Gaur S et al. (2016), Neural transcription factors bias cleavage stag...

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Genesis 2016 Jun 01;546:334-49. doi: 10.1002/dvg.22943.

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

Gaur S , Mandelbaum M , Herold M , Majumdar HD , Neilson KM , Maynard TM , Mood K , Daar IO , Moody SA .

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The decision by embryonic ectoderm to give rise to epidermal versus neural derivatives is the result of signaling events during blastula and gastrula stages. However, there also is evidence in Xenopus that cleavage stage blastomeres contain maternally derived molecules that bias them toward a neural fate. We used a blastomere explant culture assay to test whether maternally deposited transcription factors bias 16-cell blastomere precursors of epidermal or neural ectoderm to express early zygotic neural genes in the absence of gastrulation interactions or exogenously supplied signaling factors. We found that Foxd4l1, Zic2, Gmnn, and Sox11 each induced explants made from ventral, epidermis-producing blastomeres to express early neural genes, and that at least some of the Foxd4l1 and Zic2 activities are required at cleavage stages. Similarly, providing extra Foxd4l1 or Zic2 to explants made from dorsal, neural plate-producing blastomeres significantly increased the expression of early neural genes, whereas knocking down either significantly reduced them. These results show that maternally delivered transcription factors bias cleavage stage blastomeres to a neural fate. We demonstrate that mouse and human homologs of Foxd4l1 have similar functional domains compared to the frog protein, as well as conserved transcriptional activities when expressed in Xenopus embryos and blastomere explants. genesis 54:334-349, 2016. © 2016 Wiley Periodicals, Inc.

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Species referenced: Xenopus laevis
Genes referenced: chrd ctnnb1 foxd4l1 foxd4l1.2 gmnn irx1 isyna1 krt12.4 myc neurog2 sia1 sox11 sox2 tbxt tle4 zic1 zic2
???displayArticle.morpholinos??? foxd4l1.1 MO1 foxd4l1.2 MO1 zic2 MO1 zic2 MO2 zic2 MO3 zic2 MO4

Phenotypes: Xla Wt + foxd4l1.1 (fig.6.a) [+]

