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Fig 1. Normal Brg1 protein levels are essential for blastula gene expression and survival.
(A) Target sites of antisense Morpholinos on Xenopus Brg1 mRNAs. Start codon in capital letters; sequence mismatches between Xenopus species are labeled in red. (B) Radial injections of BMO1 (30ng/embryo) and BMO2 (60ng/embryo) in X. tropicalis embryos cause embryonic death from gastrulation stages on (n = 8 biological experiments), while CoMO (60ng/embryo) injected embryos survived beyond hatching stage. Please note that the majority of BMO2 injected embryos die during neurulation. The death counts were recorded at the indicated color-coded stages. NF9: late blastula; NF15: neural fold stage; NF22: late neurula; NF36: hatching. (C) Western Blot analysis of unmanipulated or radially injected X. tropicalis embryos at late blastula stage (NF9). Brg1 protein levels were quantified relative to α-tubulin and normalized to CoMO-injections. BMO1 injected embryos had a mean Brg1 protein level of 0.48, while BMO2 injected embryos had a mean Brg1 protein level of 0.6 (n = 8 experiments). (D) Pie-diagram of the microarray analysis comparing CoMO and BMO1 morphant X. tropicalis at NF9 (averaged from four independent experiments). At this stage, 10216 mRNAs classified the active gene pool; 91.4% of these were unaffected, 6.5% were down- and 2.1% were upregulated by Brg1 protein knockdown (lfdr > 0.2). (E) Validation of the responders from the GO-term ânervous system developmentâ by qRT/PCR (n = 8 independent experiments). Asterisks mark genes, which were significantly downregulated in the qRT/PCR analysis (p-value < 0.05).
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Fig 2. Targeted knockdown of Brg1 protein leads to morphological defects.
Dorsal marginal zone (DMZ) injections in X. laevis: (A) CoMO (10ng) injections have no effect on development. (B) BMO1 (10ng) injections cause stunted axes with severely reduced dorso-anterior tissues. (C) Coinjection of BMO1 and hBrg1 mRNA (1ng) restores AP-axes and anterior tissues like eyes, (n = 3 experiments). Ventral marginal zone (VMZ) injections: (D) CoMO (10ng) injection with no phenotype. (E) The majority of BMO1 (10ng) injected embryos appear wildtype-like. (E’) A smaller fraction of BMO1 morphants (10ng) show enlarged heads and eyes paired with deficits in posterior and ventral tissues. All embryos were coinjected with nlacZ mRNA as lineage tracer. (F) and (G) Phenotypic penetrance of DMZ/VMZ injected embryos (n = 3–5 experiments; *, p-value ≤ 0.03).
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Fig 3. Brg1 cooperates with maternal Wnt signaling for axis formation in X. laevis.
Panels (A-C) axial rescue of DMZ-injected BMO1 morphants (10ng) by coinjected ß-catenin mRNA (250pg). (A) DMZ-injected BMO1 morphant with severly impaired antero-posterior axis (n = 64/75 “headlessâ€); (B) overexpression of ß-catenin in DMZ does not cause gross abnormalities (n = 26/32 wt-like); (C) coinjection of BMO1 and ß-catenin rescues the truncated 1° axis (n = 12/21 heads with eyes and cement gland). Panels (E-G) show that de novo induction of a second embryonic axis by ß-Catenin (250pg) depends on endogenous Brg1 activity. (E) VMZ-injected BMO1 morphants (10ng) display wt-like morphology (n = 12/14; see also Fig 2D); (F) VMZ-injection of 250pg β-catenin mRNA induce a second body axis (n = 38/42 complete secondary heads with eyes and cement gland); (G) Coinjection of BMO1 with β-catenin mRNA blocked almost completely the induction of secondary axes (n = 18/20 single axis status; 1 double axis remaining). Correct targeting of injections was verified by fluorescence from coinjected GFP mRNA (100pg; see inserted images).
