FMRP Regulates Neurogenesis In Vivo in Xenopus laevis Tadpoles

Abstract

Fragile X Syndrome (FXS) is the leading known monogenic form of autism and the most common form of inherited intellectual disability. FXS results from silencing the FMR1 gene during embryonic development, leading to loss of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein that regulates mRNA transport, stability, and translation. FXS is commonly thought of as a disease of synaptic dysfunction; however, FMRP expression is lost early in embryonic development, well before most synaptogenesis occurs. Recent studies suggest that loss of FMRP results in aberrant neurogenesis, but neurogenic defects have been variable. We investigated whether FMRP affects neurogenesis in Xenopus laevis tadpoles that express a homolog of FMR1. We used in vivo time-lapse imaging of neural progenitor cells and their neuronal progeny to evaluate the effect of acute loss or overexpression of FMRP on neurogenesis in the developing optic tectum. We complimented the time-lapse studies with SYTOX labeling to quantify apoptosis and CldU labeling to measure cell proliferation. Animals with increased or decreased levels of FMRP have significantly decreased neuronal proliferation and survival. They also have increased neuronal differentiation, but deficient dendritic arbor elaboration. The presence and severity of these defects was highly sensitive to FMRP levels. These data demonstrate that FMRP plays an important role in neurogenesis and suggest that endogenous FMRP levels are carefully regulated. These studies show promise in using Xenopus as an experimental system to study fundamental deficits in brain development with loss of FMRP and give new insight into the pathophysiology of FXS.Key words: dendrite development; FMRP; Fragile X Syndrome; in vivo imaging; neurogenesis; Xenopus Significance StatementFragile X Syndrome (FXS) is commonly thought to arise from dysfunction of the synapse, the site of communication between neurons. However, loss of the protein that results in FXS occurs early in embryonic development, while synapses are formed relatively late. This suggests that deficits may occur earlier in neuronal development. We show that changes in FMRP expression in the brains of intact Xenopus laevis tadpoles have profound effects on neurogenesis, the generation of neurons. Therefore, neuronal function in FXS may be affected by events that have gone awry during embryonic development. These studies show promise in using Xenopus as a model of FXS and give new insight into the pathophysiology of FXS.

FMRP binding to a ranked subset of long genes is revealed by coupled CLIP and TRAP in specific neuronal cell types

“…It cannot be stated enough how important it is to avoid altering the timing or levels of experimentally tagged FMRP. In addition to years of literature in the field attesting to the deleterious effects of both under-and over-expression of FMRP in many system there are multiple reports of human intellectual disability linked to overexpression of FMRP due to genomic copy number variations [56][57][58][59]112 . Moreover, the interactions of RNA binding proteins with their RNA ligands are highly dependent on the relative stoichiometry between all possible binding partners.…”

Reversible developmental stasis in response to nutrient availability in theXenopus laevisCNS

“…6 all PH3+ cells in the tectum were included. Brightly labeled SytoxO+ cells were assayed as a measure of apoptotic cells, as described in Faulkner et al (2015). Because SytoxO labels all nuclei, to distinguish brightly labeled dying cells from healthy cells, we counted nuclei that met a Table 1 for data and Table 2 for corresponding statistics; n=500 animals total from two independent clutches, with a subset of 11-22 animals measured per group per time point.…”

“…Dying SytoxO+ cells also had a relatively small, condensed nucleus and typically colocalized with activated capase-3 (Faulkner et al, 2015). For both PH3+ and SytoxO+ cell counts, data were normalized to the day 0 control so that samples could be compared across multiple experiments from different clutches of animals.…”

“…To test whether NR or DF induced cell death, we counted the number of dying cells in the tectum using SytoxO labeling (Faulkner et al, 2015) in the three nutrition treatment groups. Fig.…”

Experience-dependent development of dendritic arbors in mouse visual cortex

“…However, not all characteristics of the arbor are determined in this way; refinement of the arbor also involves a dynamic process of growth and pruning, each with known molecular regulators. Work from the Cline lab among others have established numerous molecular regulators of dendritic outgrowth [46, 48, 49, 62–65, 70, 71, 104], including many pathways directly connected with circuit activity-dependent mechanisms promoting dendrite outgrowth [42, 46, 51, 66, 71, 105, 106]. The canonical model of this process, the Synaptotrophic Hypothesis, suggests that dendrites grow toward areas that have active inputs and are stabilized by the presence of maturing synaptic contacts [72].…”