Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway

In vivo function of CDK5 and Abl enzyme substrate 2 (Cables2), belonging to the Cables protein family, is unknown. Here, we found that targeted disruption of the entire Cables2 locus (Cables2d) caused growth retardation and enhanced apoptosis at the gastrulation stage and then induced embryonic lethality in mice. Comparative transcriptome analysis revealed disruption of Cables2, 50% down-regulation of Rps21 abutting on the Cables2 locus, and up-regulation of p53-target genes in Cables2d gastrulas. We further revealed the lethality phenotype in Rps21-deleted mice and unexpectedly, the exon 1-deleted Cables2 mice survived. Interestingly, chimeric mice derived from Cables2d ESCs carrying exogenous Cables2 and tetraploid wild-type embryo overcame gastrulation. These results suggest that the diminished expression of Rps21 and the completed lack of Cables2 expression are intricately involved in the embryonic lethality via the p53 pathway. This study sheds light on the importance of Cables2 locus in mouse embryonic development.

Mesenchymal cells engulf and clear apoptotic footplate cells in macrophageless PU.1 null mouse embryos

Apoptosis is one of the key tools used by an embryo to regulate cell numbers and sculpt body shape. Although massive numbers of cells die during development, they are so rapidly phagocytosed that very few corpses are ever seen in most embryonic tissues. In this paper, we focus on the catastrophic cell death that occurs as the developing footplate is remodelled to transform webbed regions into free interdigital spaces. In the wild-type embryo, these dead cells are rapidly engulfed and cleared by macrophages. We show that in a macrophageless mouse embryo, null for the haemopoetic-lineage-specific transcription factor, PU.1, the task of phagocytosis is taken over by ‘stand-in’ mesenchymal neighbours in a clear example of cell redundancy. However, it takes three times as many of these mesenchymal phagocytes to complete the task and, at each stage of the clearance process - in the recognition of apoptotic debris, its engulfment and finally its digestion - they appear to be less efficient than macrophages. A molecular explanation for this may be that several of the engulfment genes expressed by macrophages, including the ABC1 transporter (believed to be part of the phagocytic machinery conserved from Caenorhabditis elegans to mouse), are not upregulated by these ‘stand-in’ phagocytes.

Improving Vigor in shrunken-2 Corn Seedlings

The shrunken-2 (sh2) mutant of maize (Zea mays L.) increases sucrose and reduces starch in developing endosperm. An associated trait is poor seed and seedling vigor in seeds containing the mutation. The specific effects of sh2 mutant endosperm on embryo and seedling vigor were determined by analyzing seeds that contained either concordant wild-type or nonconcordant combinations of mutant and wild-type embryo and endosperm genotypes. The nonconcordant seed types that contained a wild-type embryo in association with a sh2 mutant endosperm or a sh2 mutant embryo in association with a wild-type endosperm were generated using the TB-3La translocation chromosome in which a wild-type Sh2 gene is attached to the centromeric portion of a B chromosome. Under stress conditions (complex stress vigor test), the seeds with mutant endosperm had lower germination, seedling fresh and dry weight, and index of conductivity than seeds with wild-type endosperm. Mutant endosperm and embryos excised from mutant endosperm imbibed more water than wild-type endosperm or embryos excised from wild-type endosperm. Because of the high concentration of osmotic solutes in the mutant endosperm, a rapid water uptake may induce a membrane disorganization. Leachate conductivities of seeds with mutant endosperm were higher than seeds with wild-type endosperm. In addition, a higher sucrose content and a lower raffinose to sucrose ratio were measured in the wild-type embryos associated with mutant endosperms than in the normal embryos excised from concordant wild-type seeds. These results suggest that a high rate of water uptake caused by the elevated concentration of osmotic solutes in seeds with mutant endosperms may affect membrane integrity during imbibition. Alternatively, the lower raffinose to sucrose ratio present in the mutant endosperm class might affect stabilization of cell membranes during seed desiccation. Embryos cultured in media containing 10% starch or no carbohydrate produced smaller seedlings than embryos cultured in 5% or 10% sucrose. Wild-type embryos excised from mutant endosperms exhibited lower germination in 0% and 5% sucrose media than embryos from concordant seed, indicating that reduced water uptake rates associated with lower external osmotic potential (10% sucrose) can improve vigor of embryos associated with sh2 mutant endosperm. The reduced vigor of embryos and seedlings that develop in association with sh2 mutant endosperm can be traced to the physiological and biochemical effects of the elevated sucrose levels present during seed formation and imbibition.

