Double Homozygous Mutants Research Articles

Adhesive Subdivisions Intrinsic to the Epithelial Somites

Developing somites express two subtypes of classic cadherin adhesion receptors, N-cadherin and cadherin-11 (cad11). To investigate the role of these adhesion molecules in somite morphogenesis, we analyzed the somites of mice whose N-cadherin and cad11 genes were disrupted. The epithelial somites of N-cadherin null mutant mice were fragmented as reported, whereas those of cad11(−/−) mice showed no structural anomaly. In mice double homozygous for N-cadherin and cad11 mutation, however, somites were further fragmented into smaller clusters than in the N-cadherin-deficient mice, suggesting that these two cadherins cooperate in the maintenance of epithelial somites. Despite the disorganization of epithelial structures, dorsoventral polarity markers were expressed in their correct patterns in all of these mutant somites. Uncx4.1, whose expression is localized only in the caudal region of each somite, was also expressed in a normal pattern in the mutant somites. However, the staining for Uncx4.1 revealed that, in the N-cadherin mutants, each somite tended to be cleaved at the border between the Uncx4.1-positive and -negative regions and that the cleaved subunits maintained the clustered state, often exhibiting epithelioid morphology. This separation of the rostral and caudal regions was observed as soon as the epithelial somites had been formed. In the N-cadherin/cad11 double-homozygous mutants, this tendency was also observed, although each half of the somite further disintegrated into randomly arranged cell clusters. These results suggest that cells of the rostral and caudal regions of each epithelial somite have an activity to aggregate independently or separate from one another and that one role of N-cadherin and cad11 is to connect the two halves into a single unit.

HNF3beta and Lim1 interact in the visceral endoderm to regulate primitive streak formation and anterior-posterior polarity in the mouse embryo.

Recent embryological and genetic experiments have suggested that the anterior visceral endoderm and the anterior primitive streak of the early mouse gastrula function as head- and trunk-organising centers, respectively. Here, we report that HNF3beta and Lim1 are coexpressed in both organising centers suggesting synergistic roles of these genes in regulating organiser functions and hence axis development in the mouse embryo. To investigate this possibility, we generated compound HNF3beta and Lim1 mutant embryos. An enlarged primitive streak and a lack of axis formation were observed in HNF3beta (-)(/)(-);Lim1(-)(/)(-), but not in single homozygous mutant embryos. Chimera experiments indicate that the primary defect in these double homozygous mutants is due to loss of activity of HNF3beta and Lim1 in the visceral endoderm. Altogether, these data provide evidence that these genes function synergistically to regulate organiser activity of the anterior visceral endoderm. Moreover, HNF3beta (-)(/)(-);Lim1(-)(/)(-) mutant embryos also exhibit defects in mesoderm patterning that are likely due to lack of specification of anterior primitive streak cells.

Synergistic activities of alpha3 and alpha6 integrins are required during apical ectodermal ridge formation and organogenesis in the mouse.

Integrins alpha6beta1 and alpha6beta4 are cell surface receptors for laminins. Integrin alpha6-null mice die at birth with severe skin blistering and defects in the cerebral cortex and in the retina. Integrin alpha3beta1 can associate with laminins and other ligands. Integrin alpha3-null mice also die at birth, with kidney and lung defects at late stages of development, and moderate skin blistering. To investigate possible overlapping functions between alpha3 and alpha6 integrins, we analyzed the phenotype of compound alpha3-/-/alpha6-/- mutant embryos. Double homozygous mutant embryos were growth-retarded and displayed several developmental defects not observed in the single mutant animals. First, limb abnormalities characterized by an absence of digit separation and the fusion of preskeletal elements were observed. Further analyses indicated a defect in the apical ectodermal ridge, an essential limb organizing center. In the double mutant, the ridge appeared flattened, and ridge cells did not show a columnar morphology. A strong reduction in ridge cell proliferation and alterations of the basal lamina underlying the ectoderm were observed. These results suggest that alpha3 and alpha6 integrins are required for the organization or compaction of presumptive apical ectodermal ridge cells into a distinct differentiated structure. Additional defects were present: an absence of neural tube closure, bilateral lung hypoplasia, and several abnormalities in the urogenital tract. Finally, an aggravation of brain and eye lamination defects was observed. The presence of novel phenotypes in double mutant embryos demonstrates the synergism between alpha3 and alpha6 integrins and their essential roles in multiple processes during embryogenesis.

