Marchak A et al. (2023), The sulfotransferase XB5850668.L is required to...

XB-ART-60189

Dev Dyn 2023 Dec 01;25212:1407-1427. doi: 10.1002/dvdy.648.

Show Gene links Show Anatomy links

The sulfotransferase XB5850668.L is required to apportion embryonic ectodermal domains.

Marchak A , Neilson KM , Majumdar HD , Yamauchi K , Klein SL , Moody SA .

???displayArticle.abstract???
BACKGROUND: Members of the sulfotransferase superfamily (SULT) influence the activity of a wide range of hormones, neurotransmitters, metabolites and xenobiotics. However, their roles in developmental processes are not well characterized even though they are expressed during embryogenesis. We previously found in a microarray screen that Six1 up-regulates LOC100037047, which encodes XB5850668.L, an uncharacterized sulfotransferase. RESULTS: Since Six1 is required for patterning the embryonic ectoderm into its neural plate, neural crest, preplacodal and epidermal domains, we used loss- and gain-of function assays to characterize the role of XB5850668.L during this process. Knockdown of endogenous XB5850668.L resulted in the reduction of epidermal, neural crest, cranial placode and otic vesicle gene expression domains, concomitant with neural plate expansion. Increased levels had minimal effects, but infrequently expanded neural plate and neural crest gene domains, and infrequently reduced cranial placode and otic vesicle gene domains. Mutation of two key amino acids in the sulfotransferase catalytic domain required for PAPS binding and enzymatic activity tended to reduce the effects of overexpressing the wild-type protein. CONCLUSIONS: Our analyses indicates that XB5850668.L is a member of the SULT2 family that plays important roles in patterning the embryonic ectoderm. Some aspects of its influence likely depend on sulfotransferase activity.

???displayArticle.pubmedLink??? 37597164
???displayArticle.pmcLink??? PMC10842325
???displayArticle.link??? Dev Dyn
???displayArticle.grants??? [+]

Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: dlx5 eya2 foxd3 ids irx1 msx1 pax2 pax3 six1 sox11 sox2 sox9 sult2a1 sult2b1 tbx1 tfap2a XB5850668
GO keywords: gastrulation [+]
???displayArticle.morpholinos??? XB5850668 MO1 XB5850668 MO2

???attribute.lit??? ???displayArticles.show???

