XB-ART-56863Dev Biol June 15, 2020; 462 (2): 165-179.
Natural size variation among embryos leads to the corresponding scaling in gene expression.
Xenopus laevis frogs from laboratory stocks normally lay eggs exhibiting extensive size variability. We find that these initial size differences subsequently affect the size of the embryos prior to the onset of growth, and the size of tadpoles during the growth period. Even though these tadpoles differ in size, their tissues, organs, and structures always seem to be properly proportioned, i.e. they display static allometry. Initial axial patterning events in Xenopus occur in a spherical embryo, allowing easy documentation of their size-dependent features. We examined the size distribution of early Xenopus laevis embryos and measured diameters that differed by about 38% with a median of about 1.43 mm. This range of embryo sizes corresponds to about a 1.9-fold difference in surface area and a 2.6-fold difference in volume. We examined the relationship between embryo size and gene expression and observed a significant correlation between diameter and RNA content during gastrula stages. In addition, we investigated the expression levels of genes that pattern the mesoderm, induce the nervous system and mediate the progression of ectodermal cells to neural precursors in large and small embryos. We found that most of these factors were expressed at levels that scaled with the different embryo sizes and total embryo RNA content. In agreement with the changes in transcript levels, the expression domains in larger embryos increased proportionally with the increase in surface area, maintaining their relative expression domain size in relation to the total size of the embryo. Thus, our study identified a mechanism for adapting gene expression domains to embryo size by adjusting the transcript levels of the genes regulating mesoderm induction and patterning. In the neural plate, besides the scaling of the expression domains, we observed similar cell sizes and cell densities in small and large embryos suggesting that additional cell divisions took place in large embryos to compensate for the increased size. Our results show in detail the size variability among Xenopus laevis embryos and the transcriptional adaptation to scale gene expression with size. The observations further support the involvement of BMP/ADMP signaling in the scaling process.
PubMed ID: 32259520
Article link: Dev Biol
Species referenced: Xenopus laevis
Genes referenced: acvr1 acvrl1 admp bmp2 bmp4 bmp7.1 bmpr1a bmpr1b cer1 chrd.1 foxd4l1.1 fst gapdh gmnn gsc lhx1 meis3 myod1 nodal3.1 nog not sia1 slc35b1 sox11 sox2 sox3 szl ventx1.2 ventx2.2 ventx3.2 wnt8a zic1 zic2 zic3
GO keywords: BMP signaling pathway
Article Images: [+] show captions
|Fig. 1. Natural size variability in Xenopus laevis. (A–C) Embryos obtained from multiple clutches (fertilizations) were photographed and measured before mid-gastrula stages. (A) The distribution in diameters of 2239 embryos (n) from 33 fertilizations (N) is shown in a violin plot. The median value is shown as a dashed line and the interquartile range is demarcated by the dotted lines. The inset shows the normal distribution of the embryo diameters. (B) The size distribution of the embryos in each clutch from a single female was plotted relative to the embryo clutches from other females (N = 33). For each clutch, a boxplot of the interquartile range and the median diameter for the clutch is shown. Whiskers mark the range from the 10th to the 90th percentile for each clutch. Clutches were ranked along the Y axis based on their median value. Clutch xvi (filled boxplot) had a median diameter close to the total median diameter of all clutches together and the significance of the size variation was calculated to it. ∗∗∗∗, p < 0.0001; ∗∗, p < 0.01; ∗, p < 0.05; ns, not significant. (C) Correlation between female snout-vent length and clutch embryo mean diameter (SEM shown). Simple linear regression suggests a lack of correlation. p-value provided is the significance of a non-zero slope. (D) Embryos were monitored from fertilization to larval stages (st. 41). Embryos from various clutches differing in size were photographed at several developmental stages to determine whether with the progression of embryogenesis the initial size differences disappear.|
|Fig. 2. RNA content as a function of size during early development. Groups of embryos from multiple clutches were collected during (A) early gastrula, (B) late gastrula, and (C) early neurula. The average embryo diameter of the clutch was determined and RNA was extracted. The volume and the average amount of RNA/embryo were calculated. At each developmental stage, the RNA content/embryo was plotted as a function of volume. N, number of clutches/RNA samples analyzed; n, number of embryos used for RNA extraction and in parenthesis the number of embryos measured to determine the average volume; r2, coefficient of determination; p-value provided is the significance of a non-zero slope.|
