Xenopus Sox11 Partner Proteins and Functional Domains in Neurogenesis

Abstract

Sox11, a member of the SoxC family of transcription factors, has distinct functions at different times in neural development. Studies in mouse, frog, chick, and zebrafish show that Sox11 promotes neural fate, neural differentiation, and neuron maturation in the central nervous system. These diverse roles are controlled in part by spatial and temporal-specific protein interactions. However, the partner proteins and Sox11-interaction domains underlying these diverse functions are not well defined. Here, we identify partner proteins and the domains of Xenopus laevis Sox11 required for protein interaction and function during neurogenesis. Our data show that Sox11 co-localizes and interacts with Pou3f2 and Neurog2 in the anterior neural plate and in early neurons, respectively. We also demonstrate that Sox11 does not interact with Neurog1, a high-affinity partner of Sox11 in the mouse cortex, suggesting that Sox11 has species-specific partner proteins. Additionally, we determined that the N-terminus including the HMG domain of Sox11 is necessary for interaction with Pou3f2 and Neurog2, and we established a novel role for the N-terminal 46 amino acids in the specification of placodal progenitors. This is the first identification of partner proteins for Sox11 and of domains required for partner-protein interactions and distinct roles in neurogenesis.

Keywords: Xenopus laevis; cell differentiation; neurogenesis; protein interaction; transcription factor.

Figure 1

Sox11, neurog2, and pou3f2 are co-expressed in distinct cell types of the neural plate. (A) WISH of sox11, pou3f2, and neurog2 at stage 12.5 (late gastrula) and stage 14 (early neurula). Embryos are dorsal view with anterior to the top. Sox11 is expressed throughout the neural plate and later in placodes. Pou3f2 expression is not detectable until the neurula stage and is strongest in the anterior neural plate. Neurog2 is expressed in the stripes of the neuronal progenitors. The scale bar is 500 microns. (B) Total number of cells across developmental stages that are sox11⁻ and neurog+ (dark gray), sox11+ and neurog+ (magenta) (top) or sox11⁻ and pou3f2+ (light gray), and sox11+ and pou3f2+ (teal) (bottom) within the neural plate (NP) at stage 12 and within the anterior neural plate (ANP), posterior neural plate (PNP), and early neurons (EN) at stage 13/14.

Figure 2

Xl Sox11 interacts with Pou3f2 and Neurog2, but not Neurog1. (A–C). Immunoprecipitation (IP) of Xl Sox11-FLAG and Pou3f2-HA (A), Neurog2-MYC (B), or Neurog1-HA (C) from in vitro translated proteins. Proteins were immunoprecipitated using either FLAG (red), HA (green), or MYC (blue) antibodies. Samples were analyzed by western blotting (WB) indicated on the right with FLAG-HRP, MYC-HRP, or HA-HRP. Input represents the total protein lysate.

Figure 3

Sox11 N-terminus is essential for protein–protein interactions. (A) Schematic of Sox11 and deletion constructs with referenced domains marked. ΔN46-Sox11-FLAG lacks the 46 amino acids (teal) upstream of the HMG domain. ΔHMG-Sox11-FLAG lacks the 72 amino acid HMG domain (orange), and ΔCterm-Sox11-FLAG lacks 265 amino acids (blue) and consists of the N-terminus and HMG domain. (C–G) Immunoprecipitation (IP) of ΔCterm-Sox11-FLAG, ΔN46-Sox11-FLAG, or ΔHMG-Sox11-FLAG with Pou3f2-HA or Neurog2-MYC (Ngn2-MYC). Proteins were generated via in vitro translation and immunoprecipitated using either FLAG (red), HA (green), or MYC (blue) antibodies. Samples were analyzed by WB with anti-FLAG-HRP, anti-HA-HRP, or anti-MYC-HRP. Inputs demonstrate each protein in the extract. (H) Graphical representation of protein co-immunoprecipitated (Sox11 variants and Pou3f2) from three replicates, one of which is represented in (B–D). (I) Graphical representation of protein co-immunoprecipitated (Sox11 variants and Neurog2) from three replicates, one of which is represented in (E–G). Co-expression bands were normalized to the input in each sample. Data represent the mean ± sem on a log₁₀ scale with percent of pulldown across 3 experimental replicates.

Figure 4

Identification of Sox11 domains required for placode and neuron formation. WISH of neurula (stage 14/15) embryos injected in one of two cells (dorsal view, anterior to the top) with Dextran as a tracer and either Δcterm-Sox11, Δn46-Sox11, Δhmg-Sox11, or Δtad-Sox11 mRNA. The right side is the injected side, and the left side serves as control expression. Embryos were analyzed for expression of tubb2 for neurons or sox3 for neural progenitors. An arrow marks the reduction in sox3 expression in placodal progenitors, and an asterisk marks the loss of expression in the trigeminal placode. Numbers in the upper right of each image denote the number of embryos with the phenotype over the total analyzed. A one-sample proportion test reveals that the null hypothesis that the overexpression of the mRNA has no effect on tubb2 expression can be rejected in all cases except for the loss of the trigeminal placode for Δhmg-Sox11 p-value = 0.564.