Transgenic models to study gonadotropin function: the role of follicle-stimulating hormone in gonadal growth and tumorigenesis.

The role of FSH in gonadal tumorigenesis and, in particular, in human ovarian cancer has been debated. It is also unclear what role the elevated FSH levels in the inhibin-deficient mouse play in the gonadal tumorigenesis. To directly assess the role of FSH in gonadal growth, differentiation, and gonadal tumorigenesis, we have generated both gain-of-function and loss-of-function transgenic mutant mice. In the gain-of-function model, we have generated transgenic mice that ectopically overexpress human FSH from multiple tissues using a mouse metallothionein-1 promoter, achieving levels far exceeding those seen in postmenopausal women. Male transgenic mice are infertile despite normal testicular development and demonstrate enlarged seminal vesicles secondary to elevated serum testosterone levels. Female transgenic mice develop highly hemorrhagic and cystic ovaries, have elevated serum estradiol and progesterone levels, and are infertile, mimicking the features of human ovarian hyperstimulation and polycystic ovarian syndromes. Furthermore, the female transgenic mice develop enlarged and cystic kidneys and die between 6-13 weeks as a result of urinary bladder obstruction. In a complementary loss-of-function approach, we have generated double-homozygous mutant mice that lack both inhibin and FSH by a genetic intercross. In contrast to male mice lacking inhibin alone, 95% of which die of a cancer cachexia-like syndrome by 12 weeks of age, only 30% of the double-mutant male mice lacking both FSH and inhibin die by 1 yr of age. The remaining double-mutant male mice develop slow-growing and less hemorrhagic testicular tumors, which are noted after 12 weeks of age, and have minimal cachexia. Similarly, the double-mutant female mice develop slow-growing, less hemorrhagic ovarian tumors, and 70% of these mice live beyond 17 weeks. The double-mutant mice demonstrate minimal cachexia in contrast to female mice lacking only inhibin, which develop highly hemorrhagic ovarian tumors, leading to cachexia and death by 17 weeks of age in 95% of the cases. The milder cachexia-like symptoms of the inhibin and FSH double-mutant mice are correlated with low levels of serum estradiol and activin A and reduced levels of aromatase mRNA in the gonadal tumors. Based on these and our previous genetic analyses, we conclude that elevated FSH levels do not directly cause gonadal tumors. However, these results suggest FSH is an important trophic modifier factor for gonadal tumorigenesis in inhibin-deficient mice.

Gli2 is required for induction of floor plate and adjacent cells, but not most ventral neurons in the mouse central nervous system.

Induction of the floor plate at the ventral midline of the neural tube is one of the earliest events in the establishment of dorsoventral (d/v) polarity in the vertebrate central nervous system (CNS). The secreted molecule, Sonic hedgehog, has been shown to be both necessary and sufficient for this induction. In vertebrates, several downstream components of this signalling pathway have been identified, including members of the Gli transcription factor family. In this study, we have examined d/v patterning of the CNS in Gli2 mouse mutants. We have found that the floor plate throughout the midbrain, hindbrain and spinal cord does not form in Gli2 homozygotes. Despite this, motoneurons and ventral interneurons form in their normal d/v positions at 9.5 to 12.5 days postcoitum (dpc). However, cells that are generated in the region flanking the floor plate, including dopaminergic and serotonergic neurons, were greatly reduced in number or absent in Gli2 homozygous embryos. These results suggest that early signals derived from the notochord can be sufficient for establishing the basic d/v domains of cell differentiation in the ventral spinal cord and hindbrain. Interestingly, the notochord in Gli2 mutants does not regress ventrally after 10.5 dpc, as in normal embryos. Finally, the spinal cord of Gli1/Gli2 zinc-finger-deletion double homozygous mutants appeared similar to Gli2 homozygotes, indicating that neither gene is required downstream of Shh for the early development of ventral cell fates outside the ventral midline.