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Fig 4. Brg1 protein knockdown diminishes gene expression in the BCNE center.
Pictures show dorsal views of whole mount RNA in situ hybridization (WMISH) at late blastulae (NF9, X. laevis) with gene expression patterns representative for the experimental condition. Injection conditions are indicated on top: CoMO (60ng/embryo), BMO1 (60ng/embryo), Rescue (BMO1 plus hBRG1 mRNA [1ng/embryo]). Reagents were radially injected into the animal hemisphere. The mRNA expression patterns of chordin, noggin and nodal-related 3 (nr3) in control morphants (panels A-C) were reduced in BMO1 morphants (panels A’–C’), but restored by coinjection of human brg1 mRNA (panels A†–Câ€). The graphs (Aâ€â€˜â€“Câ€â€˜) depict phenotypic penetrance (n = 3–4 experiments/condition). The expression of siamois was not affected by BMO1 (panels D + D’, n = 2 experiments/gene).
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Fig 5. Reduced Brg1 protein levels impair organizer function.
X. laevis embryos were radially injected with 40ng of indicated MOs and fixed at early. Representative images of the predominant morphant expression patterns are shown by WMISH in vegetal (A, B, G-L) or dorsal (D, E, N, O) side views. The Organizer region is represented by nr3 (A-C), otx2 (D-F) and foxA4 (G-I). Inserts depict deep staining in bisected embryos for nr3 or blastopore lip staining for otx2. Non-organizer mesoderm is represented by the BMP target genes vent1 (K M) and vent2 (N-P). Inserts show side views for vent1 and vegetal views for vent2 (n = 2-4 independent experiments/gene).
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Fig 6. Brg1 is required in the neuroectoderm for proper eye and brain formation.
(A-C) Orthotopic transplantations in X. laevis embryos. For experimental scheme see S8 Fig, panel A. Shown are dorsal, left and right views of single transplanted embryos at tadpole stage. (A-Aâ€) control transplanted embryo. (B-Bâ€) BMO1 morphant BCNE transplanted embryo. In (C) quantification of the transplantation results (n = 5 experiments).
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Fig 7. Downregulation of Nieuwkoop Center genes contributes to the dorso-anterior phenotype in Brg1 morphants.
At eight cell stage X. laevis embryos were injected either in the two dorso-animal blastomeres (DA) or the two dorso-vegetal blastomeres (DV) with 10 ng of the indicated MO-oligo. Nuclear LacZ mRNA was coinjected as lineage tracer. Embryos were cultivated until hatching stage (A-F) for morphological assessment or until late blastula/early gastrula stage (G-Q) for WMISH analysis. In sagittally bisected embryos the two Nieuwkoop marker genes cerberus (G, H, M, N) and hhex (J, K, O, P) were analysed after indicated injections at late blastula and early gastrula. As indicated by nlacZ staining, the expression domains and the injection domains do not overlap in DA-injected embryos. Quantification of DA-injections (L) and DV-injections (Q) (n = 5–7 biological replicates). *, p-value ≤ 0.05.
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Fig 8. Brg1 amplifies gene transcriptional activation at the MBT.
(A) Prototypic changes in mRNA pools during zygotic genome activation. (B) Global transcriptome analysis at pre-MBT and post-MBT timepoints from X. tropicalis embryos. (C) Box plot illustrating the response of RNA pools in BMO1 morphants versus control morphants. Note that overall zygotically activated genes are reduced upon Brg1 knockdown. (D) Heatmap providing mean expression levels at pre- and postMBT timepoints for genes of the GO-term “pattern specification processâ€, ranked by magnitude of activation (log2-fold change). Red asterisk mark genes that were more than 1.5-fold downregulated in BMO1 morphant embryos compared to control morphant siblings. (E) Pie Diagram for the “highly†upregulated at MBT†gene class (mRNA increases more than 5.65 fold; n = 324 genes). In response to Brg1 protein knockdown, nearly 42% of them (n = 135) were not activated to their full amplitude.
