Liane B. Russell

The mouse short ear skeletal morphogenesis locus is associated with defects in a bone morphogenetic member of the TGFβ superfamily

The mouse short ear gene is required for normal growth and patterning of skeletal structures, and for repair of bone fractures in adults. We have carried out an extensive chromosome walk in the chromosome region that surrounds this locus. Here we show that the short ear region contains the gene for a TGF beta-related protein called bone morphogenetic protein 5 (Bmp-5). This gene is deleted or rearranged in several independent mutations at the short ear locus. Mice homozygous for large deletions of the Bmp-5 coding region are viable and fertile. Mutations at the short ear locus provide an important new tool for defining the normal functions of BMPs in mammals. The specific skeletal defects seen in short-eared animals, which occur against a background of otherwise normal skeletal structures, suggest that particular aspects of skeletal morphology may be determined by individual members of a family of signaling factors that can induce the formation of cartilage and bone in vivo.

Radiation dose rate and mutation frequency.

New data have clearly confirmed the earlier finding that specific locus mutation rates obtained with chronic gamma irradiation of spermatogonia are lower than those obtained with acute x-rays. Since this result is in contrast to classical findings for Drosophila spermatozoa, and apparently contradicts one of the basic tenets of radiation genetics, it was important to determine what factors were responsible for it. Experiments undertaken for this purpose reveal the following: (i) the lower mutation frequency is due mainly to difference in dose rate of radiation, rather than quality; (ii) a dose-rate effect is not obtained in experiments with mouse spermatozoa, confirming classical findings for spermatozoa, and indicating that the explanation for intensity dependence in spermatogonia resides in some characteristic of gametogenic stage; and (iii) a dose-rate effect is found not only in spermatogonia but also in oocytes, where cell selection is improbable, indicating that the radiation intensity effect is on the mutation process itself. A threshold response for all mutations in spermatogonia and oocytes is not a necessary consequence of the findings. Plausible hypotheses consistent with the present results can lead to other predictions. From a practical point of view, the results indicate that the genetic hazards, at least under some radiation conditions, may not be as great as those estimated from the mutation rates obtained with acute irradiation. However, it should not be forgotten that even the lower mutation rates obtained with the present intensity levels are still appreciable (16).

Mammalian X-Chromosome Action: Inactivation Limited in Spread and in Region of Origin

In its simplest form the hypothesis of the single-active-X chromosome does not explain variegated-type position effects in the mouse. Inactivity appears not to involve one entire X chromosome; furthermore, even those parts of the chromosome that can change to an inactive state spread inactivation not to the entire attached piece of autosome, but along a gradient to limited distances.

Definition of functional units in a small chromosomal segment of the mouse and its use in interpreting the nature of radiation-induced mutations.

Abstract Complementation mapping of a small chromosomal region of the mouse has defined a linear series of functional units and has aided in the interpretation of the nature of radiation-induced genetic changes. Mutations used for this purpose were derived from specific-locus mutation-rate experiments and were detected by their visible phenotype in combination with the test-stocks linkage-group-2 markers d and se (recombination frequency 0.16%). A total of 235 mutants of this type have been recovered, and 102 of these were used in complementation tests involving over 800 combinations and about 40000 progeny. While the original screening for mutants employed only 2 markers, the subsequent analysis has, so far, revealed 16 complementation groups spanning 8 or 9 functional units. Involvement in a mutation of 2 or more functional units is taken to indicate chromosomal aberration. All mutations are consistent with a linear map, except for 2, which appear to “skip” units but have been shown not to result from recombinationally separable changes. These 2 mutations, 1 of which gives “conversion”-like results in recombination tests, are interpreted as small rearrangements. The remaining multi-unit mutations are, for the time being, taken to be deficiencies. Although there is evidence that homozygous deficiency of either 1 of the marked loci gives a visible effect, this by itself is no proof against the single-unit mutations being “point” mutations (perhaps single base-pair changes). A strong effect of irradiated cell stage can be demonstrated, both on the locus-spectrum ( i.e. , relative frequencies of events involving d , se , or both) and on frequency of mutations that are interpreted as aberrations. The latter ranges from 13.5% in most X-or γ-rayed spermatogonia, through 42.3% in postgonial stages, to 65.6% in oocytes. Within spermatogonia, neutron irradiation and 24-h fractionation of X-rays shifts the distribution in the direction of postgonial results. The rest of the irradiated spermatogonia closely resemble the control distribution.

