Novel animal pole‐enriched maternal mRNAs are preferentially expressed in neural ectoderm

Abstract

Background: Many animals utilize maternal mRNAs to pre‐pattern the embryo before the onset of zygotic transcription. In Xenopus laevis, vegetal factors specify the germ line, endoderm, and dorsal axis, but there are few studies demonstrating roles for animal‐enriched maternal mRNAs. Therefore, we carried out a microarray analysis to identify novel maternal transcripts enriched in 8‐cell‐stage animal blastomeres. Results: We identified 39 mRNAs isolated from 8‐cell animal blastomeres that are >4‐fold enriched compared to vegetal pole mRNAs. We characterized 14 of these that are of unknown function. We validated the microarray results for 8/14 genes by qRT‐PCR and for 14/14 genes by in situ hybridization assays. Because no developmental functions are reported yet, we provide the expression patterns for each of the 14 genes. Each is expressed in the animal hemisphere of unfertilized eggs, 8‐cell animal blastomeres, and diffusely in blastula animal cap ectoderm, gastrula ectoderm and neural ectoderm, neural crest (and derivatives) and cranial placodes (and derivatives). They have varying later expression in some mesodermal and endodermal tissues in tail bud through larval stages. Conclusions: Novel animal‐enriched maternal mRNAs are preferentially expressed in ectodermal derivatives, particularly neural ectoderm. However, they are later expressed in derivatives of other germ layers. Developmental Dynamics 243:478–496, 2014. © 2013 Wiley Periodicals, Inc.

High-throughput analysis reveals novel maternal germline RNAs crucial for primordial germ cell preservation and proper migration

“…Animal pole transcripts are expressed primarily in the Continued neural ectoderm at later developmental stages, as previously described ( Fig. S1C) (Grant et al, 2014). MetaCore (GeneGo) direct interaction pathway analysis did not reveal integrated networks among the animal pole-enriched RNAs.…”

Neural transcription factors bias cleavage stage blastomeres to give rise to neural ectoderm

“…In fact, transfer of total RNA from neural precursor blastomeres can cause epidermis precursor blastomeres to contribute to ectopic neural tubes (Hainski and Moody, 1992). Although several screens have identified animal hemisphere specific maternal transcripts (e.g., Cha et al, 2012; Bates et al, 2013; Grant et al, 2014), they have not identified the specific gene product(s) responsible for instructing cleavage stage blastomeres to autonomously express a neural fate.…”

Proteomic Characterization of the Neural Ectoderm Fated Cell Clones in the Xenopus laevis Embryo by High-Resolution Mass Spectrometry

“…Fragment ions were detected in the ion trap with rapid scan rate with the following settings: maximum IT, 50 ms; AGC, 5 × 10 4 counts; microscans, 1. Multinotch MS 3 fragmentation was triggered on the top 10 MS 2 fragments with HCD in nitrogen (65% NCE). MS 3 spectra were acquired at 15,000 FWMH in the orbitrap (maximum IT, 120 ms; AGC, 1 × 10 5 counts; microscans, 1).…”

“…Multinotch MS 3 fragmentation was triggered on the top 10 MS 2 fragments with HCD in nitrogen (65% NCE). MS 3 spectra were acquired at 15,000 FWMH in the orbitrap (maximum IT, 120 ms; AGC, 1 × 10 5 counts; microscans, 1). Fragment ions were dynamically excluded for 60 s before being reconsidered for fragmentation.…”

“…The following search parameters were applied: digestion, tryptic; number of missed cleavages, maximum 2; minimum number of unique peptides, 1; fixed modification, cysteine carbamidomethylation; variable modification, methionine oxidation; maximum mass deviation for main search of precursor masses, 4.5 ppm; de novo mass tolerance for tandem mass spectra, 0.25 Da; minimum score for modified peptides, 40; minimum δ score for modified peptides, 8. For TMT-tagged peptides additional search parameters were: sample type, reporter ion MS 3 ; label, TMT. In the NE clones, phosphorylated proteins were identified using the search parameters: variable phosphorylation of serine, threonine, and tyrosine; variable oxidation of methionine; minimum δ score for modified peptides, 8; phosphorylation localization probability filtered to >0.9 and sites probability >0.9.…”