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Supp. Fig. 1Three brg1 specific antisense Morpholinos display different translation blocking efficiencies.
In each condition, 60ng of either CoMO or BMO1, BMO2 or BMO3 were radially injected at the 2–4 cell stage into the animal pole of X. laevis embryos. At 8-cell stage, the Luciferase signals from BMO injected embryos were normalized to CoMO signal intensity (n = 3 independent biological experiments).
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Supp. F2. Apoptosis is not upregulated in BMO1 morphants.
X. laevis embryos were radially injected with CoMo or BMO1 as in Fig 1 and immunostained for activated Caspase-3 protein at early gastrula stage (NF10.5). No apoptotic cells were detected. As positive control, the embryo in panel A was injected with an expression plasmid for the proapoptotic factor FADD (60pg/embryo) into the DMZ. Dashed white line delineates the field, where FADD induced apoptosis; white asterisks indicate background staining on the blastocoel walls; inserts: dorso-vegetal views. Black dashed line marks the extent of the blastopore lip.
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Supp F3.
Functional annotation and validation of BMO1 responding genes.
Panels (A, B) show the top five GO-terms for up- or downregulated genes in X. tropicalis BMO1 morphants. Panels (C, D)—qRT/PCR based verification of microarray data in radially injected X. tropicalis embryos (n = 5–8 independent experiments/gene). Noggin was included based on its function as neural inducer, even though the microarray contained no probeset for this gene. Asterisks mark genes, which were significantly downregulated in the qRT/PCR analysis. Panel (C) Blastula signaling centers. Chordin, noggin, follistatin and bmp4 mRNAs were downregulated. Panel (D) shows germ layers markers: zic1, sox21 and gata4 were downregulated, while the upregulation of egr1 was not statistically verified. RNA in situ analysis of radially injected X. tropicalis morphant blastulae: foxd4l1 (E, F), noggin (G, H) and zic2 (I, K). (E’-K’) show corresponding sagittal sections. Whereas foxd4l1 and noggin are downregulated in BMO1 morphants, zic2 gene expression is not changed (n = two biological replicates).
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Supp F4.
Dose-dependent consequences of systemic BMO1 depletion in X. laevis.
BMO1 morpholinos were injected four times into the animal pole region at the two- to four-cell stage (“radial†injection type). Panel (A): Typical morphology of embryos injected with increasing dose of BMO1. UI–uninjected siblings. BRG1 depleted embryos get arrested in gastrulation. Top rows: brightfield image; bottom row: green fluorescence from coinjected Alexa488 dextran. Panel (B) shows representative images of embryos that have survived until heartbeat stage (NF34). While most embryos are still arrested in gastrulation, some embryos injected with the lowest BMO1 dose (20 ng) have completed gastrulation but are arrested at open neural plate stage. Axial structures and closed neural plates were never observed. Panel (C) gives quantification of the BMO1 titration (n = 3–5 biological replicates/condition).
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Supp F5.
Ventral overexpression of Brg1 induces incomplete secondary body axes.
X. laevis embryos were injected in one ventral blastomere at the four cell stage with the following reagents: (A) 100pg nlacZ mRNA. The control embryo develops a normal shape. (B) 1ng brg1 mRNA results in a truncated secondary axis. (C) Frequency of 2° axes induction. * p-value ≤ 0.05. Panels (D-I) WMISH for the muscle actin gene actc1. (D-F’) Ventrally injected embryo with 100pg nlacZ mRNA in lateral (D, E) and in dorsal view (F). (F’) is a close-up of the area marked in F, showing nlacZ stained nuclei in myocytes of the second axis. Panels (G-I’) Ventrally injected Brg1 overexpressing embryo from lateral view (G, H) and dorsal view (I). (I’) shows a close-up of the area marked in (I); the arrow points to the bifurcation of primary and secondary axes, marked by nlacZ staining.
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Supp F6. Human brg1 mRNA induces ectopic chordin expression in prospective ventral ectoderm.
