Functional evidence that Activin/Nodal signaling is required for establishing the dorsal-ventral axis in the annelid Capitella teleta

ABSTRACTThe TGF-β superfamily comprises two distinct branches: the Activin/Nodal and BMP pathways. During development, signaling by this superfamily regulates a variety of embryological processes, and it has a conserved role in patterning the dorsal-ventral body axis. Recent studies show that BMP signaling establishes the dorsal-ventral axis in some mollusks. However, previous pharmacological inhibition studies in the annelid Capitella teleta, a sister clade to the mollusks, suggests that the dorsal-ventral axis is patterned via Activin/Nodal signaling. Here, we determine the role of both the Activin/Nodal and BMP pathways as they function in Capitella axis patterning. Antisense morpholino oligonucleotides were targeted to Ct-Smad2/3 and Ct-Smad1/5/8, transcription factors specific to the Activin/Nodal and BMP pathways, respectively. Following microinjection of zygotes, resulting morphant larvae were scored for axial anomalies. We demonstrate that the Activin/Nodal pathway of the TGF-β superfamily, but not the BMP pathway, is the primary dorsal-ventral patterning signal in Capitella. These results demonstrate variation in the molecular control of axis patterning across spiralians, despite sharing a conserved cleavage program. We suggest that these findings represent an example of developmental system drift.

U1 snRNP regulates cancer cell migration and invasion in vitro

Stimulated cells and cancer cells have widespread shortening of mRNA 3’-untranslated regions (3’UTRs) and switches to shorter mRNA isoforms due to usage of more proximal polyadenylation signals (PASs) in introns and last exons. U1 snRNP (U1), vertebrates’ most abundant non-coding (spliceosomal) small nuclear RNA, silences proximal PASs and its inhibition with antisense morpholino oligonucleotides (U1 AMO) triggers widespread premature transcription termination and mRNA shortening. Here we show that low U1 AMO doses increase cancer cells’ migration and invasion in vitro by up to 500%, whereas U1 over-expression has the opposite effect. In addition to 3’UTR length, numerous transcriptome changes that could contribute to this phenotype are observed, including alternative splicing, and mRNA expression levels of proto-oncogenes and tumor suppressors. These findings reveal an unexpected role for U1 homeostasis (available U1 relative to transcription) in oncogenic and activated cell states, and suggest U1 as a potential target for their modulation.

Dpysl2 (CRMP2) is required for the migration of facial branchiomotor neurons in the developing zebrafish embryo

Dihydropyrimidinase-like family proteins (Dpysls) are relevant in several processes during nervous system development; among others, they are involved in axonal growth and cell migration. Dpysl2 (CRMP2) is the most studied member of this family; however, its role in vivo is still being investigated. Our previous studies in zebrafish showed the requirement of Dpysl2 for the proper positioning of caudal primary motor neurons and Rohon-Beard neurons in the spinal cord.In the present study, we show that Dpysl2 is necessary for the proper migration of facial branchiomotor neurons during early development in zebrafish. We generated a dpysl2 knock-out (KO) zebrafish mutant line and used different types of antisense morpholino oligonucleotides (AMO) to analyze the role of Dpysl2 in this process. Both dpysl2 KO mutants and morphants exhibited abnormalities in the migration of these neurons from rhombomers (r) 4 and 5 to 6 and 7. The facial branchiomotor neurons that were expected to be at r6 were still located at r4 and r5 hours after the migration process should have been completed. In addition, mutant phenotypes were rescued by injecting dpysl2 mRNA into the KO embryos. These results indicate that Dpysl2 is involved in the proper migration of facial branchiomotor neurons in developing zebrafish embryos.

U1 snRNP regulates cancer cell migration and invasion

Stimulated cells and cancer cells have widespread shortening of mRNA 3’-utranslated regions (3’UTRs) and switches to shorter mRNA isoforms due to usage of more proximal polyadenylation signals (PASs) in the last exon and in introns. U1 snRNA (U1), vertebrates’ most abundant non-coding (spliceosomal) small nuclear RNA, silences proximal PASs and its inhibition with antisense morpholino oligonucleotides (U1 AMO) triggers widespread mRNA shortening. Here we show that U1 AMO also modulates cancer cells’ phenotype, dose-dependently increasing migration and invasion in vitro by up to 500%, whereas U1 over-expression has the opposite effect. In addition to 3’UTR length, numerous transcriptome changes that could contribute to this phenotype are observed, including alternative splicing, and mRNA expression levels of proto-oncogenes and tumor suppressors. These findings reveal an unexpected link between U1 regulation and oncogenic and activated cell states, and suggest U1 as a potential target for their modulation.

Knockdown of Laminin gamma-3 (Lamc3) impairs motoneuron guidance in the zebrafish embryo

Background: Previous work in the zebrafish embryo has shown that laminin γ-3 (lamc3) is enriched in endothelial cells marked by expression of fli1a, but the role of Lamc3 has been unknown. Methods: We use antisense morpholino oligonucleotides, and CRISPR/Cas9 mutagenesis of F0 embryos, to create zebrafish embryos in which lamc3 expression is compromised. Transgenic imaging, immunofluorescence, and in situ hybridisation reveal that Lamc3 loss-of-function affects the development of muscle pioneers, endothelial cells, and motoneurons. Results: Lamc3 is enriched in endothelial cells during zebrafish development, but it is also expressed by other tissues. Depletion of Lamc3 by use of antisense morpholino oligonucleotides perturbs formation of the parachordal chain and subsequently the thoracic duct, but Lamc3 is not required for sprouting of the cardinal vein. F0 embryos in which lamc3 expression is perturbed by a CRISPR/Cas9 approach also fail to form a parachordal chain, but we were unable to establish a stable lamc3 null line. Lamc3 is dispensable for muscle pioneer specification and for the expression of netrin-1a in these cells. Lamc3 knockdown causes netrin-1a up-regulation in the neural tube and there is increased Netrin-1 protein throughout the trunk of the embryo. Axonal guidance of rostral primary motoneurons is defective in Lamc3 knockdown embryos. Conclusions: We suggest that knockdown of Lamc3 perturbs migration of rostral primary motoneurons at the level of the horizontal myoseptum, indicating that laminin γ3 plays a role in motoneuron guidance.