Mitochondrial inheritance and germ line determination in a bivalve species with Doubly Uniparental Inheritance (DUI)

Mitochondria are inherited maternally in most metazoans. However, in some bivalves, two mitochondrial lineages are present: one transmitted through eggs (F), the other through sperm (M). This is called Doubly Uniparental Inheritance (DUI). During male embryo development, spermatozoon mitochondria aggregate and end up in the primordial germ cells, while they are dispersed in female embryos. The molecular mechanisms of segregation patterns are still unknown. In the DUI species Ruditapes philippinarum, I examined sperm mitochondria distribution by MitoTracker, microtubule staining and TEM, and I localized germ line determinants with immunocytochemical analysis. I also analyzed the gonad transcriptome, searching for genes involved in reproduction and sex determination. Moreover, I analyzed an M-type specific open reading frame that could be responsible for maintenance/degradation of M mitochondria during embryo development. These transcripts were also localized in tissues using in situ hybridization. As in Mytilus, two distribution patterns of M mitochondria were detected in R. philippinarum, supporting that they are related to DUI. Moreover, the first division midbody concurs in positioning aggregated M mitochondria on the animal-vegetal axis of the male embryo: in organisms with spiral segmentation this zone is not involved in further cleavages, so aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area where germ plasm is transferred, suggesting their contribution in male germ line formation. The finding of reproduction and ubiquitination transcripts led to formulate a model in which ubiquitination genes stored in female oocytes during gametogenesis would activate sex-gene expression in the early embryonic developmental stages (preformation). Only gametogenetic cells were labeled by in situ hybridization, proving their specific transcription in developing gametes. Other than having a role in sex determination, some ubiquination factors could also be involved in mitochondrial inheritance, and their differential expression could be responsible for the different fate of sperm mitochondria in the two sexes.

The KIT gene in familial mastocytosis

Familial cutaneous mastocytosis is an exceptional condition of unknown etiology. In this study we report the largest series of patients with familial cutaneous mastocytosis without other manifestations (18 affected subjects from seven unrelated families), and we investigate the role of germ-line KIT mutations in the pathogenesis of the disease. The mean age at onset was 5.4 years (range from birth to 22 years), and the clinical behavior was variable over a mean follow up period of 15.1 years (range 2-36): improvement in seven, stability in eight and worsening in the remaining three patients. The pattern of inheritance was compatible with an autosomal dominant trait with incomplete penetrance; a female preponderance (14 females vs 4 males, ratio 3.5:1) was noted; among the six women who have been pregnant at least once, three experienced important clinical changes during pregnancy. No germ-line mutation was found in the exons 10, 11, and 17 of the KIT proto-oncogene, which are the most commonly mutated exons in sporadic mastocytosis. However, in the majority of affected subjects we found the Met541Leu polymorphic variant of the KIT gene, which seems to confer a growth advantage to mast cells in vitro. This observation further suggests that the Met541Leu may be a predisposing factor of cutaneous mastocytosis, although it seems to be neither necessary nor sufficient for the development of the disease.

Characterization of mitochondrial genomes in bivalve species with doubly uniparental inheritance of mitochondria

Many bivalve species possess two distinct mtDNA lineages, called F and M, respectively inherited maternally and paternally: this system is called doubly uniparental inheritance (DUI). The main experimental project of my PhD was the quantification of the two mtDNAs during the development of the DUI species Ruditapes philippinarum, from early embryos to sub-adults, using Real-Time qPCR. I identified the time interval in which M mtDNA is lost from female individuals, while it is retained in males (which are heteroplasmic through all of their life cycle). The results also suggested absence of mtDNA replication during early embryogenesis, a process constituting a bottleneck that highly reduces the copy number of mtDNA molecules in cells of developing larvae. In males this bottleneck may produce cells homoplasmic for M mtDNA, and could be considered as a first step of the segregation of M in the male germ line. Another finding was the characterization, in young clams approaching the first reproductive season, of a significant boost in copy number of F mtDNA in females and of M in males. Given the age of animals in which this mtDNA-specific growth was observed, the finding could probably be the outcome of the first round of gonads and gametes production. Other lines of research included the characterization of the unassigned regions in mt genomes of DUI bivalves. These regions can harbor signals involved in the control of replication and/or transcription of the mtDNA molecule, as well as additional open reading frames (ORFs) not related to oxidative phosphorylation. These features in DUI species could be associated to the maintenance of separate inheritance routes for the two mtDNAs. Additional ORFs are also found in other animal mt genomes: I summarized the presence of gene duplications as a co-author in a review focusing on animal mt genomes with unusual gene content.