Systematic elimination of parthenogenetic cells in mouse chimeras

The developmental potential of primitive ectoderm cells lacking paternal chromosomes was investigated by examining the distribution of parthenogenetic cells in chimeras. Using GPI-1 allozymes as marker, parthenogenetic cells were detected in most organs and tissues in adult chimeras. However, these cells were under severe selective pressure compared with cells from normal fertilized embryos. In the majority of chimeras, parthenogenetic cells in individual animals were observed in a limited number of tissues and organs and, even in these instances, their contribution was substantially reduced. Nevertheless, parthenogenetic cells were detected more consistently in some organs, especially the brain, heart, kidney and spleen. In contrast, there was apparently a systematic selection against parthenogenetic cells in some tissues, most notably in skeletal muscle, liver and pancreas. These results suggest that paternally derived genes are probably required not only for the development of extraembryonic structures but also for subsequent development of embryonic tissues derived from the primitive ectoderm lineage.     

Tissue specific loss of proliferative capacity of parthenogenetic cells in fetal mouse chimeras

Parthenogenetic cells are lost from fetal chimeras. This may be due to decreased proliferative potential. To address this question, we have made use of combined cell lineage and cell proliferation analysis. Thus, the incorporation of bromodeoxyuridine in S-phase was determined for both parthenogenetic and normal cells in several tissues of fetal day 13 and 17 chimeras. A pronounced reduction of bromodesoxyuridine incorporation by parthenogenetic cells at both developmental stages was only observed in cartilage. In brain, skeletal muscle, heart and intestinal epithelium, this reduction was either less pronounced or observed only at one of the developmental stages analysed. No difference between parthenogenetic and normal cells was observed in epidermis and ganglia. Our results show that a loss of proliferative potential of parthenogenetic cells during fetal development contributes to their rapid elimination in some tissues. The analysis of the fate of parthenogenetic cells in skeletal muscle and cartilage development demonstrated different selection mechanisms in these tissues. In skeletal muscle, parthenogenetic cells were largely excluded from the myogenic lineage proper by early post-midgestation. In primary hyaline cartilage, parthenogenetic cells persisted into adulthood but were lost from cartilages that undergo ossification during late fetal development.     

Temporal and spatial selection against parthenogenetic cells during development of fetal chimeras

The fate of parthenogenetic cells was investigated during development of fetal and early postnatal chimeras. On day 13 of embryonic development, considerable contribution of parthenogenetic cells was observed in all tissues of chimeric embryos, although selection against parthenogenetic cells seemed to start before day 13. Between days 13 and 15 of development, parthenogenetic cells came under severe selective pressure, which was most striking in tongue. The disappearance of parthenogenetic cells from tongue coincided with the beginning of myoblast fusion in this tissue. Severe selection against parthenogenetic cells was also observed in pancreas and liver, although in the latter, parthenogenetic cells were eliminated later than in skeletal muscle or pancreas. In other tissues, parthenogenetic cells may persist and participate to a considerable extent throughout the gestation period and beyond, although a significant decrease was observed in all tissues. Parthenogenetic in equilibrium fertilized chimeras were significantly smaller than their non-chimeric littermates at all developmental stages. These results suggest that the absence of paternal chromosomes is largely incompatible with the maintenance of specific differentiated cell types. Furthermore, paternally derived genes seem to be involved in the regulation of proliferation of all cell types, as indicated by the drastic growth decceleration of parthenogenetic in equilibrium fertilized chimeras and the overall decrease of parthenogenetic cells during fetal development. Chromosomal imprinting may have a role in maintaining a balance between cell growth and differentiation during embryonic development. The major exception to the selective elimination of parthenogenetic cells appear to be the germ cells; viable offspring derived from parthenogenetic oocytes were detected, sometimes at a high frequency in litters of female parthenogenetic in equilibrium fertilized chimeras.     

Postnatal development of parthenogenetic in equilibrium with fertilized mouse aggregation chimeras

Chimeras were made from parthenogenetic and fertilized cleavage-stage mouse embryos. The perinatal mortality was high. The parthenogenetic contributions to different tissues at birth ranged from 0 to 50%. No selection of parthenogenetic cells was observed in the pigmentation of the coat, but this does not exclude that such selection could act in other tissues. The weight of chimeras at birth negatively correlated to the average contribution of the parthenogenetic part. The growth rate of chimeras was lower than that of nonchimeric animals. The data presented demonstrated that, although parthenogenetic cells are not cell lethals and they can participate to some degree in normal development of most tissues, their extensive presence reduces the viability of chimeras and retards the postnatal development.     