The Shh signalling pathway in tooth development: defects in Gli2 and Gli3 mutants.

The expression of genes involved in the Sonic Hedgehog signalling pathway, including Shh, Ptc, Smo, Gli1, Gli2 and Gli3, were found to be expressed in temporal and spatial patterns during early murine tooth development, suggestive of a role in early tooth germ initiation and subsequent epithelial-mesenchymal interactions. Of these Ptc, Smo, Gli1, Gli2 and Gli3 were expressed in epithelium and mesenchyme whereas Shh was only detected in epithelium. This suggests that Shh is involved in both lateral (epithelial-mesenchymal) and planar (epithelial-epithelial) signalling in early tooth development. Ectopic application of Shh protein to mandibular mesenchyme induced the expression of Ptc and Gli1. Addition of exogenous Shh protein directly into early tooth germs and adjacent to tooth germs, resulted in abnormal epithelial invagination, indicative of a role for Shh in epithelial cell proliferation. In order to assess the possible role of this pathway, tooth development in Gli2 and Gli3 mutant embryos was investigated. Gli2 mutants were found to have abnormal development of maxillary incisors, probably resulting from a mild holoprosencephaly, whereas Gli3 mutants had no major tooth abnormalities. Gli2/Gli3 double homozygous mutants did not develop any normal teeth and did not survive beyond embryonic day 14.5; however, Gli2(-/-); Gli3(+/-) did survive until birth and had small molars and mandibular incisors whereas maxillary incisor development was arrested as a rudimentary epithelial thickening. These results show an essential role for Shh signalling in tooth development that involves functional redundancy of downstream Gli genes.

Hold up is required for establishment of oocyte positioning, follicle cell fate and egg polarity and cooperates with Egfr during Drosophila oogenesis.

In Drosophila the posterior positioning of the oocyte within the germline cluster defines the initial asymmetry during oogenesis. From this early event, specification of both body axes is controlled through reciprocal signaling between germline and soma. Here it is shown that the mutation hold up (hup) affects oocyte positioning in the egg chamber, follicle cell fate and localization of different markers in the growing oocytes. This occurs not only in dicephalic egg chambers, but also in oocytes normally located at the posterior. Generation of mosaic egg chambers indicates that hup has to be at least somatically required. Possible interactions of hup with Egfr, the Drosophila epidermal growth factor receptor homolog, have been investigated in homozygous double mutants constructed by recombination. Stronger new ovarian phenotypes have been obtained, the most striking being accumulation of follicle cells in multiple layers posteriorly to the oocyte. It is proposed that the hup gene product is a component of the molecular machinery that leads to the establishment of polarity both in follicle cell layer and oocyte, acting in the same or in a parallel pathway of Egfr.

Functional analysis in vivo of the double mutant mouse deficient in both proteolipid protein (PLP) and myelin basic protein (MBP) in the central nervous system.

Myelination is an important developmental process of the central (CNS) and peripheral nervous system (PNS). To unravel the functions of the two dominant myelin proteins in the CNS, proteolipid protein (PLP) and myelin basic protein (MBP), we generated and characterized the homozygous double mutant mouse line (plp-/-, mbp-/-), which is viable and fertile. Plasma membrane processes of oligodendrocytes deficient in PLP and MBP, but not in myelin-associated glycoprotein (MAG), spirally wrap large diameter axons, tightly adhering at their extracytosolic surfaces and forming a pseudo-compacted myelin. Neuromotor activity and coordination are considerably improved compared to the shiverer trait.