Genetics of Mammalian Sex Chromosomes

The great strides made during the past two years in the whole field of mammalian cytogenetics have, in particular, enlarged our knowledge of the role of the mammalian sex chromosomes. The following summary briefly lists the most recent discoveries in the mouse, where genetic findings have played a relatively greater role than in the other species of mammals. The male-determining property of the mammalian Y chromosome, established earlier in mouse and man, has been further confirmed by the finding of an XXY mouse, which was detected by genetic means and has been studied cytologically. This animal is a fully viable, phenotypically normal, though sterile, male. Since various doubts concerning detectability of the XXY type have been removed by the discovery of this animal, it can be concluded that the occurrence of XXY in the mouse is extremely rare. It has been shown that the X chromosome of the mouse, when it is involved in certain chromosomal rearrangements, has the power to produce variegated-type position effects, a phenomenon formerly not observed in any animal except Drosophila. The fact that the X chromosome is involved in all four of the known cases of V-type position effect in the mouse indicates that it is strongly heterochromatic, while there may be little heterochromatin on the autosomes. Recent findings have shown that the presence of two X chromosomes is necessary for the expression of the position effect in one of them. This fact, when related to various cytological findings in other species, permits the hypothesis that, in mammals, genic balance requires the action of one X in a manner which precludes realization of its heterochromatic potentialities, so that only any additional Xs present assume the properties characteristic of heterochromatin. A variety of different findings sheds light on the mechanisms that may lead to the occurrence of individuals with abnormal numbers of sex chromosomes. The XXY mouse proves, by virtue of its sex-linked marker genes, that nondisjunction can occur in the first meiotic division of a normal male (a proof not previously provided by human cases of XXY, which could have been of different origin). However, first-meiotic nondisjunction is apparently very rare in males, and there is not yet any evidence that it ever occurs in females. Data from numerous types of crosses involving five sex-linked markers yield the following results: no cases of XMXMY or OXP have occurred to date; XMXPY << XMO; OXP << XMO (where the superscripts M and P designate maternal and paternal derivation, respectively, of the X). The total frequency of XO individuals can be increased by irradiation shortly after fertilization. This treatment has yielded, in addition to XMO, several animals of the OXP constitution, a type that has not yet been found to occur spontaneously. The various findings on spontaneous and induced frequencies of mice with abnormal numbers of sex chromosomes lead to the conclusion that XO individuals are most often the result of events occurring after fertilization. Specifically, it is suggested that there exists a relatively high probability of loss of the paternally contributed sex chromosome some time between fertilization and the first cleavage(32).

Synaptonemal complex analysis of mouse chromosomal rearrangements

Electron microscopy of surface-spread spermatocytes from mice heterozygous for a tandem duplication shows the heteromorphic synaptonemal complex (SC) to comprise two lateral elements of unequal length, the longer of which is buckled out in a characteristic loop, representing the unsynapsed portion of the duplication. The loop is a regular feature of late zygotene-early pachytene nuclei; it is longest at these early stages, but, through equalization of the two axes as a consequence of synaptic adjustment, it is replaced by a normal appearing SC at late pachytene. Because equalization, as indicated by a decrease in the percent difference between axes, may begin shortly after completion of synapsis, estimates of duplication segment length are restricted to a sample selected for least adjustment. — Although the mean position of the loop is constant at various pachytene substages, individual positions vary widely from cell to cell, consistent with the behavior expected of a duplication, but not of a deletion or an inversion. The length of the segment that is duplicated is estimated to be 22% of the normal chromosome, the midpoint of the segment is mapped at 0.61 of the chromosome distal to the kinetochore, and the ends of the segment are mapped at 0.50 to 0.72. Measurements of G-banded mitotic chromosomes give comparable values: duplication length, 24%; midpoint, 0.60, and segment ends, 0.48 and 0.71. This agreement constitutes further validation of the SC/spreading method for detecting and analyzing chromosomal rearrangements at pachytene and substantiates the fidelity with which the axes and SCs represent the behavior of chromosomes in synapsis.

