Grant PA et al. (2014), Novel animal pole-enriched maternal mRNAs are p...

XB-ART-47542

Dev Dyn 2014 Mar 01;2433:478-96. doi: 10.1002/dvdy.24082.

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

Grant PA , Yan B , Johnson MA , Johnson DL , Moody SA .

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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. 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. 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.

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Species referenced: Xenopus laevis
Genes referenced: aurka denr eppk1 fam222b gdf1 gskip mief1 mtss1.2 nop14 ret srebf2 tnfaip1 uqcc6 wbp2nl

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	Fig. 1. Embryo dissection at the 8-cell stage. An 8-cell Xenopus laevis embryo was fixed in EtOH-acetic acid, and manually separated into three portions: the 4 animal blastomeres (AN, view from blastocoel); the 4 vegetal blastomeres were cut into a vegetal pole (VP) portion and an equatorial (EQ) portion. AN and VP samples were collected for microarray analyses and EQ samples were discarded.
	Fig. 2. The majority of animal transcripts enriched >3-fold over vegetal pole encode unknown genes. A pie chart identifies the proportion of genes within different gene ontologies.
	Fig. 3. qRT-PCR comparisons of AN and VP samples. Of the 11 genes identified by microarray to be animal-enriched for which successful primers were designed, 8 were detected at significantly higher levels (*P<0.05) in AN (blue) versus VP (red) samples. Numbers on Y-axis indicate fold differences relative to vegetal value (mean values from a minimum of three independent samples replicated twice). Bars represent standard error of the mean. As a control, a known VP mRNA, Vg1, also was tested, and as expected from the literature is highly enriched in the VP samples.
	Fig. 4. Location of maternal transcripts of the 14 characterized genes assayed by ISH at the 8-cell-stage cleavage embryo. All 14 are enriched in the animal blastomeres, whose equatorial cleavage furrow is indicated by an arrow. Animal pole is up and vegetal pole is down. For some embryos, ISH signal is also detected in the equatorial region (see Table 2).
	Fig. 5. Developmental time course of expression in whole embryos as detected by qRT-PCR. A: Two genes (aurka-b, Xl.54800) are most highly expressed during the maternal phase of development (st 4), gradually decrease during the onset of zygotic transcription (st 8–10), and remain at relatively low levels during the remaining stages analyzed. B: Four genes (srebf2, WBP2NL, SMCR7L, Xl.86248) are highly expressed during the maternal phase of development and gradually decrease during the onset of zygotic transcription, but rebound to higher expression levels at neurulation (st 19) through larval (st 35–38) stages. C: Two genes (Xl.15460, Xl.29665) are expressed at the lowest level maternally and at relatively high levels after neural plate stages. D: Three genes (Xl.11373, TNFAIP1, gskip) are strongly expressed maternally, with decreasing expression at early zygotic stages and strong larval expression. Y-axis indicates relative expression compared to lowest expression for that transcript (mean values from a minimum of three independent samples replicated twice). X-axis indicates developmental stage. Bars represent standard error of the mean.
	Fig. 6. Expression of the 14 characterized genes assayed by ISH in stage 8–9 blastula embryos. All 14 genes are strongly expressed in the animal cap ectoderm (top), but for some ISH signal is also detected in the equatorial region (*, also see Table 2). Animal pole is up and vegetal pole is down.
	Fig. 7. Expression patterns of animal-enriched genes from neural plate to late tail bud stages. A: Expression patterns of the seven highest ANenriched genes that were characterized. B: Expression patterns of the remaining seven AN-enriched genes that were characterized. Early (left) and late (third from right) neural plate stages are anterior views with dorsal to the top. Early (second from right) and late (far right) tail bud stages are side views with anterior to the right and dorsal to the top. ba, branchial arch; br, brain; cg, cement gland; epi, epidermis; h, heart; le, lens; li, liver; nc, neural crest; ne, nephric mesoderm; np, neural plate; olf, olfactory, oto, otocyst; ov, optic vesicle; pm, paraxial mesoderm; ppe, preplacodal ectoderm; ret; retina; sc, spinal cord; so, somites; vg, ventral gut.
