XB-ART-56609Dev Biol April 15, 2020; 460 (2): 99-107.
Xenopus embryos show a compensatory response following perturbation of the Notch signaling pathway.
As an essential feature of development, robustness ensures that embryos attain a consistent phenotype despite genetic and environmental variation. The growing number of examples demonstrating that embryos can mount a compensatory response to germline mutations in key developmental genes has heightened interest in the phenomenon of embryonic robustness. While considerable progress has been made in elucidating genetic compensation in response to germline mutations, the diversity, mechanisms, and limitations of embryonic robustness remain unclear. In this work, we have examined whether Xenopus laevis embryos are able to compensate for perturbations of the Notch signaling pathway induced by RNA injection constructs that either upregulate or inhibit this signaling pathway. Consistent with earlier studies, we found that at neurula stages, hyperactivation of the Notch pathway inhibited neural differentiation while inhibition of Notch signaling increases premature differentiation as assayed by neural beta tubulin expression. However, surprisingly, by hatching stages, embryos begin to compensate for these perturbations, and by swimming tadpole stages most embryos exhibited normal neuronal gene expression. Using cell proliferation and TUNEL assays, we show that the compensatory response is, in part, mediated by modulating levels of cell proliferation and apoptosis. This work provides an additional model for addressing the mechanisms of embryonic robustness and of genetic compensation.
PubMed ID: 31899211
PMC ID: PMC7263880
Article link: Dev Biol
Genes referenced: drosha notch1 pcna rbpj sox2 sts tubb2b
GO keywords: Notch signaling pathway
Article Images: [+] show captions
|Fig. 1. tubb2b expression in response to Notch perturbation. Embryos unilaterally injected with 1.5 ng ICD or DBM show perturbed expression of tubb2b at neurula stages on the injected (left) side (A, B, G, H). The difference in expression between sides is less stark at the tailbud stage (C, D, I, J), and minimal difference between sides is apparent at the swimming tadpole stage (E, F, K–N). All embryos shown are left-side injected. Histological analysis of tubb2b expression in Notch perturbed embryos supports whole-mount findings (G-N). Abbreviations: l, lateral longitudinal stripe of primary neurogenesis; i/m, intermediate/medial longitudinal stripes of primary neurogenesis; asc, anterior spinal cord; e, eye; fb, forebrain; hb, hindbrain; mb, midbrain; sc, spinal cord; V, cranial nerve V; IX, cranial nerve IX; X, cranial nerve X. For each stage and each condition, N = >100 for whole mount embryos and >15 for histological analyses.|
|Fig. 2. Quantification of tubb2b expression and construct persistence. qRT-PCR confirms that ICD injected embryos express less tubb2b, while DBM injected embryos express more tubb2b at the neurula stage (A). tubb2b expression remains perturbed at the tailbud stage (B), but at the swimming tadpole stage, no difference in expression level is detected when comparing perturbed embryos to vehicle-injected controls (n = 3) (C). qRT-PCR shows that while ICD (D) (n = 3) and DBM (E) (n = 3) mRNAs degrade over time, both are still detectable throughout the swimming tadpole stages. Bars represent mean ± SEM. * = p < 0.05, ** = p < 0.01.|
|Fig. 3. tubb2b expression in bilaterally perturbed embryos. Embryos were bilaterally injected at the two-cell stage with either ICD (A-C, G-I) or DBM (D-F, J-L) mRNA and reared to desired stages. ICD bilaterally injected embryos demonstrate minimal tubb2b expression at the neurula stage (A,G, arrowhead) and show morphological defects by the tailbud stage (B,H, arrowhead). DBM bilaterally injected embryos show ectopic tubb2b expression at the neurula stage (D,J, arrowhead), but appear to compensate in a gradual manner over time (E-F, K-L). GFP bilaterally injected embryos and wild type remain unperturbed (M-R). Abbreviations: i/m, intermediate/medial longitudinal stripes of primary neurogenesis; fb, forebrain; mb, midbrain; hb, hindbrain; e, eye; sc, spinal cord. For each stage, N = >50 for ICD and GFP, >10 for DBM. Embryos were obtained from a minimum of three different clutches.|
|Fig. 4. Apoptosis and proliferation in response to Notch perturbation. Total number of neural cells were measured in injected and uninjected sides of embryos over time in response to ICD injection (A) and DBM injection (B) (n = 10; * = p ≤ 0.05). Subsequently, the proportion of cells positive for TUNEL staining was measured on the injected (I) and uninjected (U) sides (C, D) (n = 5; * = p ≤ 0.05, ** = p ≤ 0.01 *** = p ≤ 0.001). Next, the proportion of neural cell expressing pcna was measured in injected and uninjected sides of embryos (E, F) (n = 5; * = p ≤ 0.05). Representative images of TUNEL staining and pcna expression in Notch perturbed embryos are shown in G and H, respectively. All embryos shown are left-side injected. Abbreviations: np, neural plate; nc, notochord; fb, forebrain; hb, hindbrain; mb, midbrain; e, eye; ot, otic vesicle.|