Maternal Ybx1 is essential for early development.

(A) Schematic of Ybx1 showing the various domains, the V83F mutation in the CSD in ybx1^sa42, and deletion of residues 197-310 in ybx1^sg8 mutants. Black box in Ybx1^sg8 indicates frameshift after residue 197 and premature stop after residue 205. (B) rYbx1^V83F lacks detectable DLE-binding activity similar to vector control, whereas rYbx1 and rYbx1^Δ197-310 peptides, and embryo lysates show binding to sqt1 probes. Western blots to detect 6xHis epitope tags show expression of recombinant Ybx1 proteins. (C) Mybx1^sa42 embryo extracts show no detectable binding to sqt1 probe compared to control extracts. (D) Schematic representation of the ybx1 genomic locus (blue) with positions of viral 2a peptide (magenta bar) and gfp (green box) indicated. Red triangles indicate Ds transposon terminal repeats. (E) DIC photomicrographs showing 16-cell, 64-cell, 1000-cell and 50% epiboly stage embryos. Mybx1^sa42 embryos are viable at 28.5°C. Mybx1^sg8 embryos cleave aberrantly after 16-cells (open arrowhead). Mybx1^sa42 embryos at 23°C, and Mybx1^sg8 embryos fail to initiate gastrulation, form syncytia (black arrowheads), and arrest. Zygotic Ybx1-GFP expression from PTg does not rescue gastrula arrest in Mybx1, whereas maternal Ybx1-GFP expression from MTg leads to normal gastrulation. Scale bar, 100 µm. (F) Histogram showing rescue of gastrulation and survival till prim5 stage of Mybx1 mutants at 23°C by two independent MTg lines (MTg #4 and MTg #6). Some embryos with zygotic expression of ybx1 (PTg) from both lines can initiate gastrulation, but none survive to prim5. Error bars show standard deviation from three experiments. Number of embryos is shown on top of the histogram.

Ybx1 is required for localization and regulated processing of sqt RNA.

(A) Control embryos at 23°C show sqt RNA localization at the 1-cell and 4-cell stage. sqt RNA transport is delayed in 1-cell Mybx1^sa42 embryos at 28.5°C, but localizes correctly by the 4-cell stage. At 23°C, sqt RNA largely remains in the yolk even at 4-cell stage and sqt RNA that reaches the blastoderm is mis-localized. Localization of sqt RNA is restored in Mybx1 by ybx1-2a-gfp MTg, but not by PTg. Localization of wnt8a RNA is normal in Mybx1^sa42 mutants at 28.5°C and 23°C. Scale bar, 100 µm. (B) Q-RT-PCRs to detect total sqt RNA levels show a mild reduction in Mybx1 compared to controls. Error bars show standard deviation from three experiments. (C) RT-PCR to detect sqt in control and Mybx1 mutants at 1-cell, 4-cell and 16-cell stages. Products are indicated on the right, and sizes on the left. Polyadenylated sqt RNA is detected from 16-cells in controls, and at 1-cell in Mybx1 mutants. Splicing of sqt intron 1 and sqt intron 2 occurs earlier in Mybx1 embryos compared to controls. PCR to detect actin is shown as control.

Ybx1 interacts with the translation initiation machinery and represses translation of sqt RNA.

(A) Western blot to detect GFP shows injected sqt-gfp is translated by 16-cells in Mybx1, whereas in controls, Sqt-GFP is detected at blastula stages, and lacZ control injection shows no Sqt-GFP. (B) Sqt-GFP protein expression is precocious and elevated in Mybx1 embryos. Error bars in B show standard deviation from three experiments. (C) Co-immunoprecipitation in embryo lysates followed by western blot analysis shows that Ybx1 interacts with eIF4E. eIF4G binds poorly with Ybx1. Faint smear in control IgG lane for eIF4G is spillover from input lane (see Figure 5-figure supplement 1C for complete blot for eIF4G). (D) Antibodies towards Ybx1, eIF4G and eIF4E pull down sqt RNA in embryos lysates. RT-PCR on the embryos lysates in panel C shows sqt RNA but not control gapdh or wnt8a RNA in RNA-IP with αYbx1 antibodies. Control IgG antibodies do not show any RT-PCR product, whereas antibodies to the translation initiation factor eIF4E can pull down sqt RNA, wnt8a and gapdh RNA, and antibodies to eIF4G detects weak bands for sqt and gapdh in the RNA-IPs. RT-PCR from whole embryo lysates is the positive control. PCR product sizes are indicated on the right.