Frequency and nature of specific-locus mutations induced in female mice by radiations and chemicals: a review

The inducibility of heritable mutations in female mammals has been measured in the mouse specific-locus test (SLT). For radiation-induced mutations, a large body of data has been accumulated that includes information about biological and physical factors that influence mutation yields. However, relatively few SLT studies in females have been conducted with chemicals to date. A single estimate of the spontaneous mutation rate in oocytes, 6/536,207, has been derived as the most appropriate one to subtract from experimental rates. This rate is highly significantly below the spontaneous mutation rate in males. Mutations recovered from females mutagenized at any time after about the 12th day post-conception are induced in non-dividing cells. In adult females, most oocytes are arrested in small follicles; maturation from this stage to ovulation takes several weeks. High-dose-rate radiations are more mutagenic in mature and maturing oocytes than in spermatogonia of the male; on the other hand, no clearly induced mutations have been recovered from irradiated arrested oocytes. Efficient repair processes have been invoked to explain the latter finding as well as the upward-curving dose-effect relation for acute irradiation, and the fact that dose protraction drastically reduces mutation yield from mature and maturing oocytes. The dose-protraction effect is much greater than that found in spermatogonia. Radiation-induced mutation rates in embryonic, fetal, and newborn females are overall lower than those in the mature and maturing oocytes of adults. A dose-protraction effect has also been demonstrated at an early developmental stage when the nuclear morphology of mouse oocytes most resembles that of the human. Of only 5 chemicals so far explored for their effect in oocytes, 2 (ethylnitrosourea, ENU, and triethylenemelamine, TEM), and possibly a third (procarbazine hydrochloride, PRC), are mutagenic--with at least one of these (ENU) mutagenic in arrested as well as maturing oocytes. However, the mutation rate is, in each case, lower than for treated male germ cells. By contrast, ENU-induced mutation yield for the maternal genome of the zygote is an order of magnitude higher than that for the zygotes paternal genome or for spermatogonia. A high proportion of mutants derived from chemical treatment of oocytes (including the oocyte genome in zygotes) are mosaics, probably owing to lesions affecting only 1 strand of the DNA. A characteristic of specific-locus mutations induced in oocytes is that they include a considerably higher percentage of large (multi-locus) lesions (LLs) than do mutations induced in spermatogonia.(ABSTRACT TRUNCATED AT 250 WORDS)

RADIATION HAZARDS TO THE EMBRYO AND FETUS

The radiosensitivity of the embryo and fetus is a matter of great practical importance to radiologists, gynecologists, and obstetricians. It is unfortunate, therefore, that the literature on this subject is diffuse and contains numerous apparently contradictory reports and opinions. Cases of grave radiation injury to the embryo or fetus have been summarized and discussed by Goldstein and Murphy in 1929, Gauss (quoting a thesis by Kraemer) in 1930, Flaskamp in 1930, and Miller, Corscaden and Harrar in 1936. Since the time of these reviews, more cases have been described by Murphy, Shirlock and Doll (1942), Jones and Neill (1944), and others. On the other hand, there are scattered reports of normal births following heavy doses of radiation: Robinson (1927) has collected 23 such cases from the earlier literature, Lacomme (1931) reports 2 and Hobbs (1950) 1. Results from recent extensive animal experiments fall into an easily comprehensible over-all picture which dispels some of the apparent contradictions in...

Whole-mammal mutagenicity tests: Evaluation of five methods☆

Transmitted genetic changes in mammals can be used to study all of the main endpoints of mutagenesis: point mutations, chromosome breakage, with or without rearrangement, and chromosome nondisjunction. Four methods most commonly employed in whole-mammal germ-line mutagenicity tests as well as an in vivo somatic prescreen, are summarized. Genetic basis, historical background, description of the test, limitations, and strengths are presented for each of the five systems. The specific-locus test using visible markers is the most reliable and practical method for detecting heritable point mutations, including small deficiencies, and does not require very large numbers of animals for risk extrapolations, if a relatively high dose can be administered without killing germ cells. For the detection of chromosome-breakage events, dominant lethals are useful to determine relative sensitivities of different germ-cell stages of the male, but the heritable-translocation test is more sensitive when the exposed cells are male meiotic and postmeiotic stages. Chromosome breakage events in the female are best revealed through a sex-chromosome loss test, which utilizes genetically marked X chromosomes. The same method can also be employed to detect nondisjunction in either sex. The in vivo somatic mutation test is useful as a prescreen for both point mutations and losses of chromosomal material. The reliability and efficiency of whole-mammal mutagenicity tests must be considered in two contexts: in the assessment of mutagenicity per se (as this applies to genetic changes transmitted to future generations), and in the use of mutagenicity as a possible indicator of carcinogenicity. In the former context, the whole-mammal tests are irreplaceable because they provide the closest practicable approach to the metabolic pathways existing in man, and because there is no array of lower-system tests that can predict the complexity of the response of the different mammalian germ-cell stages (which differ greatly with respect both to their absolute and their relative sensitivities to the induction of various genetic endpoints). In the second context, i.e., the use of mutagenicity as a screen for carcinogenicity in the exposed individual, most of the whole-mammal tests are of more limited utility, because they require several weeks or months for completion. Of the methods discussed, the spot test appears most suitable, because it provides a relatively rapid in vivo system capable of detecting both gene mutations and chromosomal changes of various kinds in somatic cells, including some that have been suggested to be involved in tumor promotion.

