Cytoglobin is a recently discovered vertebrate globin distantly related to myoglobin, and its function is unknown. Here we present the first detailed analysis of the distribution and expression of cytoglobin. Northern and Western blotting experiments show the presence of cytoglobin mRNA and protein in a broad range of tissues. Quantitative PCR demonstrates an up-regulation of cytoglobin mRNA levels in rat heart and liver under hypoxic conditions (22 and 44 h of 9% oxygen). Immunofluorescence studies with three antibodies directed against different epitopes of the protein consistently show cytoglobin in connective tissue fibroblasts as well as in hepatic stellate cells. Cytoglobin is also present in chondroblasts and osteoblasts and shows a decreased level of expression upon differentiation to chondrocytes and osteocytes. Cytoglobin is located in the cytoplasm of these cell types. Evidence against an exclusively nuclear localization of cytoglobin, as recently proposed, is also provided by transfection assays with green fluorescent protein fusion constructs, which demonstrates the absence of an active nuclear import. The differential expression of cytoglobin argues against a general respiratory function of this molecule, but rather indicates a connective tissue-specific function. We hypothesize that cytoglobin may be involved in collagen synthesis. Cytoglobin expression was also observed in some neuronal subpopulations of the central and the peripheral nervous systems. Surprisingly, cytoglobin is localized in both the cytoplasm and nucleus of neurons, indicating a possible additional role of this protein in neuronal tissues.

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Achondroplasia, the most common form of dwarfism in man, is a dominant genetic disorder caused by a point mutation (G380R) in the transmembrane region of fibroblast growth factor receptor 3 (FGFR3). We used gene targeting to introduce the human achondroplasia mutation into the murine FGFR3 gene. Heterozygotes for this point mutation that carried the neo cassette were normal whereas neo ؉ homozygotes had a phenotype similar to FGFR3-deficient mice, exhibiting bone overgrowth. This was because of interference with mRNA processing in the presence of the neo cassette. Removal of the neo selection marker by Cre͞loxP recombination yielded a dominant dwarf phenotype. These mice are distinguished by their small size, shortened craniofacial area, hypoplasia of the midface with protruding incisors, distorted brain case with anteriorly shifted foramen magnum, kyphosis, and narrowed and distorted growth plates in the long bones, vertebrae, and ribs. These experiments demonstrate that achondroplasia results from a gain-of-FGFR3-function leading to inhibition of chondrocyte proliferation. These achondroplastic dwarf mice represent a reliable and useful model for developing drugs for potential treatment of the human disease.Four fibroblast growth factor receptors (FGFRs) are known (1), and Ͼ50 mutations in three of them (FGFR1, 2, and 3) recently have been implicated in congenital skeletal and cranial disorders (reviewed in refs. 2-4). Achondroplasia, the most common form of dwarfism, was shown to be linked to a single point mutation, G380R, in the transmembrane region of FGFR3 (5, 6). FGFR3 is expressed mainly by developing bones, brain, lung, and spinal cord (7,8), and FGFR3-deficient mice show enhanced endochondral bone growth, expansion of their growth plate, and increased chondrocyte proliferation (9, 10). Thus, FGFR3 is a negative regulator of bone growth. Several experiments at the cellular level indicated that the Ach mutation (G380R) results in a constitutive activation of the receptor in a ligand-independent manner (11-13). It was suggested that this is because of stabilization of receptor dimers, a prerequisite for signal transduction in these receptors (14). This is also consistent with the constitutive activation by dimer formation described for an erbB2 (neu) receptor mutant, which carries a Val-to-Glu mutation in an analogous position to that of the FGFR3 variant in its transmembrane region (15). It is likely that, in many of the other mutations in FGFR1-3, the underlying mechanism of receptor activation is also through stabilization of receptor dimers because many of these mutations result in unpaired cysteines that may enhance inter-receptor disulfide bonds (2-4).To generate an animal model for this type of mutation and to study the role of the mutated FGFR3 in vivo, we used gene targeting to introduce the achondroplasia mutation (G380R) into murine FGFR3. This resulted in a dominant dwarf phenotype that exhibits many of the features of human achondroplasia. This is an indication from in vivo data for ...

The blind mole rat (BMR), Spalax galili, is an excellent model for studying mammalian adaptation to life underground and medical applications. The BMR spends its entire life underground, protecting itself from predators and climatic fluctuations while challenging it with multiple stressors such as darkness, hypoxia, hypercapnia, energetics and high pathonecity. Here we sequence and analyse the BMR genome and transcriptome, highlighting the possible genomic adaptive responses to the underground stressors. Our results show high rates of RNA/DNA editing, reduced chromosome rearrangements, an over-representation of short interspersed elements (SINEs) probably linked to hypoxia tolerance, degeneration of vision and progression of photoperiodic perception, tolerance to hypercapnia and hypoxia and resistance to cancer. The remarkable traits of the BMR, together with its genomic and transcriptomic information, enhance our understanding of adaptation to extreme environments and will enable the utilization of BMR models for biomedical research in the fight against cancer, stroke and cardiovascular diseases.