	FIGURE 1 Clone BG88539 encodes a sulfotransferase. Amino acid sequence of BG88539 (red font) is identical (red dots) to that of XB5850668.L (black font, NM_001097778; and to LOC100037047, BC130199.1, not shown) except at amino acids #17, #151, #219, and #229. The highly conserved sulfotransferase domain extends from residues 30-276 and includes signature sequences (red bars) for the 5′ phosphosulfate binding loop (PSB loop; aa 38-45; aa 118-128) and a C-terminal P-loop motif (aa 245-257) that are required for PAPS binding. Red arrows denote the amino acids (K40, H96) that were experimentally altered.
	FIGURE 2 Chromosomal localization of the members of SULT2 gene family including XB5850668.L in Xenopus laevis and Xenopus tropicalis, and their phylogenetic relationships. (A) The loci of SULT2 family genes on Xenopus chromosome 7. The gene cluster consists of six members on the L-subgenome and two members on the S-subgenome in the allotetraploid X. laevis, and five members in the diploid X. tropicalis. Four members were lost from the S-subgenome in X. laevis. This chromosomal region showed shared synteny between X. laevis and X. tropicalis (see also Table 1), but little synteny across species from mammals to a teleost (see also Table 2). The three groups in the Xenopus SULT2 gene family are shown by bold arrows in blue, red, and green, and their phylogenetic relationships are depicted with amino acid identity (%) above the chromosomal maps. B: Phylogenetic tree of the vertebrate SULT2 gene family. The tree was constructed with the maximum likelihood method from eight X. laevis (Xl), five X. tropicalis (Xt) and 16 other vertebrate SULT2 amino acid sequences and with nine Danio rerio (Dr) SULT1 amino acid sequences as an outgroup. Node values represent the Chi-square-based parametric values returned by the approximate likelihood ratio test. Gene and protein IDs, and annotations and other information for each member are shown in Table 1. The first two letters of the gene names denote the species: Ac, Anolis carolinensis (green anole); Dr, Danio rerio (zebrafish); Gg, Gallus gallus (chicken); Hs, Homo sapiens (human); Mm, Mus musculus (house mouse); Xl, Xenopus laevis (African clawed frog); and Xt, Xenopus tropicalis (western clawed frog). The last letter (S or L) of the gene names for Xenopus laevis indicate homeologs derived from either the S- or L-subgenome. The three groups in the Xenopus SULT2 gene family are shown by bold branches and a bracket, and the XB5850668.L gene is outlined by a red rectangle.
	FIGURE 3 Validation of efficacy and specificity of the XB5850668.L antisense morpholino oligonucleotides. (A) Sequence of a portion of the 5′UTR (to the left of the translational start ATG [underlined]) and the coding region (to the right of the ATG) of XB5850668.L mRNA. In red are the two antisense morpholino oligonucleotides (MO1, MO2) that were used to block translation of endogenous XB5850668.L mRNA. These MOs bind with no mismatches to LOC100037047 and BG88539 mRNAs, but not to other closely related variants (e.g., XM_041568315, XM_018224208). (B) Western blot of 1-egg equivalent lysates from oocytes injected with HA-tagged mRNAs containing (5′UTR-XB5850889.L-3′HA) or not containing (XB5850889.L-3′HA) the 5′UTR to which MO2 binds. Both mRNAs are efficiently translated in the absence of an equimolar mixture of MO1 + MO2. In the presence of both MOs, 5′UTR-XB5850889.L-3′HA translation is blocked, whereas XB5850889.L-3′HA translation is detected due to lack of MO2 binding but reduced due to binding of MO1. Two lanes per condition were run on each blot and assays were run in duplicate two independent times. Uninjected = lysate from oocytes that were not injected with either mRNA. (C) The percentage of embryos that showed reduced foxd3 expression in the neural crest (foxd3-NC), reduced sox11 expression in the preplacodal ectoderm (sox11-PPE) or broader expression of sox11 in the neural plate (sox11-NP). While MO1 (green bars) and MO2 (orange bars) both caused these phenotypes, the combination of an equimolar mixture of both MOs (blue bars) resulted in the greatest effect. Therefore, an equimolar combination of MO1 + MO2 was used in all knock-down experiments (see Figures 4 and 5). (D) Neural crest foxd3 expression domain at st 16 is unchanged on the side injected with a combination of both MOs and MO-insensitive mRNA (MOs + mRNA); pink dots indicate lineage-labeled nuclei on the injected side. Bar graph indicates that the foxd3 expression domain is reduced in the majority of MO-injected embryos (see also Figure 4F, G), whereas the majority of embryos have a normal foxd3 expression domain with co-injection of MO-insensitive mRNA. The percentage of embryos showing reduced (dark blue), broader (medium blue) or no change (light blue) in foxd3 expression domains are significantly different between conditions (p < .05, Chi-square test). Numbers above bars indicate number of embryos analyzed. (E) PPE six1 expression domain at st 17 is unchanged on the side injected with a combination of both MOs and MO-insensitive mRNA (MOs + mRNA); pink dots indicate lineage-labeled nuclei on the injected side. Bar graph indicates that the six1 expression domain is reduced in the majority of MO-injected embryos (see also Figure 4H, I), whereas the majority of embryos have a normal six1 expression domain with co-injection of MO-insensitive mRNA. The percentage of embryos showing reduced (dark blue), broader (medium blue) or no change (light blue) in six1 expression domains are significantly different between conditions (p < .05, Chi-square test). Numbers above bars indicate number of embryos analyzed.