Efficacious Production of Viable Germ-Line Chimeras between Embryonic Stem (ES) Cells and 8-Cell Stage Embryos

Many embryonic stem (ES) cell lines have been isolated from various mouse strains, but production of germ-line chimeras has been achieved with only strain 129. This report describes the isolation of a new ES cell line, F1/1, from a mouse blastocyst with the C57BL/6 X CBA male genotype and tests on its ability to produce germ-line chimeras by two techniques, blastocyst injection and 8-cell embryo injection. Chimera production using CD-1 blastocysts as a host was low (20%), as reported by others. But by the 8-cell embryo injection method, in which F1/1 cells were injected into the perivitelline space through a slit in the zona pellucida of 8-cell embryos, chimeric mice with extremely high chimerism were obtained at a rate of 80%. Breeding tests showed that 89% of the fertile males were germ-line chimeras and in most case, the majority of the sperms in their testes were derived from F1/1 cells. This F1/1 cell line with a different genotype from the 129 strain shows high ability to produce functional germ cells, moreover, the 8-cell embryo injection method using F1/1 cells seems to be an efficient way to produce viable germ-line chimeras.     

Development to term of mouse androgenetic aggregation chimeras.

Diploid androgenetic eggs contain two sperm-derived genomes, and only rarely develop to the early somite stage. Also, previous studies have indicated that androgenetic eggs cannot be rescued in aggregation chimeras beyond embryonic stages. Paradoxically, in blastocyst injection chimeras made with androgenetic embryonic stem (ES) cells of the 129/Sv strain, we previously obtained considerable improvement in developmental potential. Although considerable death occurred in utero, overtly normal chimeric fetuses and occasional postnatal chimeras that developed skeletal abnormalities were observed. Consequently, we have re-evaluated the developmental potential of androgenetic aggregation chimeras utilizing androgenetic eggs of the 129/Sv strain, and of the BALB/c and CD-1 strains for comparison. Regardless of strain, androgenetic aggregation chimeras were generally more inviable than previously observed with androgenetic ES cell chimeras, and often the embryoproper was abnormal even when an androgenetic contribution was detected only in the extra-embryonic membranes. This is at least a partial explanation of the greater viability of androgenetic ES cell chimeras, as ES cells do not colonize significantly certain extra-embryonic tissues. Nevertheless, in the 129/Sv strain, occasional development of chimeras to term was obtained, and one chimera that survived postnatally developed identical skeletal abnormalities to those observed previously in androgenetic ES cell chimeras. This result demonstrates that at least one example of paternal imprinting is faithfully conserved in androgenetic ES cells. Also, the postnatal chimerism shows that androgenetic eggs can give rise to terminally differentiated cell types, and are therefore pluripotent. In contrast, only possibly one BALB/c and no CD-1 androgenetic aggregation chimeras developed to term. Therefore, the developmental potential of androgenetic aggregation chimeras is to some extent dependent on mouse strain.     

Teratogenic effects of parthenogenetic cells from LTXBO mice,a strain which develops ovarian teratomas at high frequency

Summary LTXBO mice develop ovarian teratomas at high frequency. The phenotype of tumour tissues is unusual in that most contain trophoblast elements. Since the tumours are derived from parthenogenetically activated oocytes, they would not be expected to produce trophoblast. The developmental potential of parthenogenetic cells from these mice was tested in aggregation chimeras. No contribution to trophoblast tissues was observed. However, a high incidence of morphological abnormalities was seen, suggesting that the parthenogenetic cells exerted a teratogenic effect.     

