Kondo M et al. (2017), Comprehensive analyses of hox gene expression i...

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Dev Growth Differ 2017 Aug 01;596:526-539. doi: 10.1111/dgd.12382.

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Comprehensive analyses of hox gene expression in Xenopus laevis embryos and adult tissues.

Kondo M , Yamamoto T , Takahashi S , Taira M .

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From whole genome sequencing of an allotetraploid frog, Xenopus laevis, two homeologous sets (L and S) of four Hox clusters A through D (HoxA.L/S, HoxB.L/S, HoxC.L/S, and HoxD.L/S) and 13 paralogous groups (PGs) with 76 genes were identified, allowing us to carry out the first comprehensive analyses of hox gene expression in vertebrates. Expression of all hox genes during development and in adult tissues was analyzed by RNA-sequencing. The expression levels of most hox genes were similar between homeologs, but in some pairs, large differences were observed and several of these were confirmed by RT-PCR and whole mount in situ hybridization experiments. These results indicate that subfunctionalization of hox genes may have occurred since allotetraploidization. Furthermore, comprehensive analysis of hox gene expression during early development did not agree with the hypothesis of temporal collinearity especially in genes belonging to PG2 to PG10.

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Species referenced: Xenopus laevis
Genes referenced: hoxa1 hoxa10 hoxa11 hoxa13 hoxa2 hoxa3 hoxa4 hoxa5 hoxa6 hoxa7 hoxa9 hoxb1 hoxb2 hoxb3 hoxb4 hoxb5 hoxb6 hoxb7 hoxb8 hoxb9 hoxc10 hoxc11 hoxc12 hoxc13 hoxc3 hoxc4 hoxc5 hoxc6 hoxc8 hoxc9 hoxd1 hoxd10 hoxd11 hoxd13 hoxd3 hoxd4 hoxd8 hoxd9 odc1

