An essential role for transcription before the MBT in Xenopus laevis

Abstract

Most zygotic genes remain transcriptionally silent in Drosophila, Xenopus, and zebrafish embryos through multiple mitotic divisions until the midblastula transition (MBT). Several genes have been identified in each of these organisms that are transcribed before the MBT, but whether precocious expression of specific mRNAs is important for later development has not been examined in detail. Here, we identify a class of protein coding transcripts activated before the MBT by the maternal T-box factor VegT that are components of an established transcriptional regulatory network required for mesendoderm induction in Xenopus laevis, including the Nodal related ligands xnr5, xnr6, and derrière and the transcription factors bix4, and sox17α. Accumulation of phospho-Smad2, a hallmark of active Nodal signaling, at the onset of the MBT requires preMBT transcription and activity of xnr5 and xnr6. Furthermore, preMBT activation of the Nodal pathway is essential for mesendodermal gene expression and patterning of the embryo. Finally, xnr5 and xnr6 can also activate their own expression during cleavage stages, indicating that preMBT transcription contributes to a feed-forward system that allows robust activation of Nodal signaling at the MBT.

Fig. 1. Mesendodermal genes transcribed before the MBT

(A) RT-PCR was performed on embryos collected at the indicated stages. (B) Quantitative RT-PCR was performed on staged embryos and the number of transcripts/embryo was determined as described in Materials and Methods. Log₁₀[transcripts/embryo] was plotted for each gene. (C) α-amanitin (100pg/embryo) was injected into both cells at the 2-cell stage. Injected and non-injected controls were harvested at the 1000-cell stage (preMBT) for qRT-PCR analysis. The results were normalized to expression in the absence of α-amanitin (ni) for each gene. (D) Two ventral cells at the 4-cell stage were injected with rhodamine dextran as a lineage tracer, dissected into dorsal and ventral halves at the 256-cell stage, then harvested at the 1000-cell stage for qRT-PCR. MBT = 4000c; ni, not injected. Error bars represent standard error of the mean for 2 or more experiments.

Fig. 2. VegT is required for preMBT transcription

(A) VegT mRNA was depleted in oocytes with 5ng antisense phosphorthiotate oligos and embryos were recovered using the host-transfer method. Control and VegT depleted embryos were collected at the 1000-cell stage for qRT-PCR. Error bars represent the mean of triplicate measurements in qRT-PCR. (B) VegT mRNA was injected into the animal pole at the 1-cell stage. Animal caps were excised at the 256-cell stage, cultured, and harvested for qRT-PCR at the 1000-cell stage. (C) Antisense morpholino oligonucleotide (MO) targeting β-catenin (40ng/embryo) was injected laterally into two cells at the 2-cell stage. Embryos were harvested for qRT-PCR at the 1000-cell stage. ni, not injected. Error bars represent standard error of the mean for 2 or more experiments. Gene expression was normalized to ODC for each sample.

Fig. 3. preMBT Nodal expression activates Smad2/3

(A) xnr5 mRNA (5pg) or xnr1 mRNA (5pg) was injected vegetally into 1-cell embryos. Embryos were collected at the indicated stages and lysates were analyzed by Western blot for P-Smad2 and Smad2/3. “ni” indicates non-injected controls. Arrowheads indicate P-Smad2 (upper) and P-Smad3 (lower). (B) Non-injected embryos were collected at the indicated stages and subjected to immunoprecipitation with an antibody for Smad2 or for Wnt1 as a negative control (control), then immunoblotted with antibodies for P-Smad2 or Smad2/3. (C) 1-cell embryos were injected laterally with 100pg of the pol-II inhibitor α-amanitin alone or together with 5pg xnr5 mRNA, then collected for IP and immunoblot at the 4000-cell stage (MBT) as in B. (D) xnr5 and xnr6 translation was inhibited by injection in all cells of 4-cell embryos of antisense morpholino oligonucleotides (MO) directed against both xnr5 and xnr6. Xnr5/6MO “a” indicates xnr5 MOa + xnr6 MOa (15 ng each); “b” indicates xnr5 MOb + xnr6 MOb (15 ng each); ni: non-injected; CMO: control MO (30ng). Embryos were collected at the 4000-cell stage for IP and immunoblot as in B. (E) Embryos were injected ventrally at the 4-cell stage with rhodamine dextran, dissected into dorsal and ventral halves at the 256-cell stage, cultured, then harvested at the 4000-cell stage for IP and Western analysis as in B. we, whole embryo. Band intensity was determined by quantifying pixel density from a scanned film using Image J; the level of P-Smad2 was then normalized to total Smad2 and plotted.