Microinjection of cytoplasm or mitochondria derived from somatic cells affects parthenogenetic development of murine oocytes

Cloned mammals are readily obtained by nuclear transfer using cultured somatic cells; however, the rate of generating live offspring from the reconstructed embryos remains low. In nuclear transfer procedures, varying quantities of donor cell mitochondria are transferred with nuclei into recipient oocytes, and mitochondrial heteroplasmy has been observed. A mouse model was used to examine whether transferred mitochondria affect the development of the reconstructed oocytes. Cytoplasm or purified mitochondria from somatic cells derived from the external ear, skeletal muscle, and testis of Mus spretus mice or cumulus cells of Mus musculus domesticus mice were transferred into M. m. domesticus (B6SJLF1 and B6D2F1) oocytes to observe parthenogenetic development through the morula stage. All B6D2F1 oocytes injected with somatic cytoplasm or mitochondria showed delayed development when compared to oocytes injected with buffer. The developmental rates were not different among injected cell sources, with the exception of testis-derived donor cells injected into B6SJLF1 oocytes (P < 0.01). The developmental rate of B6D2F1 oocytes injected with buffer alone (98.8% survival) was different from those injected with somatic cytoplasm (60.8% survival) or somatic mitochondria (56.5% survival) (P < 0.01). Conversely, injection of ooplasm into B6D2F1 oocytes did not affect parthenogenetic development (100% survival). Our results indicate that injection of somatic cytoplasm or mitochondria affected parthenogenetic development of murine oocytes. These results have further implications for in vitro fertilization protocols employing ooplasmic transfer where primary oocyte failure is not confirmed.     

An analysis of parthogenetic cell clones in chimeric C57BL/6(PG)<-->BALB/c mice

We studied the distribution of parthenogenetic cell clones in the retinal pigment epithelium and choroid of eyes on serial sections and in the brain, kidneys, and liver by electrophoretic analysis of glucose phosphate isomerase isozymes in 12 mouse chimeras C57BL/6(PG)<-->BALB/c obtained earlier. Asymmetry was noted in the distribution of the parthenogenetic cell clones in the eye structure, just as the earlier established asymmetry in the distribution of the parthenogenetic clones of epidermal melanoblasts. A high correlation was shown between the ratio of parthenogenetic to normal cells in the retinal pigment epithelium of the right or left eyes and epidermal melanoblasts in the hair cover of the corresponding body half of the chimera. These data suggest that there is a certain relationship between the processes leading to the characteristic distribution of the ectodermal parthenogenetic clones in the retinal pigment epithelium of the right and left eyes and epidermal melanoblasts in parthenogenetic chimeras. Electrophoretic analysis did not show parthenogenetic components in the liver or kidneys of any chimera, and the parthenogenetic component was found in the brain of only two chimeras, in which a high percentage of parthenogenetic cells of ectodermal origin was noted. In these cases, asymmetry was noted in the right and left cerebral hemispheres, just as in the retinal pigment epithelium of the right and left eyes. The data obtained suggest that, during the development of the chimeras, parthenogenetic C57BL/6 cells were actively eliminated from the tissues of endodermal and mesodermal origin. In adult chimeras C57BL/6(PG)<-->BALB/c, parthenogenetic cell clones of ectodermal origin are mostly preserved.     

Androgenetic mouse embryonic stem cells are pluripotent and cause skeletal defects in chimeras: implications for genetic imprinting

The inviability of diploid androgenetic and parthenogenetic embryos suggests imprinting of paternal and maternal genes during germ cell development, and differential expression of loci depending on parental inheritance appears to be involved. To facilitate identification of imprinted genes, we have derived diploid androgenetic embryonic stem (ES) cell lines. In contrast to normal ES cells, they form tumors composed almost entirely of striated muscle when injected subcutaneously into adult mice. They also form chimeras following blastocyst injection, although many chimeras die at early postnatal stages. Surviving chimeras develop skeletal abnormalities, particularly in the rib cartilage. These results demonstrate that androgenetic ES cells are pluripotent and point to stage- and cell-specific expression of developmentally important imprinted genes.     