Identification of two loci involved in phytochrome expression in Nicotiana plumbaginifolia and lethality of the corresponding double mutant

Four Nicotiana plumbaginifolia mutants exhibiting long hypocotyls and chlorotic cotyledons under white light, have been isolated from M2 seeds following mutagenesis with ethyl methane sulphonate. In each of these mutants, this partly etiolated in white light (pew) phenotype is due to a recessive nuclear mutation at a single locus. Complementation analysis indicates that three mutants, dap5, ems28 and ems3-6-34, belong to a single complementation group called pew1, while dap1 defines the pew2 locus. The mutants at pew1 contain normal levels of immunochemically detectable apoprotein of the phytochrome that is relatively abundant in etiolated seedlings, but are deficient in spectrophotometrically detectable phytochrome, whether seedlings are grown in darkness or light. Moreover, biliverdin, a precursor of the phytochrome chromophore, restores light-regulated responses in pew1 mutants and increases their level of photoreversible phytochrome when grown in darkness. These results indicate that the pew1 locus may be involved in chromophore biosynthesis. The mutant at the pew2 locus displays no photoreversible phytochrome in etiolated seedlings, but does contain normal levels of photoreversible phytochrome when grown in the light. Biliverdin had little effect on light-regulated responses in this mutant. In addition, biliverdin did not alter the level of phytochrome in etiolated seedlings. These observations lead us to propose that this mutant could be affected in the phyA gene itself. We have also obtained the homozygous double mutant at the pew1 and pew2 loci. This double mutant is lethal at an early stage of development, consistent with a critical role for phytochrome in early development of higher plants.

The MyoD family and myogenesis: Redundancy, networks, and thresholds

The members of the MyoD family of myogenic transcriptional regulators (MyoD, Myf-5, MRF4, myogenin) have the remarkable property that, when expressed from a constitutive promoter, they can convert a large number of different cell types into muscle (reviewed by Buckingham, 1992; Emerson, 1990; Olson, 1990; Weintraub et al., 1991). In contrast, mice with homozygous gene-targeted mutations in either MyoD or Myf-5 produce fairly normal amounts of muscle (Rudnicki et al., 1992; Braun et al., 1992). This apparent paradox has now been resolved by two recent studies. In one, a simple genetic cross has shown that in the double homozygous mutants, in which both MyoD and Myfd are absent, no muscle is formed, no muscle markers are present, and no myogenin is transcribed (Rudnicki et al., 1993), indicating that MyoD and Myfd share an overlapping (redundant) function required for generating (or maintaining) muscle cell identity and activating myogenin. In the other study (Hasty et al., 1993; Nabeshimaet al., 1993), mice with a homozygous targeted mutation of myogenin contain myoblasts since these embryos produce normal amounts of MyoD RNA, but these cells fail to differentiate fully, indicating that myogenin has a unique function in the transition from a determined myoblast to a fully differentiated myotube. These results suggest a simple epistatic relation, shown in Figure 1. While MyoD and Myf-5 are postulated to be responsible for myoblast determination, markers for the determined myoblast state are limited; in fact, besides the expression of MyoD or Mfl-5, the only other reliable marker known is the capacity of cells to differentiate into myotubes when challenged. Thus, one wonders how MyoD and My65 actually establish a determined state, especially since known inhibitors of their transcription function (e.g., Id, Jun, Myc, etc.) are present at high levels in myoblasts. Perhaps other functions of MyoD and Myf-5 define the myoblast state or perhaps the myoblast form (which is unknown) of MyoD and Myf-5 (e.g., a homodimer or a heterodimer with Jun or Id) is capable of activating certain noncanonical promoters not yet identified. That there are indeed myoblast functions has now become very clear from recent knockout experiments. In myogenin knockouts, myoblasts are present in the appropriate positions and numbers in the embryo; they just fail to differentiate into muscle. However, in the MyoLIMyf-5 double knockout mice, cells fail to occupy the muscle compartments. Thus, myogenin-independent myoblast identity seems to be definable by the generation of approximately the right number of cells in the right positions of the body under the direction of MyoD-Myf-5 function. Regulation of Myogenic Genes In Vitro and In Vivo Tissue culture experiments also support the notion that Minireview

Photoreceptor-specific mRNAs in mice carrying different allelic combinations at the rd and rds loci