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	Fig. 1. The Hox clusters of Xenopus lae-vis and X. tropicalis. (A) Schematic repre-sentations of the four and eight Hoxclusters of X. tropicalis and X.laevis,respectively. hox genes of 13 paralogousgroups (PGs) are present. even-skippedhomeobox (evx) genes are present onHoxA and HoxD clusters. The four pairsof homeologous Hox clusters of X. laevisconsist of clusters from its two subge-nomes, and are designated as L and S.hoxb2p.L is a pseudogene (Session et al.2016). Positions of micro RNA genesmir10a (blue arrowheads) and mir10b (redarrowheads) are also shown. (B) Positionsof Hox clusters on X. laevis chromo-somes. Hox clusters are present on corre-sponding chromosomes of the twosubgenomes at corresponding positions.
	Fig. 2. Expression of hox genes. (A–D) The expression of all 76 hox genes from four pairs of Hox clusters in X. laevis was analysed byRNA-seq analyses during developmental stages (stage (st) 8–40). The average values of TPM from two clutches are plotted for eachgene. Only the PG number is indicated where there is no corresponding gene in that cluster. Between some homeologs, a large differ-ence in expression levels are observed (for example, hoxb5.L and hoxb5.S [arrows]). Note that hoxb4.L is expressed maternally (as-terisk). (E) Expression proﬁles during development and in adult tissues of hoxb2, hoxb4, hoxb5, and hoxb6 homeologs. Average TPMvalues are plotted. oo12: oocyte stages I and II; oo34: oocyte stages III and IV; oo56: oocyte stages V and VI; st: stage.
	Fig. 2. Expression of hox genes. (A–D) The expression of all 76 hox genes from four pairs of Hox clusters in X. laevis was analysed byRNA-seq analyses during developmental stages (stage (st) 8–40). The average values of TPM from two clutches are plotted for eachgene. Only the PG number is indicated where there is no corresponding gene in that cluster. Between some homeologs, a large differ-ence in expression levels are observed (for example, hoxb5.L and hoxb5.S [arrows]). Note that hoxb4.L is expressed maternally (as-terisk). (E) Expression proﬁles during development and in adult tissues of hoxb2, hoxb4, hoxb5, and hoxb6 homeologs. Average TPMvalues are plotted. oo12: oocyte stages I and II; oo34: oocyte stages III and IV; oo56: oocyte stages V and VI; st: stage.
	Fig. 3. Expression of hoxb2, hoxb4, and hoxb5 homeologs. (A) Expression analysis of hoxb2p.L, hoxb2.S, hoxb4.L, and hoxb4.S. RT-PCR using primers speciﬁc to each gene was performed against cDNA from eggs (egg) and stage 8–40 embryos. As controls, a house-keeping gene, odc, was ampliﬁed using reverse-transcribed (RT+) or untreated (RT-) RNA as templates. (B) WISH analyses of hoxb2 andhoxb5 homeologs. Results for stage 20 (hoxb2 homeologs) and stage 31 (hoxb5 homeologs) are shown. The coloring reaction was per-formed for the same length of time to compare between homeologs.
	Fig. 4. Temporal expression of hox genes during developmental stages 8 to 40. (A) Expression levels of 50 genes that had TPM morethan 5.0 at some point was normalized so that the peak TPM is 1. By clustering analysis, genes were grouped into eight “expressiongroups” (ExpG). hoxb4.L was exceptional, since the egg showed the highest value, but to observe zygotic expression, it was normalizedto the value at stage 40. Nevertheless, because of its unique pattern, this was not included in any ExpG. (B) Expression proﬁles of eachExpG. The patterns are mostly overlapping. (C) Summary of expression proﬁles. Four of the six PG1 (labial) genes are categorized inExpG1 (the other two are ExpG4), and expressed early, and PG11 to 13 genes in the posterior group are expressed late (ExpG8) or notuntil after stage 40, whereas the pb, central, and posterior PGs 9 and 10 showed no temporal collinearity. Asterisks indicate homeologsthat belong to the same ExpG.
	Fig. 4. Temporal expression of hox genes during developmental stages 8 to 40. (A) Expression levels of 50 genes that had TPM morethan 5.0 at some point was normalized so that the peak TPM is 1. By clustering analysis, genes were grouped into eight “expressiongroups” (ExpG). hoxb4.L was exceptional, since the egg showed the highest value, but to observe zygotic expression, it was normalizedto the value at stage 40. Nevertheless, because of its unique pattern, this was not included in any ExpG. (B) Expression proﬁles of eachExpG. The patterns are mostly overlapping. (C) Summary of expression proﬁles. Four of the six PG1 (labial) genes are categorized inExpG1 (the other two are ExpG4), and expressed early, and PG11 to 13 genes in the posterior group are expressed late (ExpG8) or notuntil after stage 40, whereas the pb, central, and posterior PGs 9 and 10 showed no temporal collinearity. Asterisks indicate homeologsthat belong to the same ExpG.
	Fig. 4. Temporal expression of hox genes during developmental stages 8 to 40. (A) Expression levels of 50 genes that had TPM morethan 5.0 at some point was normalized so that the peak TPM is 1. By clustering analysis, genes were grouped into eight “expressiongroups” (ExpG). hoxb4.L was exceptional, since the egg showed the highest value, but to observe zygotic expression, it was normalizedto the value at stage 40. Nevertheless, because of its unique pattern, this was not included in any ExpG. (B) Expression proﬁles of eachExpG. The patterns are mostly overlapping. (C) Summary of expression proﬁles. Four of the six PG1 (labial) genes are categorized inExpG1 (the other two are ExpG4), and expressed early, and PG11 to 13 genes in the posterior group are expressed late (ExpG8) or notuntil after stage 40, whereas the pb, central, and posterior PGs 9 and 10 showed no temporal collinearity. Asterisks indicate homeologsthat belong to the same ExpG.
	Fig. 5. Expression of hox genes in adulttissues. (A) Expression levels of 43 genesthat had TPM more than 2.5 at somepoint were normalized so that the peakTPM is 1. By clustering analysis, thegenes were grouped into eight, dependingon the tissue(s) with the most expression.The genes in the “kidney+” group showedexpression in kidney and some other tis-sue, whereas the “kidney” group geneswere expressed exclusively in kidney. (B)Summary of expression proﬁles. Asterisksindicate homeologs that show a similarpattern of expression.
	Fig. 6. Summary of expression proﬁles.Single asterisks indicate homeologs thatbelong to the same ExpG during develop-mental stages and show a similar patternof expression in adult tissues. Doubleasterisks indicate homeologs in which theexpression of one of the two is notdetected or is relatively low.
	Fig. S1. Phylogenetic tree of the X. laevis and X.tropicalis hox genes.
	Fig. S2. Phylogenetic relationships of six gene sets inwhich evolutionary rate difference was detectedbetween the L and S genes.
	Fig. S3. Expression proﬁles of all 76 X. laevis hoxgenes.
	Fig. S3. Expression proﬁles of all 76 X. laevis hoxgenes.
	Fig. S3. Expression proﬁles of all 76 X. laevis hoxgenes.
	Fig. S3. Expression proﬁles of all 76 X. laevis hoxgenes.
	Fig. S3. Expression proﬁles of all 76 X. laevis hoxgenes.