Validation of the in vivo somatic mutation method in the mouse as a prescreen for germinal point mutations.

The in-vivo somatic mutation method developed by us in an earlier X-ray experiment was tested for its usefulness in chemical mutagenesis work, specifically in the prescreening for germinal point mutations. In order to explore possible parallelisms, the 7 compounds chosen for study, as well as the genetic markers used, were those with which large-scale specific-locus mutation-rate experiments in germcells had been conducted in the past or were in progress. From 1–3 dose levels were tested for each compound. On day 101/4 after copulation of C57BL females with T males, a single injection of the test compound was administered, and about 2000 offspring altogether were subsequently scored for survival, morphology, and presence of spots of various types. In accordance with our earlier results we found 3 types of spots: white nearmidline ventral spots (WMVS) which probably result from killing of melanocyte precursor cells; spots resulting from misdifferentiation; and the remainder, which probably result from expression of the recessive by one of several mechanisms (RS). Induction of teratogenic effects, which were stage-specific rather than agent-specific, generally paralleled induction of WMVSs. Both are interpreted as resulting from cell killing. Induction of RSs did not always parallel induction of WMVSs, but roughly paralleled relative frequencies of specific-locus mutations induced in spermatogonia by the same compounds. Even though the in vivo somatic-mutation method probably detects genetic changes additional to point mutations, the results indicate that it may be a useful prescreen for germinal specific-locus mutations, provided care is taken to distinguish between the 3 types of spots, only one of which (RS) is indicative of expression of the recessive. Die in vivo-Methode der Induktion somatischer Mutationen, die wir in einem früheren Röntgenstrahlen-Versuch entwickelten, wurde auf ihre Anwendungsmöglichkeit auf dem Gebiet der Chemomutagenese im Hinblick auf ihre Eignung als „Screening“-Test für Punktmutationen in Keimzellen untersucht. Um festzustellen, ob Parallelen zwischen diesem Testsystem und dem „Specific locus“-Test bestehen, wurden 7 Prüfsubstanzen und genetische Marker ausgewählt, für die in der letzten Zeit umfangreiche Untersuchungen über die Mutationsrate in Keimzellen im Rahmen von spezifischen Locus-Testen durchgeführt wurden oder noch laufen. Pro Substanz wurden 1–3 Dosierungen getestet. C57BL-Weibchen erhielten 101/4 Tage nach der Paarung mit T-Männchen eine einmalige Injektion der jeweiligen Testsubstanz. Insgesamt wurden ca. 2000 F1-Tiere auf Überlebensrate, Morphologie und Fellflecke verschiedener Art untersucht. Übereinstimmend mit unseren früheren Untersuchungen wurden drei verschiedene Arten von Fellflecken gefunden: Weiße ventrale nahe der Mittellinie gelegene Flecke (WMVS), die wahrscheinlich auf das Absterben von Melanocyten-Vorläufer-Zellen zurückzuführen sind; Flecke, die auf Störungen bei der Differenzierung zurückzuführen sind und einen dritten Typ, der wahrscheinlich auf der Expression eines rezessiven Gens durch einen von mehreren Mechanismen beruht (RS). Generell verlief die Induktion teratogener Effekte, die mehr Stadienspezifisch als Substanz-spezifisch waren, der Induktion von WMVSs parallel. Beide werden als Folge des Absterbens von Zellen gedeutet. Die Induktion von RSs verlief nicht stets parallel zur Induktion von WMVSs, zeigte jedoch gewisse Parallelen zur relativen Häufigkeit von durch dieselben Substanzen in Spermatogonien hervorgerufenen „Specific locus“-Mutationen. Obwohl die „in vivo somatic mutation method“ möglicherweise zusätzlich zu Punktmutationen noch andere genetische Mutationen erfaßt, deuten die Befunde doch darauf hin, daß diese Methode als „Screening“-Test auf „Specific locus“-Mutationen in Keimzellen geeignet sein könnte. Voraussetzung hierfür ist, daß sorgfältig zwischen den 3 Fellflecktypen unterschieden wird, von denen nur der RS-Typ ein Indiz für die