Nodal signaling is deregulated in Mybx1 embryos.

(A) Phosphorylated-Smad2 is detected at the 64-cell stage in Mybx1 embryos. (B) Phospho-Smad2 levels are elevated in Mybx1 embryos at cleavage stages, compared to controls. (C) Quantitative real-time RT-PCR shows that Nodal target (sqt, gsc, ntl, bon) and YSL gene expression (mxtx2) is elevated in Mybx1 compared to controls, whereas expression of lefty2, the Wnt target, boz, ventral mesoderm gene vox, FGF target spry4, and the enveloping layer (EVL) marker cldE, is either not significantly altered or marginally reduced. (D) Whole mount in situ hybridization shows expanded YSL domains of sqt, gsc, and mxtx2 in Mybx1 embryos; the cldE expression domain is not significantly different from controls, and vox is not detectable. Scale bar, 100 µm. (E) Mybx1 mutants have expanded YSL. DAPI staining to detect nuclei and E-cadherin immunostaining to detect membranes shows 1 tier of YSL nuclei (undergoing division) in control embryos. In Mybx1^sa42and Mybx1^sg8 embryos, multiple tiers of YSL are observed (yellow boxed area). Bottom panels show higher magnification of yellow, boxed area. Cell membranes are clearly demarcated in control embryos, but appear fragmented in Mybx1 mutants. Scale bars, 100 µm. (F) Histogram showing YSL nuclei numbers in Mybx1 and control embryos, with or without ybx1 transgenes at 23°C. ~75% of control embryos have no YSL nuclei and only 25% show 1-6 YSL nuclei, whereas ~80% of Mybx1 embryos and Mybx1 embryos with PTg, show 7 or more YSL nuclei, and ~25% show >20 YSL nuclei. Mybx1 embryos with ybx1 MTg show reduced numbers of YSL nuclei. Number of embryos scored is indicated above the histogram. Error bars in B and C show standard deviation from three experiments.

YSL and gastrulation defects in Mybx1 mutants can be rescued by blocking Nodal signaling.

(A) DAPI-stained nuclei and E-cadherin immunostained membranes are clearly demarcated in lacZ and lefty1 RNA-injected control embryos. E-cadherin staining appears fragmented and number of YSL nuclei is increased in Mybx1 mutant embryos injected with control lacZ RNA (white arrowhead). In Mybx1 embryos injected with lefty1 RNA, the number of YSL nuclei is restored to normal levels and membrane staining does not appear fragmented. Bottom panels show higher magnification of area boxed in yellow; scale bar, 100 µm. (B) Histogram showing rescue of gastrulation and survival till prim5 stage by injection of lefty1 RNA but not lacZ RNA in Mybx1 mutants that were subjected to temperature shift at 23°C. Error bars show standard deviation from three experiments. Number of embryos is shown on top of the histogram. (C) Histogram showing gastrulation and % survival in embryos from ybx1^sa42/sa42;sqt^cz35/+ crosses and Mybx1 mutants subjected to temperature shift at 23°C. Most embryos from ybx1^sa42/sa42;sqt^cz35/+ crosses initiate and complete gastrulation in comparison to Mybx1 ^sa42 mutants. (D) Histogram showing survival at 23°C and genotypes of embryos from matings of ybx1^sa42/sa42;sqt^cz35/+, in comparison to Mybx1^sa42 embryos which do not survive at 23°C. The expected % for each genotype is 25% for Mybx1^sa42;sqt^cz35/cz35 and Mybx1^sa42;sqt^+/+, and 50% for Mybx1^sa42;sqt^cz35/+. All Mybx1^sa42;sqt^cz35/cz35 embryos (which have no Sqt signaling) survive, whereas many Mybx1^sa42;sqt^cz35/+ and Mybx1^sa42;sqt^+/+ do not survive at 23°C. Number of embryos scored is indicated above the histogram, and % observed for each genotype is indicated to the right of the colored bars.