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	Figure 1: Explants can be made from cleavage stage blastomeres of known fates. (A) When 16-cell Xenopus embryos cleave in a regular pattern, the dorsal midline blastomeres (D11) will faithfully give rise to neural plate (np) and the ventral midline blastomeres (V11) will give rise to epidermis (epi). (B) The same embryo has gone through one cell division, i.e., is at the 32-cell stage. (C) The four daughters (outlined in B) of the midline D11 blastomeres (labeled in A) have been manually dissected and transferred to explant culture.
	Figure 2: There is no detectable dorsal-ventral difference in mRNA localization of four neural transcription factors at cleavage stages. In situ hybridization detects the indicated mRNAs at the same relative intensities in the dorsal (top) and ventral (bottom) 16- to 32-cell animal blastomeres, indicating a lack of localization. Not shown: 1) there is no signal in vegetal hemisphere blastomeres; 2) similar results were observed when animal cap fragments were processed for ISH to ensure probe penetrance in the yolk-ladened cells; and 3) sense probes did not shown signal in either animal or vegetal hemispheres.
	Figure 3: Neural transcription factors ectopically induce neural plate genes in ventral-animal (V11) blastomere explants. (A) The percentage of V11 explants scored positive for neural plate (sox2, zic1) or epidermis (K81) mRNAs after ectopic expression of one of the four neural transcription factors (Foxd4l1, Zic2, Sox11, Gmnn). The number above each bar indicates the number of explants scored. Asterisk () indicates a significant difference (p<0.05, Chi-square) compared to uninjected, control V11 explants. (B) Examples of explants microinjected with each neural transcription factor and assayed for each neural plate gene. For sox2, all Foxd4l1- and all Gmnn-expressing explants are positive. For other panels, arrows indicate the positively stained explants. Pink cells are labeled with the nÎ²Gal lineage tracer. (C) Examples of explants assayed for an epidermis gene (K81). In uninjected, control V11 explants, K81 is expressed in all surface cells of the explant, whereas in neural transcription factor-expressing explants (Zic2, Gmnn), K81 expression (arrows) is only in nÎ²Gal-negative cells. (D) The percentage of V11 explants scored positive for mesoderm genes (bra, chd) after ectopic expression of one of the four neural transcription factors. The number above each bar indicates the number of explants scored. Asterisk () indicates a significant difference (p<0.05, Chi-square) compared to uninjected, control V11 explants. (E) Examples of explants microinjected with each neural transcription factor and assayed for each mesoderm gene. Arrows indicate the positively stained explants.
	Figure 4: Neural transcription factors increase the frequency of neural plate gene expression in dorsalanimal (D11) blastomere explants. (A) The percentage of D11 explants scored positive for neural plate (sox2, zic1) or epidermis (K81) gene expression after increasing Foxd4l1 or Zic2 levels by microinjecting additional mRNA, or after decreasing endogenous levels by microinjecting translation blocking MOs (FoxMOs, ZicMOs). The number above each bar indicates the number of explants scored. Asterisk () indicates a significant difference (p<0.05, Chi-square) compared to uninjected, control D11 explants. (B) Examples of explants assayed for sox2 or K81. In uninjected, control D11 explants, sox2 is autonomously expressed in ~40% of explants. In either Foxd4l1- or Zic2-expressing D11 explants this frequency increases to nearly every explant. Conversely, in MO-injected D11 explants the frequency is reduced significantly below control. Arrows point out positively stained explants. In both Foxd4l1- and Zic2-expressing explants, K81 is expressed (arrows) only in nÎ²Gal-negative cells. (C) The percentage of D11 explants scored positive for mesoderm genes (bra, chd) after increasing Foxd4l1 or Zic2 levels by microinjecting additional mRNA. The number above each bar indicates the number of explants scored. Asterisk () indicates a significant difference (p<0.05, Chi-square) compared to uninjected, control D11 explants. (D) Examples of explantsassayed for bra or chd. Arrows point out positively stained explants.
	Figure 5AB: CLUSTAL analysis displayed in ESpript comparing frog and human Foxd4/Foxd4-like protein functional domains. (A) The three Xenopus laevis proteins (Foxd4l1.2, Foxd4l1.1a, Foxd4l1.1b), the two Xenopus tropicalis proteins (Foxd4l1.2, Foxd4l1.1) and the two human proteins (FOXD4L1, FOXD4) each contain an â€œacidic blobâ€ domain in the N-terminal portion of the protein (between green arrows). Red blocks indicate identical amino acids (aa), yellow blocks indicate conserved aa, and bold aa within a yellow block indicate aa of the same type (e.g., acidic, hydrophobic, etc.). (B) The Eh-1 domain (between blue arrows), which binds the transcriptional repressor Groucho/Grg/TLE, is conserved in all of the proteins except human FOXD4.