	FIGURE 4 Loss of endogenous XB5850668.L alters gene expression in each ectodermal domain. (A) The expression domains of neural border genes were differentially affected by loss of XB5850668.L. Those associated later in development with the lateral neural plate and neural crest (pax3, anterior view; msx1, side views) were predominantly broader on the MO side of the embryo compared to the control side (ctrl) of the same embryo. Those associated later in development with epidermis and PPE (dlx5, anterior view; tfap2a, side views) were predominantly reduced in size and intensity on the MO side of the embryo compared to the control side (ctrl) of the same embryo. Arrow in dlx5 embryo points out the major domain (on the control side) that was analyzed. Dorsal is to the top of each image. (B) Percentage of embryos in which neural border gene expression domains were reduced in size (dark blue), broader (medium blue), or did not change (light blue) when analyzed at the end of gastrulation (st 13). The number of embryos analyzed for each gene is at the top of each bar. (C) The expression domains of three neural plate genes (sox2, sox11, irx1) were broader upon loss of XB5850668.L (MO) compared to the control side (ctrl) of the same embryo. Anterior views with dorsal to the top. (D) Percentage of embryos in which neural plate expression domains were reduced in size (dark blue), broader (medium blue) or did not change (light blue) when analyzed at neural plate stages (st 16–18). The number of embryos analyzed for each gene is at the top of each bar. (E) The expression domain of epidermis-specific keratin (krt12.4) was reduced, that is, further from the midline, on the MO side of the embryo (95.45%, n = 22). Anterior view with dorsal to the top. (F) The expression domains of three genes associated with neural crest (pax3, foxd3, sox9) were predominantly reduced in size on the XB5850668.L MO side of the embryo compared to the control side (ctrl) of the same embryo. For pax3, the most noticeable staining at this stage was in the hatching gland precursors. The black arrow on the sox9 embryo denotes the control otic placode domain and the red arrow denotes the otic placode domain on the MO side. Anterior views with dorsal to the top. Inset shows sox9 otic placode expression in a different embryo, demonstrating a reduced domain on the MO side (red arrow) compared to control side (black arrow). Lateral views with dorsal to the top. (G) Percentage of embryos in which neural crest expression domains were reduced (dark blue), broader (medium blue) or did not change (light blue) when analyzed at neural plate stages (st 16–18). The number of embryos analyzed for each gene is at the top of each bar. (H) The expression domains of three PPE genes (six1, anterior view; sox11, side views; irx1, side views) were reduced on the XB5850668.L MO side of the embryo compared to the control side (ctrl) of the same embryo. For sox11 and irx1, the black arrows denote the control PPE domain and the red arrows denote the PPE domain on the MO side. Dorsal is to the top of each image. (I) Percentage of embryos in which PPE expression domains were reduced (dark blue), broader (medium blue) or did not change (light blue) when analyzed at neural plate stages (st 16–18). The number of embryos analyzed for each gene is at the top of each bar.
	FIGURE 5 XB5850668.L is required for otic vesicle gene expression. (A) Knockdown of XB5850668.L translation on one side of the larva (asterisk, right panel) results in reduced expression of six1 in the otic vesicle (OV, blue circle) compared to the control side of the same embryo (left panel). (B) Knockdown of XB5850668.L translation on one side of the larva (asterisk, right panel) results in reduced expression of irx1 in the OV (blue circle) compared to the control side of the same embryo (left panel). (C) Knockdown of XB5850668.L translation on one side of the larva (asterisk, right panel) results in reduced expression of sox9 in the OV (blue circle) compared to the control side of the same embryo (left panel). (D) Knockdown of XB5850668.L translation on one side of the larva (asterisk, right panel) results in reduced expression of eya2 in the OV (blue circle) compared to the control side of the same embryo (left panel). (E) Knockdown of XB5850668.L translation on one side of the larva (asterisk, right panel) results in reduced expression of pax2 in the OV (blue circle) compared to the control side of the same embryo (left panel). (F) Knockdown of XB5850668.L translation on one side of the larva (asterisk, right panel) results in reduced expression of dlx5 in the OV (blue circle) compared to the control side of the same embryo (left panel). (G) Percentage of embryos in which OV expression domains of genes were reduced (dark blue), broader (medium blue) or did not change (light blue) when analyzed at larval stages (st 28–32). The number of embryos analyzed for each gene is on top of each bar.