Several retinal mRNAs encoding photoreceptor-specific proteins have been examined in congenic lines of mice carrying different allelic combinations at the rd and rds loci, to determine how mRNA expression is affected by the presence of both the rd and rds genes together or by the presence of one or two rd and rds alleles in the visual cells. Slot blots with retinal RNA from 9 to 30 days old C3H +/+, +/+; rd/+, +/+; rd rd , +/+; +/+, +/ rds; +/+, rds rds ; and the rd rd , rds rds double homozygous mutant mice were hybridized successively to several [ 32P]cDNA probes encoding proteins involved in phototransduction. The increases and decreases in the levels of mRNA coding for opsin, the α-subunit of transducin, 48 kDa protein and the β-subunit of cGMP-phosphodiesterase in the heterozygous and homozygous rds and rd mouse retinas reflected the growth and degeneration of the photoreceptor cells. The heterozygous rd mouse ( rd/+, +/+) retina expressed cGMP-phosphodiesteraseβ mRNA levels halfway between those in the control (+/+, +/+) and homozygous ( rd rd , +/+) mice, indicating a possible dosage effect. The double homozygous mutant ( rd rd , rds rds ) showed intermediate levels between those observed in the two homozygotes for all mRNAs studied, suggesting a possible interaction between the rd and rds genes.

Genetic Control of Root Hair Development in Arabidopsis thaliana.

Visual examination of roots from 12,000 mutagenized Arabidopsis seedlings has led to the identification of more than 40 mutants impaired in root hair morphogenesis. Mutants from four phenotypic classes have been characterized in detail, and genetic tests show that these result from single nuclear recessive mutations in four different genes designated RHD1, RHD2, RHD3, and RHD4. The phenotypic analysis of the mutants and homozygous double mutants has led to a proposed model for root hair development and the stages at which the genes are normally required. The RHD1 gene product appears to be necessary for proper initiation of root hairs, whereas the RHD2, RHD3, and RHD4 gene products are required for normal hair elongation. These results demonstrate that root hair development in Arabidopsis is amenable to genetic dissection and should prove to be a useful model system to study the molecular mechanisms governing cell differentiation in plants.

ENZYME ACTIVITY IN EMS-INDUCED NULL MUTATIONS OF DUPLICATED GENES ENCODING PHOSPHOGLUCOSE ISOMERASES IN CLARKIA

The duplication of the nuclear gene encoding the cytosolic isozyme of phosphoglucose isomerase (PGI; EC 5.3.1.9) originated within Clarkia, a genus of annual plants native to California. Previous immunological studies showed that species with and without the duplication have the same levels of cytosolic PGI activity (relative to that of the plastid PGI isozyme), as well as similar levels of cytosolic PGI protein. In the present study, we characterized seven EMS-induced null activity mutations in both duplicate PGI genes. The mutations reduced PGI activity levels in direct proportion to the normal contribution of each gene. Homozygous mutants at Pgi-3 had 64% of wild-type activity, whereas those at Pgi-2 had only 36%. The effects of the mutations at the two loci were additive, as shown by further reductions in activity in certain progeny classes in F(2) progenies between them. The homozygous double null mutant class was not recovered and is presumably lethal. All of the mutants appear to be CRM+. The results account for the previously observed differences in in vivo accumulation of the duplicate isozymes in numerous Clarkia species. The results further show that PGI activity is not directly regulated by metabolic factors and suggest that the reduced PGI levels in Clarkias with the duplication probably evolved by regulatory changes in transcription or translation. The study also demonstrates a novel method to evaluate whether a particular enzyme activity is essential.

Establishment of an albumin-deficient and fatty strain of rats.

An albumin-deficient and fatty strain of rats (AFR) was established from crosses between albumin-deficient rats (NAR) and fatty Zucker rats. AFRs have double homozygous mutant genes (alb/alb, fa/fa). The AFRs are heavier than the fatty Zucker rats and they have enlarged livers and adrenal glands. In addition, AFRs show more severe hyperlipidemia than fatty Zucker rats. This strain of rats may serve as a model of human obesity and hyperlipidemia.