Embryonic origin of skeletal muscle cells in the iris of the duck and quail

Summary The origin of skeletal muscle cells in avian iris muscle was investigated by quantitative analysis of heterochromatin profiles at the electron-microscopic level in irides of six types of quail-duck chimeras. Each of the following tissues was transplanted into the head region from quail to duck between stages 9 and 10: cranial neural crest; trunk neural crest; midbrain and adjacent mesoderm; forebrain; forebrain without neural crest; and forebrain without neural crest and mesoderm. The average ratio of heterochromatin profile to nucleus profile in iris skeletal muscle cells was high (quail type) in the dorsal iris, but low (duck type) in the ventral iris of the chimeras resulting from isotopic transplantation of cranial neural crest. Heterotopic transplantation of trunk neural crest to cranial position resulted in failure of development of skeletal muscle cells in the dorsal iris, but not in the appearance of skeletal muscle cells in the ventral iris. The average ratio of heterochromatin profile to nucleus profile in iris skeletal muscle cells was high in the chimeras resulting from transplantation of midbrain region and the chimeras resulting from transplantation of forebrain region, intermediate in the chimeras resulting from transplantation of forebrain region without neural crest, and low in the chimeras resulting from transplantation of forebrain region without neural crest and mesoderm. These results indicate that the skeletal muscle cells in the dorsal iris are of cranial neural crest origin while those in the ventral iris are not, and could possibly arise from cranial mesoderm.     

The development potential of parthenogenetically derived cells in chimeric mouse embryos: implications for action of imprinted genes

Parthenogenetic embryos of mice die shortly after implantation and characteristically contain poorly developed extraembryonic tissue. To investigate the basis of the abnormal development of parthenotes, we combined them with normal embryos to produce chimeras and examined the distribution of the parthenogenetically derived cells during preimplantation and early postimplantation development. The parthenogenetic embryos were derived from a transgenic mouse line bearing a large insert, which allowed these cells to be identified in histological sections using in situ hybridization. At the blastocyst stage, the parthenogenetic embryos contributed cells to the trophectoderm (TE) and inner cell mass (ICM) of chimeras. By 6.5 days, however, in almost every embryo, parthenogenetically derived cells were not detected in the extraembryonic trophoblast tissue descended from the TE. In contrast, parthenogenetically derived cells could contribute to all descendants of the ICM of 6.5-and 7.5-day chimeras, including the extraembryonic visceral and parietal endoderm. Quantitative analysis of the degree of chimerism in the embryonic ectoderm at 6.5-7.5 days indicated that parthenogenetically derived cells could contribute as extensively as normal cells. These results indicate that normal trophoblast development requires gene expression from the paternally inherited genome before 6.5 days of embryogenesis. Tissues of the ICM lineage, however, apparently can develop independently of the paternal genome at least to 7.5 days of embryogenesis. Comparison of these results with those of others suggests that the influence of imprinted genes is manifested at different times and in a variety of tissues during development.     

Fate of haploid parthenogenetic cells in mouse chimeras during development

The developmental capability of haploid parthenogenetic cells was investigated by studies on haploid parthenogenetic in equilibrium fertilized mouse chimeras. Two chimeras were born. One female chimera was smaller at birth and grew slower than its littermates. The distribution of haploid-derived cells in the chimeras was analyzed 11 months after their birth. Cells derived from haploid embryos were found only in the brain, eyes, pigment cells in hair follicles, and spleen, in which they constituted 30%, 20%, 10%, and less than 5%, respectively, of the cells. The correlation between the parthenogenetic contribution to the brain and growth retardation is discussed. All of the cells examined in these chimeric organs (brain and eyes) contained a diploid amount of DNA, suggesting that diploidization of the haploid parthenogenetic cells occurred during development. Possibly, the haploid state is not sufficient for cell growth, even in chimeras with fertilized embryos.     

The Perfect Host: A Mouse Host Embryo Facilitating More Efficient Germ Line Transmission of Genetically Modified Embryonic Stem Cells

There is a continual need to improve efficiency in creating precise genetic modifications in mice using embryonic stem cells (ESCs). We describe a novel approach resulting in 100% germline transmission from competent injected ESCs. We developed an F1 mouse host embryo (Perfect Host, PH) that selectively ablates its own germ cells via tissue-specific induction of diphtheria toxin. This approach allows competent microinjected ESCs to fully dominate the germline, eliminating competition for this critical niche in the developing and adult animal. This is in contrast to conventional methods, where competition from host germ cells results in offspring derived from host cells and ESCs, necessitating extensive breeding of chimeras and genotyping to identify germline. The germline transmission process is also complicated by variability in the actual number of ESCs that colonize the germline niche and the proportion that are germline competent. To validate the PH approach we used ESC lines derived from 129 F1, BALB/cByJ, and BTBR backgrounds as well as an iPS line. Resulting chimeric males produced 194 offspring, all paternally derived from the introduced stem cells, with no offspring being derived from the host genome. We further tested this approach using eleven genetically modified C57BL/6N ESC lines (International Knockout Mouse Consortium). ESC germline transmission was observed in 9/11 (82%) lines using PH blastocysts, compared to 6/11 (55%) when conventional host blastocysts were used. Furthermore, less than 35% (83/240) of mice born in the first litters from conventional chimeras were confirmed to be of ESC-origin. By comparison, 100% (137/137) of the first litter offspring of PH chimeras were confirmed as ESC-derived. Together, these data demonstrate that the PH approach increases the probability of germline transmission and speeds the generation of ESC derived animals from chimeras. Collectively, this approach reduces the time and costs inherent in the production of genetically modified animals.     

