Barbara Meyer

Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development.

The cytosolic protein APAF1, human homolog of C. elegans CED-4, participates in the CASPASE 9 (CASP9)-dependent activation of CASP3 in the general apoptotic pathway. We have generated by gene trap a null allele of the murine Apaf1. Homozygous mutants die at embryonic day 16.5. Their phenotype includes severe craniofacial malformations, brain overgrowth, persistence of the interdigital webs, and dramatic alterations of the lens and retina. Homozygous embryonic fibroblasts exhibit reduced response to various apoptotic stimuli. In situ immunodetection shows that the absence of Apaf1 protein prevents the activation of Casp3 in vivo. In agreement with the reported function of CED-4 in C. elegans, this phenotype can be correlated with a defect of apoptosis. Our findings suggest that Apaf1 is essential for Casp3 activation in embryonic brain and is a key regulator of developmental programmed cell death in mammals.

Disruption of the murine homeobox gene Cdx1 affects axial skeletal identities by altering the mesodermal expression domains of Hox genes

Cdx1 is expressed along the embryonic axis from day 7.5 postcoitum until day 12, by which time the anterior limit of expression has regressed from the hindbrain level to the forelimb bud region. To assign a functional role for Cdx1 in murine embryonic development, we have inactivated the gene via homologous recombination. Viable fertile homozygous mutant mice were obtained that show anterior homeotic transformations of vertebrae. These abnormalities were concomitant with posterior shifts of Hox gene expression domains in the somitic mesoderm. The presence of putative Cdx1-binding sites in Hox gene control regions as well as in vitro transactivation of Hoxa-7 indicates a direct regulation.

Fras1 deficiency results in cryptophthalmos, renal agenesis and blebbed phenotype in mice.

Loss of tight association between epidermis and dermis underlies several blistering disorders and is frequently caused by impaired function of extracellular matrix (ECM) proteins. Here we describe a new protein in mouse, Fras1, that is specifically detected in a linear fashion underlying the epidermis and the basal surface of other epithelia in embryos. Loss of Fras1 function results in the formation of subepidermal hemorrhagic blisters as well as unilateral or bilateral renal agenesis during mouse embryogenesis. Postnatally, homozygous Fras1 mutants have fusion of the eyelids and digits and unilateral renal agenesis or dysplasia. The defects observed in Fras1−/− mice phenocopy those of the existing bl (blebbed) mouse mutants, which have been considered a model for the human genetic disorder Fraser syndrome. We show that bl/bl homozygous embryos are devoid of Fras1 protein, consistent with the finding that Fras1 is mutated in these mice. In sum, our data suggest that perturbations in the composition of the extracellular space underlying epithelia could account for the onset of the blebbed phenotype in mouse and Fraser syndrome manifestation in human.

Efficient poly A trap approach allows the capture of genes specifically active in differentiated embryonic stem cells and in mouse embryos

Special vectors have been constructed that allow the trapping of genes in mouse embryonic stem (ES) cells. These vectors generally contain the neomycin phosphotransferase (neo) gene for selection and the β ‐galactosidase (β‐gal) gene as a marker. Promoterless vectors can be used to identify genes that are active in undifferentiated ES cells. To also have access to genes that are inactive in totipotent ES cells, we constructed a polyadenylation (poly A) trap vector in which the expression of a poly A less neo gene is driven by a constitutive promoter, whereas the expression of β‐gal depends on the trapped sequences. We demonstrate here that this vector integrates with a high frequency into transcription units and that it traps genes with very different expression patterns in vitro and in vivo. The vector integrates efficiently into transcription units that are inactive in undifferentiated ES cells and which can be activated through in vitro differentiation. Furthermore, in vivo expression patterns demonstrate that this vector integrates into genes that exhibit a highly specific temporal and spatial expression pattern during embryogenesis. Dev. Dyn. 1998;212:326–333.

Gene trap: a way to identify novel genes and unravel their biological function

The gene trap methodology is a powerful tool to characterize novel genes and analyze their importance in biological phenomena. It is based on the use of mouse embryonic stem cells and reporter vectors designed to randomly integrate into the genome, tagging an insertion site and generating a mutation. Theoretically, all the 100 000 genes present in the mouse genome could be tagged and functionally inactivated at the same time. Here we describe the basic concepts and perspectives of this methodology and show some results obtained by the gene trap approach used to study molecular cascades in basic cell biology and in developmental processes.

Inactivation of a Testis-specific Lis1 Transcript in Mice Prevents Spermatid Differentiation and Causes Male Infertility

Lis1 protein is the non-catalytic component of platelet-activating factor acetylhydrolase 1b (PAF-AH 1B) and associated with microtubular structures. Hemizygous mutations of the LIS1 gene cause type I lissencephaly, a brain abnormality with developmental defects of neuronal migration. Lis1 is also expressed in testis, but its function there has not been determined. We have generated a mouse mutant (LIS1GT/GT) by gene trap integration leading to selective disruption of a Lis1 splicing variant in testis. Homozygous mutant males are infertile with no other apparent phenotype. We demonstrate that Lis1 is predominantly expressed in spermatids, and spermiogenesis is blocked when Lis1 is absent. Mutant spermatids fail to form correct acrosomes and nuclei appear distorted in size and shape. The tissue architecture in mutant testis appears severely disturbed displaying collapsed seminiferous tubules, mislocated germ cells, and increased apoptosis. These results provide evidence for an essential and hitherto uncharacterized role of the Lis1 protein in spermatogenesis, particularly in the differentiation of spermatids into spermatozoa.