	Figure 5C: Xenopus Grg4 binds to the Foxd4/Foxd4-like proteins from different species. Myc-tagged versions of Foxd4/Foxd4-like proteins from Xenopus, mouse and human were expressed in Xenopus oocytes either alone or along with HA-tagged wild-type Xenopus Grg4. Co-immunoprecipitation (IP) and Western blot (WB) analyses of oocyte lysates expressing HA- and Myc-tagged constructs are indicated (Top left and right panels). All four constructs bind with Grg4 with the following avidities XlFoxd4l1>mFoxd4>hFOXD4L1>hFOXD4. The control panels (2nd from top) show that the IPs contain similar levels of Grg4 (left) or Foxd4/Foxd4-like proteins (right), as do the direct lysates (bottom 2 panels on both left and right).
	Figure 6: Similar to Xenopus Foxd4l1, human FOXD4 and FOXD4L1 can affect the expression of neural plate genes when mis-expressed in Xenopus embryos. (A) Left panel: Ectopic expression of Xenopus Foxd4l1, human FOXD4 and human FOXD4L1 in the V11 lineage results in a high frequency of ectopic expression of gmnn and zic2. However, unlike Xenopus Foxd4l1, neither human protein ectopically induces sox11. In control embryos none of these genes are expressed in the V11 lineage (far right bar). The number above each bar indicates the number of embryos scored. Asterisk () indicates a significant difference (p<0.05, Chi-square) compared to ectopic expression of Xenopus Foxd4l1. Middle panel: an example of ectopic induction of gmnn (dark blue stain) in the animal cap ectoderm (ac). Right panel: an example of lack of induction of sox11 in the animal cap ectoderm (ac); endogenous sox11 expression can be seen in the neural plate (np). Red dots indicate the lineage-labeled cells expressing the microinjected mRNAs. Embryos are animal cap views with dorsal to the top. (B) Left panel: Expressing Xenopus Foxd4l1, human FOXD4 or human FOXD4L1 in the D11 lineage up-regulates gmnn and zic2 within that lineage at high frequencies. This change in gene expression is never seen in control embryos (far right bar). The number above each bar indicates the number of embryos scored. Asterisk () indicates a significant difference (p<0.05, Chi-square) compared to ectopic expression of Xenopus Foxd4l1. Middle panel: an example of up-regulation of zic2 (dark blue stain) within the lineage-labeled clone (red dots) compared to endogenous levels in the neural plate (np). Embryo is a dorsal view with animal to the top and blastopore lip marked by arrow. (C) Left panel: Expressing Xenopus Foxd4l1, human FOXD4 or human FOXD4L1 in the D11 lineage down-regulates sox2, sox11 and zic1 within that lineage; for sox11 and zic1 the frequencies are significantly less than Xenopus Foxd4l1 (asterisks; p<0.05, Chi-square). This change in gene expression is never seen in control embryos (far right bar). The number above each bar indicates the number of embryos scored. Middle panel: an example of reduced sox2 expression in the lineage-labeled clone (red dots) compared to endogenous expression in the adjacent neural plate (np). Right panel: an example of reduced zic1 expression in the lineage-labeled clone (red dots) compared to endogenous expression in the adjacent neural plate (np). Embryos are dorsal views with animal to the top and blastopore lips marked by arrows.
	Figure 7: Mammalian Foxd4/Foxd4-like homologues can affect the expression of neural plate genes in Xenopus blastomere explants. (A) The percentage of V11 explants that express neural plate genes (sox2, zic1). The mammalian genes cause ectopic expression at frequencies significantly greater than uninjected controls (, p<0.05, Chi-square), but are not as effective as Xenopus protein (# indicates a significant difference [p<0.05, Chi-square] compared to Xenopus Foxd4l1-expressing explants). (B) The percentage of D11 explants that express neural plate genes (sox2, zic1). The mFoxd4 and hFOXD4 increase sox2 expression at frequencies significantly greater than uninjected controls (), but are not as effective as Xenopus protein (# indicates a significant difference [p<0.05, Chi-square] compared to Xenopus Foxd4l1-expressing explants).
	Figure 8: A scheme for how neural cell fate may be acquired step-wise. At the 16-cell stage, dorsal-animal blastomeres (blue) contain molecules, such as nTFs, that bias them to a neural cell fate by an unknown mechanism (indicated by â€œ?â€). At blastula stages, maternal Î²-catenin becomes nuclear in the dorsal marginal cells that are the descendants of the dorsal-animal blastomeres (blue) to directly activate Siamois/Twin. These transcription factors in turn directly activate the zygotic expression of some some nTFs (foxd4l1, gmnn, zic2) in these same cells. During gastrulation, Siamois/Twin directly activate signaling factors in the Organizer mesoderm that diffuse towards the animal pole to promote nTF expression in the nascent neural ectoderm (blue). The nTFs then up-regulate early neural genes (e.g., sox2, zic1), which in turn act upstream of neural differentiation factors (e.g., irx1, ngnr1) and markers of committed neurons (e.g., tubb2b).
	foxd4l1.1 (forkhead box D4-like 1, gene 1) gene expression in Xenopus laeivs embryo, NF stage 6 (32-cell), lateral view, dorsal up, ventral down.
	zic2 (Zic family member 2) gene expression in Xenopus laeivs embryo, NF stage 6 (32-cell), lateral view, dorsal up, ventral down.
	sox11 (SRY-box 11) gene expression in Xenopus laeivs embryo, NF stage 6 (32-cell), lateral view, dorsal up, ventral down.
	gmnn (geminin, DNA replication inhibitor) gene expression in Xenopus laeivs embryo, NF stage 6 (32-cell), lateral view, dorsal up, ventral down.

References [+] :

Abrams, Early zebrafish development: it's in the maternal genes. 2009, Pubmed