Influence of paternally imprinted genes on development.

The parental origin of chromosomes is critical for normal development in the mouse because some genes are imprinted resulting in a predetermined preferential expression of one of the alleles. Duplication of the paternal (AG: androgenones) or maternal (GG/PG: gynogenones/parthenogenones) genomes will result in an excess or deficiency of gene dosage with corresponding phenotypic effects. Here, we report on the effects of paternally imprinted genes on development following introduction of the AG inner cell mass into normal blastocysts. There was a striking increase in embryonic growth by up to 50%, and a characteristic change in embryonic shape, partly because of the corresponding increase in length of the anterior-posterior axis. These changes, between e12-e15, were proportional to the contribution from AG cells to the embryo. However, a contribution of AG cells in excess of 50% was invariably lethal as development progressed to e15. A limited number of chimeras were capable of full-term development provided there was a relatively low contribution from AG cells. The distribution of AG cells in chimeras was not uniform, especially later in development when there was a disproportionate presence of AG cells in the mesodermally derived tissues. Their contribution was consistently greater in the heart and skeletal muscle, but was considerably lower in the brain. Chimeras detected after birth were either dead or developed severe abnormalities of the skeletal elements, particularly of the ribs which were enlarged, distorted and fused, with greatly increased cartilaginous material with an absence of normal ossification. These phenotypic effects in chimeras are reciprocal to those observed in the presence of GG/PG cells, which resulted in a substantial size reduction approaching 50%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epigenotype switching of imprintable loci in embryonic germ cells

 Expression of imprinted genes is dependent on their parental origin. This is reflected in the heritable differential methylation of parental alleles. The gametic imprints are however reversible as they do not endure for more than one generation. To investigate if the epigenetic changes in male and female germ line are similar or not, we derived embryonic germ (EG) cells from primordial germ cells (PGCs) of day 11.5 and 12.5 male and female embryos. The results demonstrate that they have an equivalent epigenotype. First, chimeras made with EG cells derived from both male and female embryos showed comparable fetal overgrowth and skeletal abnormalities, which are similar to but less severe than those induced by androgenetic embryonic stem (ES) cells. Thus, EG cells derived from female embryos resemble androgenetic ES cells more than parthenogenetic cells. Furthermore, the methylation status of both alleles of a number of loci in EG cells was similar to that of the paternal allele in normal somatic cells. Hence, both alleles of Igf2r region 2, Peg1/Mest, Peg3, Nnat were consistently unmethylated in EG cells as well as in the primary embryonic fibroblasts (PEFs) rescued from chimeras. More strikingly, both alleles of p57kip2 that were also unmethylated in EG cells, underwent de novo methylation in PEFs to resemble a paternal allele in somatic cells. The exceptions were the H19 and Igf2 genes that retained the methylation pattern in PEFs as seen in normal somatic tissues. These studies suggest that the initial epigenetic changes in germ cells of male and female embryos are similar. Received: 1 September 1997 / Accepted: 15 October 1997     

Developmental Potential of Haploid-derived Parthenogenetic Cells in Mouse Chimeric Embryos1

Studies were made on the contribution of haploid-derived parthenogenetic cells to haploid parthenogenetic ? fertilized chimeric embryos on day 9 and 10 of pregnancy. In most cases, the contribution of haploid-derived parthenogenetic cells to embryonic tissues was higher than that to extraembryonic tissues. The contribution of haploid-derived cells to embryonic tissues of some chimeras was more than 90%. Chromosomal analysis showed that actively dividing cells in most chimeric embryos contained about 40 chromosomes, indicating that they were diploidized, as haploid parthenogenetic blastocysts have about 20 chromosomes. Results suggested that haploid-derived parthehogenetic cells in chimeric embryos diploidized spontaneously after the blastocyst stage. These cells were capable of differentiating into most cell types of embryonic tissues, but scarcely differentiated into extraembryonic tissues of day 9 embryos. The fate of haploid-derived parthenogenetic cells during postimplantational development was similar to that of diploid parthenogenetic cells that had been diploidized experimentally in the one-cell stage.     

