Author Summary Many animals have sex-specific combinations of chromosomes. In humans, for example, females have two X chromosomes while males have one X and one Y. In most species with XX:XY systems, the Y chromosome is degenerate and gene-poor while the X encodes a large number of functional genes. A variety of systems have evolved to ensure that males with one X chromosome and females with two X chromosomes have the same gene expression level for X-linked genes. The vinegar fly D. melanogaster has at least two dosage compensation systems: one that acts early in development, and another active in later stages. In this paper, we determine expression levels for thousands of genes in male and female embryos at different developmental stages in two species, D. pseudoobscura and D. miranda, that have unusually large fractions of their genomes in X or X-like chromosomes. We show that dosage compensation is established slowly during embryogenesis, and that in these species, dosage compensation appears to be absent in early development. This may be due to a lineage-specific loss or gain of compensation mechanism, or possibly because the machinery of dosage compensation cannot effectively handle the increased demand in these species. Sex chromosome dosage differences between females and males are a significant form of natural genetic variation in many species. Like many species with chromosomal sex determination, Drosophila females have two X chromosomes, while males have one X and one Y. Fusions of sex chromosomes with autosomes have occurred along the lineage leading to D. pseudoobscura and D. miranda. The resulting neo-sex chromosomes are gradually evolving the properties of sex chromosomes, and neo-X chromosomes are becoming targets for the molecular mechanisms that compensate for differences in X chromosome dose between sexes. We have previously shown that D. melanogaster possess at least two dosage compensation mechanisms: the well- characterized MSL-mediated dosage compensation active in most somatic tissues, and another system active during early embryogenesis prior to the onset of MSL-mediated dosage compensation. To better understand the developmental constraints on sex chromosome gene expression and evolution, we sequenced mRNA from individual male and female embryos of D. pseudoobscura and D. miranda, from similar to 0.5 to 8 hours of development. Autosomal expression levels are highly conserved between these species. But, unlike D. melanogaster, we observe a general lack of dosage compensation in D. pseudoobscura and D. miranda prior to the onset of MSL-mediated dosage compensation. Thus, either there has been a lineage-specific gain or loss in early dosage compensation mechanism(s) or increasing X chromosome dose may strain dosage compensation systems and make them less effective. The extent of female bias on the X chromosomes decreases through developmental time with the establishment of MSL-mediated dosage compensation, but may do so more slowly in D. miranda than D. pseudoobscura. These results also prompt a number of questions about whether species with more sex-linked genes have more sex-specific phenotypes, and how much transcript level variance is tolerable during critical stages of development.