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The tumor suppressor gene p53 controls cellular response to a variety of stress conditions, including DNA damage and hypoxia, leading to growth arrest and͞or apoptosis. Inactivation of p53, found in 40 -50% of human cancers, confers selective advantage under hypoxic microenvironment during tumor progression. The mole rat, Spalax, spends its entire life cycle underground at decidedly lower oxygen tensions than any other mammal studied. Because a wide range of respiratory adaptations to hypoxic stress evolved in Spalax, we speculated that it might also have developed hypoxia adaptation mechanisms analogous to the genetic͞epige-netic alterations acquired during tumor progression. Comparing Spalax with human and mouse p53 revealed an arginine (R) to lysine (K) substitution in Spalax (Arg-174 in human) in the DNAbinding domain, identical to known tumor associated mutations. Multiple p53 sequence alignments with 41 additional species confirmed that Arg-174 is highly conserved. Reporter assays uncovered that Spalax p53 protein is unable to induce apoptosisregulating target genes, resulting in no expression of apaf1 and partial expression of puma, pten, and noxa. However, cell cycle arrest and p53 stabilization͞homeostasis genes were overactivated by Spalax p53. Lys-174 was found critical for apaf1 expression inactivation. A DNA-free p53 structure model predicts that Arg-174 is important for dimerization, whereas Spalax Lys-174 prevents such interactions. Similar neighboring mutations found in human tumors favor growth arrest rather than apoptosis. We hypothesize that, in an analogy with human tumor progression, Spalax underwent remarkable adaptive p53 evolution during 40 million years of underground hypoxic life.

Blind subterranean mole rats (Spalax, Spalacidae) evolved adaptive strategies to cope with hypoxia that climaxes during winter floods in their burrows. By using real-time PCR, we compared gene expression of erythropoietin (Epo), a key regulator of circulating erythrocytes, and hypoxia-inducible factor 1␣ (HIF-1␣), Epo expression inducer, in the kidneys of Spalax and white rats, Rattus norvegicus. Our results show significantly higher, quicker, and longer responses to different O 2 levels in Spalax compared with Rattus. (i) In normoxia, both Spalax and Rattus kidneys produce small amounts of Epo. Maximal expression of Rattus Epo is noticed after a 4-h hypoxia at 6% O 2. Under these conditions, Spalax Epo levels are 3-fold higher than in Rattus. After 24 h of 10% O 2, Spalax Epo reaches its maximal expression, remarkably 6-fold higher than the maximum in Rattus; (ii) the HIF-1␣ level in normoxia is 2-fold higher in Spalax than in Rattus. Spalax HIF-1␣ achieves maximal expression after 4-h hypoxia at 3% O 2, a 2-fold increase compared with normoxia, whereas no significant change was detected in Rattus HIF-1␣ at any of the conditions studied; (iii) at 6% O2 for 10 h, in which Rattus cannot survive, Epo and HIF-1␣ levels in Spalax galili, living in heavily flooded soils, are higher than in Spalax judaei, residing in light aerated soil. We suggest that this pattern of Epo and HIF-1␣ expression is a substantial contribution to the adaptive strategy of hypoxia tolerance in Spalax, evolved during 40 million years of evolution to cope with underground hypoxic stress.

Binding of cellular growth factors to their receptors constitutes a highly specific interaction and the basis for cell and tissue‐type specific growth and differentiation. A unique feature of fibroblast growth factor (FGF) receptors is the multitude of structural variants and an unprecedented degree of cross‐reactivity between receptors and their various ligands. To examine receptor‐ligand specificity within these families of growth factors and receptors, we used genetic engineering to substitute discrete regions between Bek/FGFR2 and the closely related keratinocyte growth factor receptor (KGFR). We demonstrate that a confined, 50 amino acid, variable region within the third immunoglobulin‐like domain of Bek and KGFR exclusively determines their ligand binding specificities. Replacing the variable region of Bek/FGFR2 with the corresponding sequence of KGFR resulted in a chimeric receptor which bound KGF and had lost the capacity to bind basic FGF. We present evidence that the two variable sequences are encoded by two distinct exons that map close together in the mouse genome and follow a constant exon, suggesting that the two receptors were derived from a common gene by mutually exclusive alternative mRNA splicing. These results identify the C‐terminal half of the third immunoglobulin‐like domain of FGF receptors as a major determinant for ligand binding and present a novel genetic mechanism for altering receptor‐ligand specificity and generating receptor diversity.