Effect of canine surfactant protein (SP-A) on the respiratory burst of phagocytic cells

Cells obtained from bronchoalveolar lavage, or neutrophils of peripheral blood of dog, were incubated with the canine surfactant‐associated protein A (SP‐A). A significant decrease of the production of Superoxide anion was observed after subsequent stimulation with phorbol‐12‐myristate‐13‐acetate (PMA) as measured by the lucigenin‐dependent chemiluminesence (CL). Several other proteins used for control experiments did not decrease lucigenin‐dependent CL, indicating a specific effect of SP‐A on phagocytes. Treatment of SP‐A with collagenase prior to incubation with neutrophils destroyed the depleting effect on oxygen radical production of PMA‐stimulated cells. We propose that SP‐A acts as a regulatory factor of the respitatory burst of alveolar macrophages and neutrophils in the lungs. The inhibitory effect of SP‐A is down‐regulated by collagenase released from stimulated alveolar macrophages.

A COMPARATIVE STUDY OF SPECIES IN THE STEPHANODISCUS NIAGARAE-COMPLEX AND A DESCRIPTION OF S. HETEROSTYLUS SP. NOV.

The Stephanodiscus niagarae-complex is comprised of large, planktonic, morphologically variable species with dissimilarly shaped valves (heterovalvy). The species are characterized by a set of subtle morphological features causing controversy in species distinction. In this paper we present a comparative study based on a re-investigation of the original material for the following species: S. niagarae, S. rotula, S. aegyptlacus, S. neoastraea, S. galileensis and S. agassizensis. New material from Farmoor Reservoir (England), Loch Neagh (Ireland) and a lake in the Rift Valley (East-Africa) was also analyzed. The position of the valve face fultoportulae is used as a character to differentiate two groups. In one group the position of the fultoportulae is the same on each valve face, whereas in the other group the positions are different. A third group is lacking fultoportulae. A new species, S. heterostylus is described based on material from Farmoor.

Fas-associated factor (FAF1) is required for the early cleavage-stages of mouse embryo

FAF1 was initially isolated as a Fas-associated factor and was subsequently found to interact with a subset of additional proteins that are involved in many cellular events including Fas-mediated apoptosis, heat shock signalling pathways and ubiquitin-dependent processes. Here, we describe that the 74-kDa FAF1 is ubiquitously expressed, while the expression of its post-translational-processed 49-kDa isoform is restricted to post-meiotic male germ cells. In ovary, FAF1 protein is localized predominantly in the cytoplasm of oocytes in all follicle stages. To determine the function of FAF1 in vivo, we analysed a mouse mutant line in which a gene trap vector was inserted in the Faf1 locus. The mutation disrupts the Faf1 and leads to lethality of the Faf1(GT/GT) embryos near the 2-cell stage. Analysis of FAF1 expression revealed that the protein is present in early preimplantation stages, while embryonic expression of Faf1 mRNA becomes appreciable at 4-cell stage. These results indicate that the death of Faf1(GT/GT) at the 2-cell stage may coincide with the depletion of maternal FAF1 in these embryos. Thus, our results indicate that the FAF1 gene product is necessary for early embryonic development.

Genetic ablation of the mammillary bodies in the Foxb1 mutant mouse leads to selective deficit of spatial working memory

Mammillary bodies and the mammillothalamic tract are parts of a classic neural circuitry that has been implicated in severe memory disturbances accompanying Korsakoffs syndrome. However, the specific role of mammillary bodies in memory functions remains controversial, often being considered as just an extension of the hippocampal memory system. To study this issue we used mutant mice with a targeted mutation in the transcription factor gene Foxb1. These mice suffer perinatal degeneration of the medial and most of the lateral mammillary nuclei, as well as of the mammillothalamic bundle. Foxb1 mutant mice showed no deficits in such hippocampal‐dependent tasks as contextual fear conditioning and social transmission of food preference. They were also not impaired in the spatial reference memory test in the radial arm maze. However, Foxb1 mutants showed deficits in the task for spatial navigation within the Barnes maze. Furthermore, they showed impairments in spatial working memory tasks such as the spontaneous alternation and the working memory test in the radial arm maze. Thus, our behavioural analysis of Foxb1 mutants suggests that the medial mammillary nuclei and mammillothalamic tract play a role in a specific subset of spatial tasks, which require combined use of both spatial and working memory functions. Therefore, the mammillary bodies and the mammillothalamic tract may form an important route through which the working memory circuitry receives spatial information from the hippocampus.