Elsevier

Gene Expression Patterns

Volume 10, Issue 6, September 2010, Pages 265-273
Gene Expression Patterns

Combination of in silico and in situ hybridisation approaches to identify potential Dll1 associated miRNAs during mouse embryogenesis

https://doi.org/10.1016/j.gep.2010.06.002Get rights and content

Abstract

MicroRNAs (miRNAs) have regulatory functions during vertebrate embryogenesis. They are short ∼21 bp long endogenously expressed single-stranded RNAs, which preferentially bind to complementary sequences in the 3′ untranslated regions (UTR) of mRNAs and typically down-regulate the respective target mRNAs by translational repression or enhanced mRNA degradation. The Notch ligand Delta-like 1 (Dll1) is expressed in a highly dynamic pattern and has pleiotropic functions during embryogenesis and in adult tissues. Here, we report an interspecies in silico analysis to identify 16 miRNAs, which potentially bind to the mouse, human and chicken Dll1 3′UTRs. To analyze whether these miRNAs could regulate Dll1 gene expression during somitogenesis and neurogenesis, we performed a systematic whole mount in situ hybridisation screen, followed by radioactive in situ hybridisation on sections, using LNA modified DNA probes in mouse embryos. We find that 7 miRNAs (miR-34a, miR-103, miR-107, miR-130a, miR-130b, miR-449a and miR-449c) are expressed in developing somites, limbs, restricted regions of the brain and neural tube between 9.5 dpc and 12.5 dpc. This suggests that these miRNAs could possibly target the Dll1 3′UTR in these regions. The other miRNAs are not expressed or below the detection limit and thus are unlikely to regulate Dll1 at the analyzed embryonic stages.

Section snippets

Results and discussion

MicroRNAs (miRNAs) are short ∼21 bp long, endogenous, single-stranded RNAs, that base-pair to specific sites in the 3′ untranslated regions (UTRs) of protein coding mRNAs, typically leading to translational repression of their respective targets or enhanced mRNA degradation (Bartel, 2004). miRNAs are transcribed from the genome and processed by the RNAse III enzyme Dicer. Complete inactivation of Dicer in mice leads to early embryonic lethality (Bernstein et al., 2003). Later functions for Dicer

Conclusion

It is noteworthy, that we did not observe significant differences between miR-103/miR-107 and miR-130a/miR-130b expression patterns, respectively, during the examined developmental stages. This may be due to the fact that the sequences of processed miR-103/miR-107 on the one hand and miR-130a/miR-130b on the other hand differ only in one, respectively, two nucleotide(s). Thus, it cannot be excluded that cross-hybridisation may at least to some extent contribute to the observed similarities in

In silico analysis of 3′UTRs

Transcript sequences were analyzed using the MicroCosm Targets Version 5.1. Transcripts used were ENSMUST00000014917, ENST00000366756 and ENSGALT00000037704 for mouse, human and chicken Dll1, respectively. For Dll3 transcript ENSMUST00000050191 was analyzed. For Jagged1, Jagged2 and Dll4 transcripts ENSMUST00000028735, ENSMUST00000075827 and ENSMUST00000102517 were used, respectively. We took it as a requirement for our analysis of evolutionary conservation that a corresponding miRNA for a

Acknowledgements

This work was supported by the Marie Curie training network of novel animal models for medical purposes “CLONET” and the Helmholtz Alliance on Systems Biology (CoReNe). Special thanks go to the animal caretaker team of the Helmholtz Center Munich.

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