Precursor-product relationship between vitellogenin and the yolk proteins as derived from the complete sequence of a Xenopus vitellogenin gene.

In Xenopus laevis four estrogen-responsive genes are expressed simultaneously to produce vitellogenin, the precursor of the yolk proteins. One of these four genes, the gene A2, was sequenced completely, as well as cDNAs representing 75% of the coding region of the gene. From this data the exon-intron structure of the gene was established, revealing 35 exons that give a transcript of 5,619 bp without the poly A-tail. This A2 transcript encodes a vitellogenin of 1,807 amino acids, whose structure is discussed with respect to its function. At the nucleic acid as well as at the protein level no extensive homologies with any sequences other than vitellogenin were observed. Comparison of the amino acid sequence of the vitellogenin A2 molecule with biochemical data obtained from the different yolk proteins allowed us to localize the cleavage products on the vitellogenin precursor as follows: NH2 - lipovitellin I - phosvitin (or phosvette II - phosvette I) - lipovitellin II - COOH.

Vertebrate and nematode genes coding for yolk proteins are derived from a common ancestor.

One of the most obvious characteristics of the egg cells of oviparous animals is their large size resulting to a major extent from the deposition of nutritional reserves, mainly constituted of yolk proteins. In general, these are derived from a precursor called vitellogenin, which undergoes posttranslational modifications during secretion and during transport into and storage within the oocytes. Comparative analysis of the structural organization of the vitellogenin gene and of its product in different species shows that the vitellogenin gene is very ancient and that in vertebrates the gene may have more resemblance to the earliest gene than in invertebrates.

Loss of egg yolk genes in mammals and the origin of lactation and placentation

Embryonic development in nonmammalian vertebrates depends entirely on nutritional reserves that are predominantly derived from vitellogenin proteins and stored in egg yolk. Mammals have evolved new resources, such as lactation and placentation, to nourish their developing and early offspring. However, the evolutionary timing and molecular events associated with this major phenotypic transition are not known. By means of sensitive comparative genomics analyses and evolutionary simulations, we here show that the three ancestral vitellogenin-encoding genes were progressively lost during mammalian evolution (until around 30-70 million years ago, Mya) in all but the egg-laying monotremes, which have retained a functional vitellogenin gene. Our analyses also provide evidence that the major milk resource genes, caseins, which have similar functional properties as vitellogenins, appeared in the common mammalian ancestor approximately 200-310 Mya. Together, our data are compatible with the hypothesis that the emergence of lactation in the common mammalian ancestor and the development of placentation in eutherian and marsupial mammals allowed for the gradual loss of yolk-dependent nourishment during mammalian evolution

Parental influences on pathogen resistance in brown trout embryos and effects of outcrossing within a river network.

Phenotypic plasticity can increase tolerance to heterogeneous environments but the elevations and slopes of reaction norms are often population specific. Disruption of locally adapted reaction norms through outcrossing can lower individual viability. Here, we sampled five genetically distinct populations of brown trout (Salmo trutta) from within a river network, crossed them in a full-factorial design, and challenged the embryos with the opportunistic pathogen Pseudomonas fluorescens. By virtue of our design, we were able to disentangle effects of genetic crossing distance from sire and dam effects on early life-history traits. While pathogen infection did not increase mortality, it was associated with delayed hatching of smaller larvae with reduced yolk sac reserves. We found no evidence of a relationship between genetic distance (W, FST) and the expression of early-life history traits. Moreover, hybrids did not differ in phenotypic means or reaction norms in comparison to offspring from within-population crosses. Heritable variation in early life-history traits was found to remain stable across the control and pathogen environments. Our findings show that outcrossing within a rather narrow geographical scale can have neutral effects on F1 hybrid viability at the embryonic stage, i.e. at a stage when environmental and genetic effects on phenotypes are usually large.

Evolution and expression of vitellogenin genes.

A major event during the growth period of oocytes in nonmammalian animals is the accumulation of yolk. The genes coding for the yolk protein precursor, known as vitallogenin, are well characterized in a few vertebrate and invertebrate species. Studies on the evolution of these genes and on the regulatory mechanisms involved in their time, tissue- and hormone-specific expression are presented and discussed in this review.