	FIGURE 6 Increasing XB5850668.L has minimal effects on ectodermal gene expression domains. (A) The expression domains of neural border genes were mostly unaffected by increased XB5850668.L. However, a small percentage of those associated later in development with the lateral neural plate and neural crest (pax3, msx1, anterior views) showed reduced intensity of expression on the mRNA-injected side of the embryo (pink lineage-labeled nuclei) compared to the control side (ctrl) of the same embryo. dlx5, which later in development is associated with epidermis and PPE, was unaffected by increased XB5850668.L (anterior view); the same was found for tfap2a expression (shown in 2B). Dorsal is to the top of each image. (B) Percentage of embryos in which neural border expression domains were reduced (dark blue), broader (medium blue), or did not change (light blue) when analyzed at the end of gastrulation (st 13). The number of embryos analyzed for each gene at the top of each bar. (C) The expression domains of neural plate genes were mostly unaffected by increased XB5850668.L. However, a small percentage of embryos showed broader neural plate domains on the mRNA-injected side of the embryo (pink lineage-labeled nuclei) compared to the control side (ctrl) of the same embryo. Arrows in sox11 show its PPE expression domain on the control (black) and mRNA-injected (red) sides. In this case, increased XB5850668.L resulted in a narrower sox11 PPE domain. Anterior views with dorsal to the top. (D) Percentage of embryos in which neural plate expression domains were reduced (dark blue), broader (medium blue) or did not change (light blue) when analyzed at neural plate stages (st 16–18). The number of embryos analyzed for each gene is at the top of each bar. (E) The expression of epidermis-specific keratin (krt12.4) was reduced on the mRNA-injected side in only 25% of embryos; there was no change in expression in 75% of embryos (n = 56). Anterior view with dorsal to the top. (F) The expression domains of neural crest genes were mostly unaffected by increased XB5850668.L (see 6H for sox9). However, small percentages of embryos showed a broader domain (pax3, foxd3) or reduced staining intensity (sox9) on the mRNA-injected side of the embryo (pink lineage-labeled nuclei) compared to the control side (ctrl) of the same embryo. For pax3, both the neural crest (bracket) and hatching gland (arrow) domains were affected. Arrows in sox9 show its otic placode domain on the control (black) and mRNA-injected (red) sides, which frequently was reduced (see also 6H, I). Anterior views with dorsal to the top. (G) Percentage of embryos in which neural crest expression domains were reduced (dark blue), broader (medium blue) or did not change (light blue) when analyzed at neural plate stages (st 16–18). The number of embryos analyzed for each gene is at the top of each bar. (H) The expression domains of PPE/placode genes were mostly unaffected by increased XB5850668.L. However, small percentages of embryos showed a broader domain (sox11) or a reduced domain (sox11 in 6C; sox9 and also 6F) on the mRNA-injected side of the embryo (pink lineage-labeled nuclei) compared to the control side (ctrl) of the same embryo. Arrows indicate PPE/placode domains on the control (black) and mRNA-injected (red) sides. Anterior views with dorsal to the top. (I) Percentage of embryos in which PPE expression domains were reduced (dark blue), broader (medium blue) or did not change (light blue) when analyzed at neural plate stages (st 16–18). The number of embryos analyzed for each gene is at the top of each bar.
	FIGURE 7 XB5850668.L gain-of-function has minimal effects on otic vesicle gene expression. Percentage of embryos in which OV expression domains were reduced (dark blue), broader (medium blue) or did not change (light blue) when analyzed at larval stages (st 28–32). The number of embryos analyzed for each gene is inside the bars.
	FIGURE 8 Mutation of amino acids in the PAPS binding (H40I) or catalytic (H96F) domains tend to attenuate effects of wild type XB5850668.L gain-of-function on ectodermal gene expression domains. The percentage of embryos in which the expression domain was reduced (dark blue), broader (medium blue) or did not change (light blue) after injections of mRNAs (100 pg) encoding wild type (wt) or mutated versions (K40I, H96F, double = K40I + H96F) of XB5850668.L. The number of embryos analyzed is inside each bar. * indicates mutant mRNA phenotype frequencies that were significantly different from wt at the p < .05 level (Chi square test). (A) sox2 neural plate (np) expression domain changes. (B) sox11 neural plate (np) expression domain changes. (C) irx1 neural plate (np) expression domain changes. (D) foxd3 neural crest (nc) expression domain changes. (E) sox9 neural crest (nc) expression domain changes. (F) six1 preplacodal (ppe) expression domain changes. (G) sox11 preplacodal (ppe) expression domain changes. (H) irx1 preplacodal (ppe) expression domain changes. (I) sox9 otic placode (op) expression domain changes.
	FIGURE 9 Mutation of amino acids in the PAPS binding (H40I) or catalytic (H96F) domains tend to attenuate effects of wild type XB5850668.L gain-of-function on otic vesicle gene domains. Percentage of embryos in which an OV gene expression domain was reduced (dark blue), broader (medium blue) or did not change (light blue) after injections of mRNAs (100 pg) encoding wild type (wt) or mutated versions (K40I, H96F, double = K40I + H96F) of XB5850668.L. The number of embryos analyzed for each gene is inside the bars. * indicates mutant mRNA phenotype frequencies that were significantly different from wt at the p < .05 level (Chi square test). (A) six1 OV expression domain changes. (B) irx1 OV expression domain changes. (C) sox9 OV expression domain changes. (D) eya2 OV expression domain changes. (E) pax2 OV expression domain changes. (F) dlx5 OV expression domain changes. (G) tbx1 OV expression domain changes.

References [+] :

Acampora, Craniofacial, vestibular and bone defects in mice lacking the Distal-less-related gene Dlx5. 1999, Pubmed