Homeostatic balance between dorsal and cactus proteins in the Drosophila embryo

The maternal-effect gene dorsal encodes the ventral morphogen that is essential for elaboration of ventral and ventrolateral fates in the Drosophila embryo. Dorsal belongs to the rel family of transcription factors and controls asymmetric expression of zygotic genes along the dorsoventral axis. The dorsal protein is cytoplasmic in early embryos, possibly because of a direct interaction with cactus. In response to a ventral signal, dorsal protein becomes partitioned into nuclei of cleavage-stage syncytial blastoderms such that the ventral nuclei have the maximum amount of dorsal protein, and the lateral and dorsal nuclei have progressively less protein. Here we show that transgenic flies containing the dorsal cDNA, which is driven by the constitutively active hsp83 promoter, exhibits rescue of the dorsal- phenotype. Transformed lines were used to increase the level of dorsal protein. Females with dorsal levels roughly twice that of wild-type produced normal embryos, while a higher level of dorsal protein resulted in phenotypes similar to those observed for loss-of-function cactus mutations. By manipulating the cactus gene dose, we found that in contrast to a dorsal/cactus ratio of 2.5 which resulted in fully penetrant weak ventralization, a cactus/dorsal ratio of 3.0 was acceptable by the system. By manipulating dorsal levels in different cactus and dorsal group mutant backgrounds, we found that the relative amounts of ventral signal to that of the dorsal-cactus complex is important for the elaboration of the normal dorsoventral pattern. We propose that in a wild-type embryo, the activities of dorsal and cactus are not independently regulated; excess cactus activity is deployed only if a higher level of dorsal protein is available. Based on these results we discuss how the ventral signal interacts with the dorsal-cactus complex, thus forming a gradient of nuclear dorsal protein.

Probing gene activity in Drosophila embryos

The segmentation pattern of the Drosophila wild-type embryo is characterized by a number of easily identifiable cuticular structures. They include skeletal elements of the involuted head and ventral denticle belts that define by size, pattern and orientation the anterior part of the three thoracic and eight abdominal segments. Further landmarks such as sensory organs and the posterior tracheal endings (‘Filzkörper’), in combination with the denticle belts, allow one to unequivocally determine the polarity and quality of each segment in preparations of the larval cuticle (see Fig. 1D). The segmentation pattern of Drosophila is established at about blastoderm stage and it requires both maternally and zygotically active genes. Genetic analysis has identified a number of genes with zygotic activity that regulate key steps during pattern formation. Mutations in these genes cause specific defects in the segmental pattern of the embryo that allow the definition of classes of segmentation genes required for the subdivision of the embryo into segmental units (Nüsslein-Volhard & Wieschaus, 1980).

Interaction of granule, Purkinje and inferior olivary neurons in Lurcher chimaeric mice

Heterozygous lurcher (+/Lc) mutant mice lose 100% of their Purkinje cells (PCs), 90% of their granule cells, and 75% of their inferior olivary neurons. In order to determine the primary site of Lc gene action, lurcher↔ wild-type aggregation chimaeras were produced. The cerebella of the three chimaeras examined were intermediate or normal in size compared to +/Lc and wild-type cerebella. The PCs were reduced in number. Using the β-glucuronidase locus (Gus) as a cell marker, all of the PCs present were identified as having descended from the wild-type embryo. It appears that all of the +/Lc PCs degenerated. Hence, the Lc gene acts directly on PCs to cause their degeneration. The inferior olivary nuclei of the chimaeras seemed to have fewer neurons than wild-type but more than +/Lc animals. As revealed by β-glucuronidase histochemistry, both +/+and +/Lc cells were present, and the ratio of genotypes was similar to the ratio seen in other regions of the brain. The evidence suggests that the death of olivary neurons in lurcher is secondary to another defect, probably the loss of PCs. β-glucuronidase is not an accutate cell marker for granule cells, and so no conclusion concerning the action of the Lc gene on granule cells could be made with these chimaeras.