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a substantial number of transcripts will be missed in somatic cell RNA at less than 1
billion mapped reads. Because of cost limitations, most RNA-Seq studies on staged
early embryos are based on 10–100 times fewer mapped reads per stage; these stud-
ies are informative about large scale changes in gene expression but not sufficiently
powered to rule out the expression of low abundance or undetected transcripts. As a
practical example, increasing sequencing depth to 1.5 billion reads identified numer-
ous new lncRNAs in early zebrafish not detected in earlier studies (Pauli et al. 2012 ).
Detecting new gene expression in early embryos is also complicated by highly
dynamic cytoplasmic polyadenylation and deadenylation of up to ~25 % of mRNAs
during early development (Dworkin et al. 1985 ; McGrew et al. 1989 ; Graindorge
et al. 2006 ; Paranjpe et al. 2013 ). Detection methods that depend on oligo-dT prim-
ing alone cannot distinguish between changes in polyadenylation of maternal RNAs
and changes in RNA abundance. This concern can be addressed using ribosome
depleted RNAs, metabolic labeling of nascent RNAs, sequencing of introns, identi-
fication of paternal polymorphisms, and/or validating by qRT-PCR using random-
primed cDNA, as in the above studies. However, in general, distinct transcriptomes
are represented in libraries prepared by polyA enrichment versus ribo-depletion,
with more low abundance genes detected using ribosome depletion compared to
oligo-dT priming (Li et al. 2014 ; SEQC/MAQC-III 2014 ).
Accurate staging of multicellular embryos is a potential concern, especially when
comparing stages close to the MBT and when comparing different clutches, different
rearing temperatures, or related but genetically distinct species (see Yanai et al.
2011 ; Harvey et al. 2013 ). For example, the first cell cycle in zebrafish is 45 min,
compared with 15 min cleavage cycles subsequently; therefore the use of natural
matings can introduce significant variation if staged samples are collected based on
time alone (Langley et al. 2014 ; Steven Harvey, personal communication). This con-
cern can be readily alleviated by using small numbers of embryos and visually stag-
ing embryos during cleavage stages, as clearly described (Heyn et al. 2014 ; Karla
Neugebauer, personal communication). In addition, several of the studies cited
above did not examine pre-MBT stages later than the 64-cell stage of zebrafish or
X. tropicalis, and therefore likely missed pre-MBT transcription detected by others
between early cleavage stages and the MBT.
9.3.4 Function of Pre-MBT Gene Expression
The above expression analyses demonstrate that new transcription before the MBT is
a conserved phenomenon in vertebrates (as in invertebrates) and suggest specific
groups of zygotic genes that regulate early developmental events may be similarly
regulated. However, many of these pre-MBT genes are initially present at low levels
of expression, and the concern that this represents transcriptional noise has been raised.
The evidence that early transcription is specifically regulated includes: (1) A limited
set of genes is reproducibly transcribed before the MBT; (2) pre-MBT transcription is
restricted to specific blastomeres for some genes (e.g., bozozok, Xnr5, Xnr6); (3)
M. Zhang et al.