Rab7 is required for mesoderm patterning and gastrulation in Xenopus

Abstract

Early embryogenesis requires tightly controlled temporal and spatial coordination of cellular behavior and signaling. Modulations are achieved at multiple levels, from cellular transcription to tissue-scale behavior. Intracellularly, the endolysosomal system emerges as an important regulator at different levels – but in vivo studies are rare. In the frog Xenopus, little is known about developmental roles of endosomal regulators, or their potential involvement in signaling, especially true for late endosomes. Here, we chose to analyze a hypothesized role of Rab7 in this context, a small GTPase known for its role as a late endosomal regulator. First, rab7 showed strong maternal expression. Following localized zygotic transcript enrichment in the mesodermal ring and neural plate, it was found in tailbud stage neural ectoderm, notochord, pronephros, eyes and neural crest tissues. Inhibition resulted in strong axis defects caused by a requirement of rab7 for mesodermal patterning and correct gastrulation movements. To test a potential involvement in growth factor signaling, we analyzed early Wnt-dependent processes in the mesoderm. Our results suggest a selective requirement for ligand-induced Wnt activation, implicating a context-dependent role of Rab7.

FGF-mediated establishment of left-right asymmetry requires Rab7 function in the dorsal mesoderm in Xenopus

“…also Schneider et al, 2019 ). Further, our recent findings of defects in mesoderm patterning and subsequent gastrulation movements after loss of Rab7, which resulted in severe dorsal/posterior truncations at tailbud stage, were also highly reminiscent of a loss of FGF receptor 1 mediated signaling ( Amaya et al, 1991 , 1993 ; Kreis et al, 2021 ). Based on these parallels, we hypothesized that Rab7 was required for FGF-mediated specification and/or maintenance of mesodermal and somitic fates during gastrulation, i.e., upstream of MAPK cascade activation.…”

“…Our previous analyses of the role of Rab7 in mesoderm patterning during gastrulation suggested a regulatory role for pre-patterning and specification of the LRO or its precursor tissues as well ( Kreis et al, 2021 ). To address the hypothesized impact of Rab7 deficiency on later LRO function and LR asymmetry, we injected Xenopus embryos at the four-cell stage with reduced amounts of a rab7 translation blocking Morpholino oligomere (TBMO; cf.…”

“…To address the hypothesized impact of Rab7 deficiency on later LRO function and LR asymmetry, we injected Xenopus embryos at the four-cell stage with reduced amounts of a rab7 translation blocking Morpholino oligomere (TBMO; cf. Kreis et al, 2021 ) into the dorsal lineage, i.e. targeting the future LRO (see Figure 1A for experimental setup; cf.…”

“…First, to understand the basis of the LR asymmetry phenotype, we analyzed foxj1 at early gastrula stages to check SM specification, and tekt2 , a Foxj1 target gene highlighting the differentiated LRO during neurulation (see Figure 2A for experimental setup; Stubbs et al, 2008 ). We have previously shown that inhibition of Rab7 (using higher MO concentrations) strongly blocked foxj1 expression, i.e., LRO precursor formation ( Kreis et al, 2021 ). In our low-dose MO approach used here, morphant embryos always showed activation of foxj1 during gastrulation, but approximately one third of them displayed reduced expression ( Figures 2B–E ).…”

“…In our recent work we analyzed the role of the late endosome master regulator Rab7 for early embryonic development in Xenopus . We demonstrated that rab7 was required for involution and axial elongation during gastrulation by regulating mesodermal specification ( Kreis et al, 2021 ). While organizer induction and DV patterning were unaltered in Rab7-deficient embryos, foxj1 expression in the SM was strongly inhibited.…”