Expression eines rezessiven Mechanismus darstellt.The in-vivo somatic mutation method developed by us in an earlier X-ray experiment was tested for its usefulness in chemical mutagenesis work, specifically in the prescreening for germinal point mutations. In order to explore possible parallelisms, the 7 compounds chosen for study, as well as the genetic markers used, were those with which large-scale specific-locus mutation-rate experiments in germcells had been conducted in the past or were in progress. From 1–3 dose levels were tested for each compound. On day 101/4 after copulation of C57BL females with T males, a single injection of the test compound was administered, and about 2000 offspring altogether were subsequently scored for survival, morphology, and presence of spots of various types.In accordance with our earlier results we found 3 types of spots: white nearmidline ventral spots (WMVS) which probably result from killing of melanocyte precursor cells; spots resulting from misdifferentiation; and the remainder, which probably result from expression of the recessive by one of several mechanisms (RS). Induction of teratogenic effects, which were stage-specific rather than agent-specific, generally paralleled induction of WMVSs. Both are interpreted as resulting from cell killing. Induction of RSs did not always parallel induction of WMVSs, but roughly paralleled relative frequencies of specific-locus mutations induced in spermatogonia by the same compounds. Even though the in vivo somatic-mutation method probably detects genetic changes additional to point mutations, the results indicate that it may be a useful prescreen for germinal specific-locus mutations, provided care is taken to distinguish between the 3 types of spots, only one of which (RS) is indicative of expression of the recessive.ZusammenfassungDie in vivo-Methode der Induktion somatischer Mutationen, die wir in einem früheren Röntgenstrahlen-Versuch entwickelten, wurde auf ihre Anwendungsmöglichkeit auf dem Gebiet der Chemomutagenese im Hinblick auf ihre Eignung als „Screening“-Test für Punktmutationen in Keimzellen untersucht.Um festzustellen, ob Parallelen zwischen diesem Testsystem und dem „Specific locus“-Test bestehen, wurden 7 Prüfsubstanzen und genetische Marker ausgewählt, für die in der letzten Zeit umfangreiche Untersuchungen über die Mutationsrate in Keimzellen im Rahmen von spezifischen Locus-Testen durchgeführt wurden oder noch laufen. Pro Substanz wurden 1–3 Dosierungen getestet. C57BL-Weibchen erhielten 101/4 Tage nach der Paarung mit T-Männchen eine einmalige Injektion der jeweiligen Testsubstanz. Insgesamt wurden ca. 2000 F1-Tiere auf Überlebensrate, Morphologie und Fellflecke verschiedener Art untersucht.Übereinstimmend mit unseren früheren Untersuchungen wurden drei verschiedene Arten von Fellflecken gefunden: Weiße ventrale nahe der Mittellinie gelegene Flecke (WMVS), die wahrscheinlich auf das Absterben von Melanocyten-Vorläufer-Zellen zurückzuführen sind; Flecke, die auf Störungen bei der Differenzierung zurückzuführen sind und einen dritten Typ, der wahrscheinlich auf der Expression eines rezessiven Gens durch einen von mehreren Mechanismen beruht (RS). Generell verlief die Induktion teratogener Effekte, die mehr Stadienspezifisch als Substanz-spezifisch waren, der Induktion von WMVSs parallel. Beide werden als Folge des Absterbens von Zellen gedeutet. Die Induktion von RSs verlief nicht stets parallel zur Induktion von WMVSs, zeigte jedoch gewisse Parallelen zur relativen Häufigkeit von durch dieselben Substanzen in Spermatogonien hervorgerufenen „Specific locus“-Mutationen. Obwohl die „in vivo somatic mutation method“ möglicherweise zusätzlich zu Punktmutationen noch andere genetische Mutationen erfaßt, deuten die Befunde doch darauf hin, daß diese Methode als „Screening“-Test auf „Specific locus“-Mutationen in Keimzellen geeignet sein könnte. Voraussetzung hierfür ist, daß sorgfältig zwischen den 3 Fellflecktypen unterschieden wird, von denen nur der RS-Typ ein Indiz für die Expression eines rezessiven Mechanismus darstellt.