Measuring potential effects of the developmental burden associated with the vertebrate notochord

Abstract

The notochord functions primarily as a supporting tissue to maintain the anteroposterior axis of primitive chordates, a function that is replaced entirely by the vertebral column in many vertebrates. The notochord still appears during vertebrate embryogenesis and plays a crucial role in the developmental pattern formation of surrounding structures, such as the somites and neural tube, providing the basis for the vertebrate body plan. The indispensable role of the notochord has often been referred to as the developmental burden and used to explain the evolutionary conservation of notochord; however, the existence of this burden has not been successfully exemplified so far. Since the adaptive value of target tissues appears to result in the evolutionary conservation of upstream structures through the developmental burden, we performed comparative gene expression profiling of the notochord, somites, and neural tube during the mid-embryonic stages in turtles and chicken to measure their evolutionary conservation. When compared with the somites and neural tube, overall gene expression profiles in the notochord showed significantly lower or merely comparable levels of conservation. However, genes involved in inductive signalings, such as the sonic hedgehog (Shh) cascade and the formation of functional primary cilia, showed relatively higher levels of conservation in all the three structures analyzed. Collectively, these results suggest that shh signals are critical as the inductive source and receiving structures, possibly constituting the inter-dependencies of developmental burden.

Keywords: developmental burden; evolution; gene expression profile; notochord; phylotypic period.

Figure 1

Embryos and structures targeted for the laser‐micro dissection‐based RNAseq. (a) Stage HH16 of the chicken embryo (left) and stage TK11 of turtle embryo (right). Images were modified and adapted from our previous study. (b) 3D reconstructed image of the chicken embryo at stage HH16 made by Avizo. Only neural tissues (blue), somites (green), notochord (red), and blood vessels (pink) are shown. Purple lines represent target anteroposterior levels. Representative images (hematoxylin and eosin‐stained) of anterior and posterior sections are shown on the right. As anterior somites have already started to differentiate in this stage, somite‐derived regions were also collected (green‐dashed line). Scale bars = 100 μm [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Evolutionary conservation of gene expression profiles in the notochord, neural tube, and somites between chicken and turtles. Evolutionary distances between gene expression profiles of chicken and turtles were evaluated by 1 – Spearman correlation coefficients of 1:1 orthologs. Bar plots on the left represent sections from the anterior level, and bar plots on the right represent sections from the posterior level. N = 3. Different structures were dissected from the same individual for each biological replicate. Error bar: SD, p values: Dunnett's test (two‐tailed)

Figure 3

Expression levels of shh‐related genes in the posterior notochord. Left: Gene expression levels (TPM) in posterior notochords of chicken and turtles are shown as a scatter plot. Shh‐related genes (46 genes) are colored in red, and other genes in the genomic background are colored in light blue (12,233 genes). Each plot represents average expression levels of biological replicates within each species. The gray zone represents signal ratio chicken–turtle less than twofold. Right: Pie charts represent the ratio of shh‐related genes within the twofold range (up), and the ratio of genomic background (down). Deviations in ratios represent SD. The differences in the ratio of genes within the twofold range and genes outside the twofold range were statistically significant between shh‐related genes and the genomic background (Student's t test, n = 9) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

GO terms enriched in notochord–neural tube–somites conserved genes. 1:1 orthologous genes with conserved expression levels (within twofold change between turtles and chicken) in the notochord, neural tube, and somites were identified, and then subsets of genes that show conserved expressions in the late embryonic phase (TK27 for turtles and HH38 for chicken) were subtracted. These genes were further analyzed for the enrichment of GO terms by comparing them to those of genomic background, and their effect sizes are shown in the bar plot. Only GO terms with statistical significance (two‐sided Fisher's exact test with Holm‐corrected alpha levels), both in anterior and posterior structures, are shown (see also Tables S3 and S4 for more detail). The X axis represents times enrichment over genomic frequency