|Fig. 3. Changes in marginal zone gene expression in response to size changes as a function of developmental stage. Pairs of embryo clutches differing in size were collected (N = 18) and processed in parallel during (A) early/mid-gastrula (st. 10.5), (B) late gastrula (st. 12), and early (C) neurula (st. 14–15). Gene expression levels were determined by qPCR and the relative expression level between large and small embryos after correcting for the amount of RNA/embryo. In all three graphs the order of the genes and their categorization as DMZ, LMZ, and VMZ (dorsal, lateral and ventral marginal zone respectively) were retained from early gastrula although with development the expression domains change. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ns, not significant.|
|Fig. 4. Blastopore gene expression scales proportionally with embryo size. Groups of embryos of a wide range of sizes from multiple clutches were fixed during mid-gastrula (st. 11) for wnt8a analysis (A–E) or early gastrula (st. 10.25) for chrd.1 (F–J), gsc (K–O) and admp (P–T) analysis by in situ hybridization with gene-specific probes. For each gene, a group of mix-sized embryos was processed for analysis. Based on calculated circumference, the lower 45th percentile comprised the small embryos, and the upper 45th percentile were the large embryos. The middle 10% were omitted from the analysis. For each embryo, the angle describing the expression domain, the embryo diameter and the diameter of the blastopore were measured. Plots comparing the calculated circumference (B, G, L, Q), the angle describing the expression domain (C, H, M, R), the calculated arc of the expression domain (D, I, N, S), and the percent circumference covered by the expression domain (arc)(E, J, O, T) between small and large embryos are shown. ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ns, not significant.|
|Fig. 5. Temporal expression pattern of the neural genes. The temporal pattern of expression of the neural genes was determined by quantitative real-time RT-PCR (qPCR) with primers specific for each gene. The relative expression level was calculated normalizing to the stage 19 sample. (A) Temporal expression pattern for nog, sox3, zic2, and gmnn (A); for foxd4l1.1, zic1, and zic3 (B); and for fst, meis3, sox11, and sox2 (C).|
|Fig. 6. Expression scaling among neural genes. RNA samples were collected from pairs of embryo clutches differing in size. The embryos were collected during (A) early/mid-gastrula (st. 10.5), (B) late gastrula (st. 12), and (C) early neurula (st. 14–15) stages. qPCR was performed for members of the early neural network. Results were normalized to the amount of RNA/embryo, i.e. embryo equivalents. ∗, p <0.05; ∗∗, p <0.01; ∗∗∗, p <0.001; ∗∗∗∗, p <0.0001; ns, not significant.|
|Fig. 7. Scaling of neural expression domains with embryo size. Analysis of the spatial expression domains of foxd4l1.1 (A–G), sox2 (H–N), and zic1 (O–U) in embryos of different sizes. Representative embryos illustrating the measurement of the foxd4l1.1 (A), sox2 (H), and zic1 (O) expression domains. Analysis of the expression domain in embryos at early/mid-gastrula (st. 10–11; B-D, I–K, P-R) and late gastrula (st. 12; E-G, L-N, S–U) were hybridized in situ with probes specific for foxd4l1.1 (B–G), sox2 (I–N), and zic1 (P–U). For each embryo, the area of gene expression and the diameter of the embryo were measured. From these parameters, the surface area of the embryo (B, E, I, L, P, S), the area of the expression domain (C, F, J, M, Q, T), and the percent of the surface area covered by the expression domain (D, G, K, N, R, U) were calculated. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ns, not significant.|
|Fig. 8. Cell density and size in small and large embryos. (A) Phalloidin stained neural plate. (B) DAPI stained neural plate. (C) Cell density in a comparable area of the anterior neural plate was calculated in phalloidin and DAPI stained large and small embryos at early neurula stages (st. 14). (D) The long axis of cells within the neural plate of large and small embryos was measured in phalloidin stained embryos. ns, not significant (p > 0.05, two-sided t-test).|
|Fig. 9. Consequences of scaling of the BMP morphogen gradient with size. Schematic representation of the size-scaled BMP gradient and the effects on the organizer domain as determined by the arc length along the circumference and the induced neural domain.|