Genetic analysis of alpha 4 integrin functions in the development of mouse skeletal muscle

It has been suggested, on the basis of immunolocalization studies in vivo and antibody blocking experiments in vitro, that alpha 4 integrins interacting with vascular cell adhesion molecule 1 (VCAM-1) are involved in myogenesis and skeletal muscle development. To test this proposal, we generated embryonic stem (ES) cells homozygous null for the gene encoding the alpha 4 subunit and used them to generate chimeric mice. These chimeric mice showed high contributions of alpha 4- null cells in many tissues, including skeletal muscle, and muscles lacking any detectable (< 2%) alpha 4-positive cells did not reveal any gross morphological abnormalities. Furthermore, assays for in vitro myogenesis using either pure cultures of alpha 4-null myoblasts derived from the chimeras or alpha 4-null ES cells showed conclusively that alpha 4 integrins are not essential for muscle cell fusion and differentiation. Taking these results together, we conclude that alpha 4 integrins appear not to play essential roles in normal skeletal muscle development.     

Differential requirement for utrophin in the induced pluripotent stem cell correction of muscle versus fat in muscular dystrophy mice

Duchenne muscular dystrophy (DMD) is an incurable degenerative muscle disorder. We injected WT mouse induced pluripotent stem cells (iPSCs) into mdx and mdx∶utrophin mutant blastocysts, which are predisposed to develop DMD with an increasing degree of severity (mdx < mdx∶utrophin). In mdx chimeras, iPSC-dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Dystrobrevin was observed in dystrophin-positive and, at a lesser extent, utrophin-positive areas. In the mdx∶utrophin mutant chimeras, although iPSC-dystrophin was also supplied to the muscle sarcolemma, mice still displayed poor skeletal muscle histopathology, and negligible levels of dystrobrevin in dystrophin- and utrophin-negative areas. Not only dystrophin-expressing tissues are affected by iPSCs. Mdx and mdx∶utrophin mice have reduced fat/body weight ratio, but iPSC injection normalized this parameter in both mdx and mdx∶utrophin chimeras, despite the fact that utrophin was compromised in the mdx∶utrophin chimeric fat. The results suggest that the presence of utrophin is required for the iPSC-corrections in skeletal muscle. Furthermore, the results highlight a potential (utrophin-independent) non-cell autonomous role for iPSC-dystrophin in the corrections of non-muscle tissue like fat, which is intimately related to the muscle.     

Skeletal myogenic progenitors in the endothelium of lung and yolk sac

We previously showed that clonable skeletal myogenic cells can be derived from the embryonic aorta but become very rare in the more mature and structured fetal aorta. The aim of this study was to investigate whether, during fetal and postnatal development, these myogenic progenitors progressively disappear or may rather associate with the microvascular district, being thus distributed to virtually all tissues. To test this hypothesis, we used F1 embryos (or mice) from a transgenic line expressing a striated muscle-specific reporter gene (LacZ) crossed with a transgenic line expressing a different endothelial-specific reporter genes (GFP). Endothelial cells were isolated from yolk sac (at E11) and lung (at E11, E17, P1, P10, and P60), two organs embryologically unrelated to paraxial mesoderm, rich in vessels, and devoid of skeletal muscle. Endothelial cells, purified by magnetic bead selection (CD31/PECAM-1(+)) or cell sorting (Tie2-GFP(+)) were then challenged for their skeletal myogenic potential in vitro and in vivo.The results demonstrated that both yolk sac and lung contain progenitor cells, which express endothelial markers and are endowed with a skeletal myogenic potential that they reveal when in the presence of differentiating myoblasts, in vitro, and regenerating muscle, in vivo.The number (or potency to generate skeletal muscle) of these vessels associated cells decreases rapidly with age and is very low in mature animals, possibly correlating with reduced regenerative capacity of adult mammalian tissues.