Molecular changes underlying mammalian phenotypic innovation

Mammals are characterized by specific phenotypic traits that include lactation, hair, and relatively large brains with unique structures. Individual mammalian lineages have, in turn, evolved characteristic traits that distinguish them from others. These include obvious anatom¬ical differences but also differences related to reproduction, life span, cognitive abilities, be¬havior. and disease susceptibility. However, the molecular basis of the diverse mammalian phenotypes and the selective pressures that shaped their evolution remain largely unknown. In the first part of my thesis, I analyzed the genetic factors associated with the origin of a unique mammalian phenotype lactation and I studied the selective pressures that forged the transition from oviparity to viviparity. Using a comparative genomics approach and evolutionary simulations, I showed that the emergence of lactation, as well as the appear¬ance of the casein gene family, significantly reduced selective pressure on the major egg-yolk proteins (the vitellogenin family). This led to a progressive loss of vitellogenins, which - in oviparous species - act as storage proteins for lipids, amino acids, phosphorous and calcium in the isolated egg. The passage to internal fertilization and placentation in therian mam¬mals rendered vitellogenins completely dispensable, which ended in the loss of the whole gene family in this lineage. As illustrated by the vitellogenin study, changes in gene content are one possible underlying factor for the evolution of mammalian-specific phenotypes. However, more subtle genomic changes, such as mutations in protein-coding sequences, can also greatly affect the phenotypes. In particular, it was proposed that changes at the level of gene reg¬ulation could underlie many (or even most) phenotypic differences between species. In the second part of my thesis, I participated in a major comparative study of mammalian tissue transcriptomes, with the goal of understanding how evolutionary forces affected expression patterns in the past 200 million years of mammalian evolution. I showed that, while com¬parisons of gene expressions are in agreement with the known species phylogeny, the rate of expression evolution varies greatly among lineages. Species with low effective population size, such as monotremes and hominoids, showed significantly accelerated rates of gene expression evolution. The most likely explanation for the high rate of gene expression evolution in these lineages is the accumulation of mildly deleterious mutations in regulatory regions, due to the low efficiency of purifying selection. Thus, our observations are in agreement with the nearly neutral theory of molecular evolution. I also describe substantial differences in evolutionary rates between tissues, with brain being the most constrained (especially in primates) and testis significantly accelerated. The rate of gene expression evolution also varies significantly between chromosomes. In particular, I observed an acceleration of gene expression changes on the X chromosome, probably as a result of adaptive processes associated with the origin of therian sex chromosomes. Lastly, I identified several individual genes as well as co-regulated expression modules that have undergone lineage specific expression changes and likely under¬lie various phenotypic innovations in mammals. The methods developed during my thesis, as well as the comprehensive gene content analyses and transcriptomics datasets made available by our group, will likely prove to be useful for further exploratory analyses of the diverse mammalian phenotypes.

In vivo fate mapping with SCL regulatory elements identifies progenitors for primitive and definitive hematopoiesis in mice.

One of the principal issues facing biomedical research is to elucidate developmental pathways and to establish the fate of stem and progenitor cells in vivo. Hematopoiesis, the process of blood cell formation, provides a powerful experimental system for investigating this process. Here, we employ transcriptional regulatory elements from the stem cell leukemia (SCL) gene to selectively label primitive and definitive hematopoiesis. We report that SCL-labelled cells arising in the mid to late streak embryo give rise to primitive red blood cells but fail to contribute to the vascular system of the developing embryo. Restricting SCL-marking to different stages of foetal development, we identify a second population of multilineage progenitors, proficient in contributing to adult erythroid, myeloid and lymphoid cells. The distinct lineage-restricted potential of SCL-labelled early progenitors demonstrates that primitive erythroid cell fate specification is initiated during mid gastrulation. Our data also suggest that the transition from a hemangioblastic precursors with endothelial and blood forming potential to a committed hematopoietic progenitor must have occurred prior to SCL-marking of definitive multilineage blood precursors.

Vitellogenesis and the vitellogenin gene family.

Vitellogenin is synthesized under estrogen control in the liver, extensively modified, transported to the ovary, and there processed to the yolk proteins lipovitellin and phosvitin. In the frog Xenopus laevis there are at least four distinct but related vitellogenin genes. The two genes A1 and A2 have a 95 percent sequence homology in their messenger RNA coding regions, and contain 33 introns that interrupt the coding region (exons) at homologous positions. Sequences and lengths of analogous introns differ, and many introns contain repetitive DNA elements. The introns in these two genes that have apparently arisen by duplication have diverged extensively by events that include deletions, insertions, and probably duplications. Rapid evolutionary change involving rearrangements and the presence of repeated DNA suggests that the bulk of the sequences within introns may not have any specific function.

Sequence homologies within the 5' end region of the estrogen-controlled vitellogenin gene in Xenopus and chicken.

In oviparous vertebrates vitellogenin, the precursor of the major yolk proteins, is synthesized in the liver of mature females under the control of estrogen. We have established the organization and primary structure of the 5' end region of the Xenopus laevis vitellogenin A2 gene and of the major chicken vitellogenin gene. The first three homologous exons have exactly the same length in both species, namely 53, 21 and 152 nucleotides, and present an overall sequence homology of 60%. In both species, the 5'-non-coding region of the vitellogenin mRNA measures only 13 nucleotides, nine of which are conserved. In contrast, the corresponding introns of the Xenopus and the chicken vitellogenin gene show no significant sequence homology. Within the 500 nucleotides preceding the 5' end of the genes, at least six blocks with sequence homologies of greater than 70% were detected. It remains to be demonstrated which of these conserved sequences, if any, are involved in the hormone-regulated expression of the vitellogenin genes.