(A)X. laevis embryos were injected at the 4 cell stage in one ventral blastomere with either 500pg or 1ng human brg1 mRNA. At late Blastula stage (NF9) the embryos were fixed and stained for chordin mRNA. At this stage, chordin is normally expressed in the dorsal BCNE signaling center in prospective neuroectoderm. The ventral overexpression of human brg1 mRNA induces a second chordin expression zone on the ventral side in prospective epidermis. nlacZ mRNA was coinjected as lineage tracer. Panel (B) gives quantification (n = 2 biological replicates/condition).
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Supp F7. Mesodermal marker genes in X. laevis BMO1 morphant gastrulae.
Radially injected X. laevis embryos with CoMO or BMO1 (40ng/embryo) were stained for mRNAs indicated on the left (vegetal views, dorsal on top). Each marker was analyzed in 2–4 independent experiments, and classified into normal or reduced expression. Numbers in panels tell the number of embryos with the shown expression pattern; the graphs on the right translate this information in % penetrance.
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Supp F8. Orthotopic BCNE center transplantation reveals autonomous requirement for Brg1 in head formation.
(A) The experimental scheme of BCNE center transplantation (X. laevis). Note that the BCNE region is labeled with different colors in both the donor and host embryos to facilitate orthotopic grafting. (B-D) images represent dorsal views of BCNE transplanted tadpoles as merged brightfield/Alexagreen-fluorescent views. C and D show embryos with transplanted Brg1 morphant BCNE. After recording, these embryos were singly subjected to WMISH against otx2 mRNA. (B’-D’) Rows detail otx2 mRNA staining seen from left, right side and dorsal view. In wt embryos, otx2 is expressed in forebrain (including retina and olfactory epithelium [fb]), midbrain (mb) and hindbrain (hb) areas. Note the symmetric expression in the wildtype transplant, and the amorphous structure of the otx2-positiv BMO1 morphant tissue. (E) Quanitification of otx2 mRNA pattern in WT (n = 17) and BMO1 morphant (n = 24) transplants. Differences for the retina stain were significant with *, p ≤ 0,007.
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Supp. F9. Dorso-animal and dorso-vegetal control injections.
(A-D)X. laevis embryos injected at the 8 cell stage dorso-vegetally with either CoMO or BMO1 analysed for mRNA staining of BCNE genes chordin (A, B) and noggin (C, D). (E) Quantification of the two markers. (F-I) display the mRNA pattern of cerberus (F, G) and hhex (H, I) in dorso-animally injected embryos with either CoMO or BMO1. Note the partial overlap of chordin and noggin expression domain with the DV-injected area. (K) Quantification of chordin and noggin mRNA expression. *, p-value ≤ 0.05.
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Supp F10. Brg1 is required for the transcriptional burst at the MBT.
Panel (A) displays experimental scheme of sample collection for genome-wide comparison of preMBT versus postMBT transcriptomes (X. tropicalis). (B) The preMBT sample closest to the MBT (see sample #3 in red) was identified by qRT/PCR analysis for the marker gene gs17. Gs17 mRNA levels of the “early†samples 1–5 were normalized to the value of four cell stage embryos (n = 3 biological replicates). The postMBT sample (here #2 in red) was chosen as the one being harvested 40 min before the appearance of the blastoporus pigmentation lip in the sibling cohorts. This time point correlates with the late blastula stage used for the BMO1 microarray analysis of Fig 1. (C) The top 4 enriched GO-terms in the class of highly upregulated genes at MBT. (D) Heatmap providing mean expression levels at the pre- and postMBT timepoints for genes of the GO-term “nervous system developmentâ€, ranked by amplitude of mRNA increase (log2 fold-change). Genes that were downregulated in the BMO1-morphant transcriptome, compared to the control morphant state are marked with an asterisk. Like in the term “pattern specification process†only genes that show a marked upregulation of mRNA after MBT were affected by the Brg1 protein knockdown.
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