	Fig. 7. Expression patterns of animal-enriched genes from neural plate to late tail bud stages. A: Expression patterns of the seven highest ANenriched genes that were characterized. B: Expression patterns of the remaining seven AN-enriched genes that were characterized. Early (left) and late (third from right) neural plate stages are anterior views with dorsal to the top. Early (second from right) and late (far right) tail bud stages are side views with anterior to the right and dorsal to the top. ba, branchial arch; br, brain; cg, cement gland; epi, epidermis; h, heart; le, lens; li, liver; nc, neural crest; ne, nephric mesoderm; np, neural plate; olf, olfactory, oto, otocyst; ov, optic vesicle; pm, paraxial mesoderm; ppe, preplacodal ectoderm; ret; retina; sc, spinal cord; so, somites; vg, ventral gut.
	Fig. 8. Expression patterns of animal-enriched genes at late larval stages. A: Expression patterns of the five highest AN-enriched genes that were characterized. B: Expression patterns of the five next highest AN-enriched genes that were characterized. C: Expression patterns of the remaining four AN-enriched genes that were characterized. Left: Side views of embryos with anterior to the right and dorsal to the top. Right: Transverse vibratome sections of whole mount ISH preparations showing internal histology. ba, branchial arch; br, brain; cg, cement gland; epi, epidermis; h, heart; le, lens; li, liver; nc, neural crest; ne, nephric mesoderm; np, neural plate; olf, olfactory, oto, otocyst; ov, optic vesicle; pm, paraxial mesoderm; ppe, pre-placodal ectoderm; ret; retina; sc, spinal cord; so, somites; vg, ventral gut.
	Fig. 8. Expression patterns of animal-enriched genes at late larval stages. A: Expression patterns of the five highest AN-enriched genes that were characterized. B: Expression patterns of the five next highest AN-enriched genes that were characterized. C: Expression patterns of the remaining four AN-enriched genes that were characterized. Left: Side views of embryos with anterior to the right and dorsal to the top. Right: Transverse vibratome sections of whole mount ISH preparations showing internal histology. ba, branchial arch; br, brain; cg, cement gland; epi, epidermis; h, heart; le, lens; li, liver; nc, neural crest; ne, nephric mesoderm; np, neural plate; olf, olfactory, oto, otocyst; ov, optic vesicle; pm, paraxial mesoderm; ppe, pre-placodal ectoderm; ret; retina; sc, spinal cord; so, somites; vg, ventral gut.
	Fig. 8. Expression patterns of animal-enriched genes at late larval stages. A: Expression patterns of the five highest AN-enriched genes that were characterized. B: Expression patterns of the five next highest AN-enriched genes that were characterized. C: Expression patterns of the remaining four AN-enriched genes that were characterized. Left: Side views of embryos with anterior to the right and dorsal to the top. Right: Transverse vibratome sections of whole mount ISH preparations showing internal histology. ba, branchial arch; br, brain; cg, cement gland; epi, epidermis; h, heart; le, lens; li, liver; nc, neural crest; ne, nephric mesoderm; np, neural plate; olf, olfactory, oto, otocyst; ov, optic vesicle; pm, paraxial mesoderm; ppe, pre-placodal ectoderm; ret; retina; sc, spinal cord; so, somites; vg, ventral gut.
	aurka (aurora kinase A) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	denr (density-regulated protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	mief1 (mitochondrial elongation factor 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	nop14 (NOP14 nucleolar protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	wbp2nl (WBP2 N-terminal like) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	fam222b (family with sequence similarity 222, member B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	gskip (GSK3B interacting protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	srebf2 (sterol regulatory element binding transcription factor 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	tnfaip1 (tumor necrosis factor, alpha-induced protein 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.