Genetic analysis of nitrate reductase-deficient tobacco plants regenerated from mutant cells

Fifteen nitrate reductase (NR)-deficient mutants that had been selected from amphihaploid cell cultures of Nicotiana tabacum and regenerated to fertile amphidiploid plants were genetically analyzed through crosses. All the 15 mutants proved to be allelic. Segregation among F2 progeny from crosses between mutant and wild-type plants and among testcross progeny showed the regenerated plants to be homozygous double mutants. The NR deficiency is conferred by two unlinked recessive nuclear mutations, which thus define a pair of duplicate loci (nia1 nia2). These loci were identified as the structural genes for the apoprotein of NADH-NR. — Two mutants (Nia28, Nia30) were characterized further. Both Nia28 and Nia30 plants had less than 2% of wild-type NR activity, were resistant to chlorate and incapable of sustained growth on nitrate or in soil. However, they grew normally on media containing ammonium succinate (with or without nitrate). Growth test showed that Nia28 seedlings do not utilize nitrate, whereas Nia30 seedlings utilize nitrate at a very small rate. — The examination of single mutants (nia1-28/28 and nia2-28/28) revealed that either of the two loci is able to produce wild-type levels of NR activity. NR activity and chlorate sensitivity respond to nia + gene dosage only at the very early seedling stage. At later developmental stages, both the basal and the nitrate induced levels of NR activity were found to be independent of the number of nia + genes, indicating complete compensation of gene dosage effects by regulatory mechanism.

Genetic interaction in the retinal degeneration of mice

In the retina of mice, homozygous for the rd (retinal degeneration) gene, rapid loss of visual cells starting from around 10 days after birth reduces the outernuclear layer to a single row at the age of 21 days. In the mouse, homozygous for the rds (retinal degeneration slow) gene, the retina fails to develop the receptor outer segments and lacks rhodopsin, and a very slow loss of visual cells starting from two weeks after birth results in progressive reduction of the outer nuclear layer. In order to examine the influence of absence of rhodopsin on the degeneration caused by the rd gene, mice homozygous for both the rd and the rds genes were produced by appropriate breeding. In the double homozygous mutants, the visual cells remain lacking in receptor outer segments, but ultrastructural changes in the inner segments and visual cell death as indicated by the appearance of pycnotic nuclei start at the same time as in the rd genotype. However, the rate of degeneration is considerably slowed down in the double homozygotes and the thickness of the outer nuclear layer at 14, 21 and 28 days remains consistently thicker than in the retina of rd mice. A single row of outer nuclei, as seen in the rd retina at 21 days, is seen in the double homozygotes at 2 months. The relevance of the present findings in relation to the therapeutic use of vitamin A in retinitis pigmentosa is discussed.

Establishment of an albumin-deficient and jaundiced strain of rats.

An albumin deficient and jaundiced rat (AJR) strain was established from crosses between Albumin-Deficient rats (NAR) and jaundiced Gunn rats. AJRs have a double homozygous mutant trait (alb/alb, j/j), and systemic jaundice and various neurological signs are observed 5--7 days after birth, kernicterus occurs and they die within three weeks after birth. This strain of rats may serve as a model of human kernicterus and also be useful in elucidating the mechanism of bilirubin metabolism.

Cycloheximide resistance can be mediated through either ribosomal subunit.

Two cycloheximide-resistant mutants of Tetrahymena thermophila were analyzed to determine the site of their cycloheximide resistance. The mutations in both strains had been previously shown to be genetically dominant and located at separate loci (denoted Chx-A and Chx-B). Strains carrying these mutations were readily distinguished by the extent to which they were resistant to the drug. The homozygous double mutant was more resistant than either single mutant. Cell-free extracts of wild type and of the three mutant strains, assayed for protein synthetic activity by both runoff of natural mRNA and poly(U)-dependent phenylalanine polymerization, demonstrated that in vitro the mutants were all more resistant than the wild type. Further fractionation of the cell-free systems into ribosomes and supernates localized cycloheximide resistance to the ribosome for both Chx-A and Chx-B homozygotes. Ribosome dissociation and pairwise subunit mixing in the in vitro system indicated that ribosome resistance was conferred by the 60S subunit from one strain whereas resistance in the other strain was mediated through the 40S subunit. This was further confirmed by reconstruction of all four cycloheximide-resistance "phenotypes" by mixing ribosomal subunits from appropriate strains. This finding suggests that the mechanisms by which these mutations confer resistance to cycloheximide are different.