an elaborate, cross-linked protein structure covalently bound to hydrophobic lipid externally and aggregated keratin internally (1). Formation of this structure is poorly understood.The complexity of keratinocyte terminal differentiation and the importance of precisely timed and compartmentalized processing is illustrated by the maturation of the stratum corneum protein filaggrin. Filaggrin is synthesized as a high molecular mass precursor comprising multiple subunits that are sequentially processed and modified in temporally and spatially regulated steps by diverse proteases and enzymes to produce mature filaggrin subunits. Mature filaggrin is thought to be important in the aggregation and collapse of the keratin network leading to flattening of the keratinocyte and the destruction of the nucleus in granular layer (terminally differentiating) keratinocytes (2). Filaggrin is also incorporated into the cornified envelope (2). Premature or aberrant filaggrin processing can be catastrophic, leading to disruption of cornified envelope integrity and skin barrier function (3-5) and is far more damaging than reduced filaggrin levels that, in contrast, lead only to mild skin defects (6).Possible regulators of protein processing and trafficking during terminal differentiation are heat shock proteins (Hsps). 2Hsps have diverse roles as cellular chaperones, anti-apoptotic factors, stress-protective proteins, and cytoskeletal stabilizers (7,8). Human HspB1 (Hsp27) (9 -11) and the mouse HspB1...

Telomerase activity was identified in extracts from several different mouse cell lines. Addition of telomeric TTAGGG repeats was specific to telomeric oligonucleotide primers and sensitive to pretreatment with RNase A. In contrast to the hundreds of repeats synthesized by the human and Tetrahymena telomerase enzymes in vitro, mouse telomerase synthesized only one or two TTAGGG repeats onto telomeric primers. The products observed after elongation of primers with circularly permuted (TTAGGG)3 sequences and after chain termination with ddATP or ddTTP indicated that mouse telomerase pauses after the addition of the first dG residue in the sequence TTAGGG. The short length of the products synthesized by mouse telomerase was not due to a diffusible inhibitor in the mouse extract, because the human telomerase continued to synthesize long products when mixed with mouse fractions. Primer challenge experiments showed that the human enzyme synthesized long TTAGGG repeats processively in vitro, whereas the mouse telomerase appeared to be much less processive. The identification of short telomerase reaction products in mouse extracts suggests that extracts from other organisms may also generate only short products. This knowledge may aid in the identification of telomerase activity in organisms where activity has not yet been detected.Telomerase is a highly specialized DNA polymerase which synthesizes telomeric repeat sequences de novo both in vitro and in vivo (reviewed in ref. 1). Net telomere elongation by telomerase may balance the loss of sequences from chromosome ends at each round of DNA replication (2,3). Telomerase is a ribonucleoprotein in which the RNA component provides the template for the synthesis of telomeric repeats (4, 5). Telomerase activity has been identified in the ciliates Tetrahymena, Euplotes, and Oxytricha and in transformed human cells (for review see refs. 1 and 6). In vitro, Tetrahymena telomerase is highly processive, synthesizing hundreds of telomeric (TTGGGG)n repeats (7). Telomere lengthening in Tetrahymena in vivo appears to be much less processive than primer elongation by telomerase in vitro (8), suggesting that telomere length could be regulated in part by telomerase processivity. The large number of yeast genes which affect telomere length (9-13) suggest that length regulation may be a complex process involving telomerase, telomere-binding proteins, and other components.Telomere length regulation is implicated in both cellular senescence and the immortalization of human cells. In primary somatic cells, telomeric (TTAGGG)n repeats are lost with age both in vivo and in vitro. This shortening has been proposed to play a role in signaling the cell cycle exit characteristic of senescent cells (14, 15), although a causal role has not been demonstrated. In contrast to primary human cells, telomere length in immortalized cell lines is stably maintained (16,17). Recent data (18) have shown that telomerase activity was not detectable in primary human cells when telomeres were shortening. ...

Desmosomal adhesion is important for the integrity and protective barrier function of the epidermis and is disregulated during carcinogenesis. Strong adhesion between keratinocytes is conferred by the desmosomal cadherins, desmocollin (Dsc) and desmoglein. These constitute two gene families, members of which are differentially expressed in epidermal strata. It has been suggested that this stratum-specific expression regulates keratinocyte differentiation. We tested this hypothesis by misdirecting the expression of the basally abundant desmosomal cadherins Dsc3a and Dsc3b to suprabasal differentiating keratinocytes in transgenic mice. No phenotype was apparent until adulthood, when mice developed variable ventral alopecia and had altered keratinocyte differentiation within affected areas. The follicular changes were reminiscent of changes in transgenic mice with an altered ␤-catenin stability. Stabilized ␤-catenin and increased ␤-catenin transcriptional activity were demonstrated in transgenic mice prior to the phenotypic change and in transgenic keratinocytes as a consequence of transgene expression. Hence, a link between desmosomal cadherins and ␤-catenin stability and signaling was demonstrated, and it was shown that desmocollin cadherin expression can affect keratinocyte differentiation. Furthermore, the first function for a "b-type" desmocollin cadherin was demonstrated.Desmosomes and adherens junctions are multiprotein adhesive complexes located at epithelial membranes. Both impart adhesion through transmembrane cadherins linked via armadillo family members to the cytoskeleton. At adherens junctions, the armadillo members ␤-catenin and plakoglobin (␥-catenin) are involved in linking classical cadherins to the actin network. Plakoglobin is also a desmosomal component, as it participates with desmoplakin in linking desmosomal cadherins to the keratin intermediate filament cytoskeleton. Additionally, ␤-catenin and plakoglobin have signaling and transcriptional regulatory roles in the cytoplasm and nucleus, where Wnt signaling induces the transient stabilization of ␤-catenin, resulting in nuclear translocation and the regulation of downstream genes in association with the Lef1/Tcf (lymphoid enhancer factor/T-cell factor) transcription factors (reviewed in reference 29). However, a role for desmosomes in cell signaling is still being debated (10, 12).Desmosomal cadherins comprise two families, desmocollins (Dsc) and desmogleins (Dsg), each consisting of multiple isoforms. Isoforms 1 and 3 of Dsc and Dsg are expressed in the stratified layers of the epidermis in a reciprocal graded fashion (32; reviewed in references 9 and 19). Isoform 3 expression is strongest in the basal, proliferative layer, with levels decreasing as keratinocytes differentiate. Isoform 1 levels peak in the upper granular layer, with decreasing levels in the mid-spinous layer, where different cadherins are mixed within individual desmosomes. Dsc2 and Dsg2 are expressed weakly in epidermal basal layers, and recently discovered isoforms homolog...