Fig. 4. preMBT Nodal signaling is required for mesendoderm induction

(A) Embryos were treated with control medium (nt) or SB5 (200µM) from the 4-cell stage to stage 10 and harvested for Western blot for P-Smad2/3 or Smad2/3. (B) Embryos were treated with SB5 from the indicated stages until the onset of gastrulation and cultured until stage 40. Frequency of phenotype is indicated in the lower right corner. (C) Embryos were treated with SB5 beginning at the indicated stages, cultured until stage 10, and analyzed by in situ hybridization (ISH) for mesendodermal gene expression (left). “Control” indicates DMSO (vehicle) only. Embryos were also harvested at early gastrula stage (stage 10) for qRT-PCR for the indicated mesendodermal markers and for the ectodermal marker foxi1e (right panel). All samples were normalized to expression levels in untreated embryos at stage 10 (control). “Control” indicates DMSO; “SB5” indicates treatment from 4-cell until gastrula (stage 10). (D) SB5 was added at the 4-cell stage; embryos were washed at the indicated stages, transferred to control medium, and analyzed by ISH for mesendodermal gene expression at stage 10, as in (C). Histogram on right shows qRT-PCR for mesendodermal and ectodermal markers, as in (C). Note that the “control” and “SB5” (4-cell until stage 10) qRT-PCR data in panel D are identical to those shown in panel C, as qRT-PCR for both groups was performed at the same time.

Fig. 5. Rapid inhibition of Nodal signaling by SB5

(A) xnr5 mRNA (20 pg) was injected at the 2-cell stage. SB5 (200uM) or vehicle (0.2% DMSO) was added at the 128 cell-stage and embryos were collected for immunoblot at the indicated stages. The left panels are immunoblots with an antibody specific for phospho-Smad2/3 and the right panels show total Smad2/3 as a loading control. Double arrowheads indicate the Smad2 and Smad3 bands. (B) SB5 or vehicle was added to uninjected embryos at the 1000-cell stage and embryos were collected at the indicated times for detection of phospho-Smad2 by immunoprecipitation with anti-Smad2 antibodies followed by immunoblot with either the phospho-Smad2 specific antibody (left panel) or an antibody for total Smad2/3 (right panel). +20, +40, +60 and +80 indicate minutes after the MBT.

Fig. 6. Smad2 rescues mesendodermal gene expression when activated before but not after the MBT

Embryos were injected with a hormone inducible, activated form of Smad2 (GR-tSmad2) at the 2-cell stage (along with FLDx as a lineage tracer) and then treated with SB5 beginning at the 4-cell stage. GR-tSmad2 was activated by addition of dexamethasone (dex) before the MBT (stage 7) or after the MBT (stage 9) and embryos were cultured until the onset of gastrulation and then collected for analysis of mesendodermal gene expression by ISH (A) or qRT-PCR (B), as described in Fig. 4. (In panel A, the insets show FLDx immunostaining to reveal the progeny of injected blastoemeres and the numbers in the upper left indicate the frequency of the displayed phenotype/expression pattern). Error bars represent standard deviation for a representative experiment.

Fig. 7. Nodal signaling contributes to xnr5/6 expression

(A) SB5 (200 µM) was added at the 4-cell stage and embryos were fixed for ISH at MBT (stage 8.5). Whole-embryos were photographed in vegetal views, dorsal to the top (vegetal view). Embryos bisected along the dorsal/ventral axis prior to ISH are shown with dorsal side to the right (lateral view). The frequency of the expression pattern for each group is indicated in the lower right corner. DMSO was used as a control. (B) To inhibit Nodal signaling, the dominant-negative Nodal receptor (DN-ALK4) was injected (1 ng), along with fluorescent dextran (insets, rust-colored staining), into the 2 right-hand blastomeres of 4-cell embryos. Embryos were fixed at the indicated times and analyzed by ISH. Fluorescent dextran injection alone is shown as control. The number of cells expressing xnr5 or xnr6 in injected clones was counted and significance was determined using the Kruskal-Wallis test. (C) 8-cell embryos were injected in one animal cell with GR-tSmad2 (400 pg) and fluorescent dextran. GR-tSmad2 was activated by addition of dex for a two-hour window beginning at the indicated stages. Embryos were fixed at the end of the window and analyzed by ISH.

Fig. 8. xnr5 and xnr6 can activate Smad2 and regulate their own expression before the MBT

(A) 8-cell embryos were injected in one animal cell with fluorescent dextran alone (control) or with xnr1 (50 pg), xnr5 (20pg), or xnr6 (20 pg) mRNAs. Whole mount ISH for xnr5, xnr6, and chd was performed on embryos fixed at the indicated stages. (B) Embryos from A were analyzed by qRT-PCR at stage 8 (preMBT). (C) 8-cell embryos were injected in one animal cell with GFP-Smad2 (200pg) alone or with xnr5 mRNA (50pg). Embryos were fixed at the 512-cell stage, processed for anti-GFP immunostaining, and sectioned for fluorescence imaging. DAPI was used to reveal nuclei. The graph shows the percent of DAPI positive nuclei that also contain GFP-Smad2. (D) Embryos injected as in C were fixed at various stages as indicated and stained for xnr6 expression. ui, uninjected.