Massé KL et al. (2007), Anxa4 Genes are Expressed in Distinct Organ Sys...

XB-ART-39389

Organogenesis 2007 Oct 01;32:83-92. doi: 10.4161/org.3.2.4945.

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Anxa4 Genes are Expressed in Distinct Organ Systems in Xenopus laevis and tropicalis But are Functionally Conserved.

Massé KL , Collins RJ , Bhamra S , Seville RA , Jones EA .

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Anxa4 belongs to the multigenic annexin family of proteins which are characterized by their ability to interact with membranes in a calcium-dependent manner. Defined as a marker for polarized epithelial cells, Anxa4 is believed to be involved in many cellular processes but its functions in vivo are still poorly understood. Previously, we cloned Xanx4 in Xenopus laevis (now referred to as anxa4a) and demonstrated its role during organogenesis of the pronephros, providing the first evidence of a specific function for this protein during the development of a vertebrate. Here, we describe the strict conservation of protein sequence and functional domains of anxa4 during vertebrate evolution. We also identify the paralog of anxa4a, anxa4b and show its specific temporal and spatial expression pattern is different from anxa4a. We show that anxa4 orthologs in X. laevis and tropicalis display expression domains in different organ systems. Whilst the anxa4a gene is mainly expressed in the kidney, Xt anxa4 is expressed in the liver. Finally, we demonstrate Xt anxa4 and anxa4a can display conserved function during kidney organogenesis, despite the fact that Xt anxa4 transcripts are not expressed in this domain. This study highlights the divergence of expression of homologous genes during Xenopus evolution and raises the potential problems of using X. tropicalis promoters in X. laevis.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: anxa4 mmut

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	Figure 4. Comparison of in situ expression patterns of the three orthologs in X. laevis and tropicalis embryos. (A) anxa4 probes from each the Xenopus orthologs identify distinct in situ expression domains on X. laevis embryos. anxa4a and anxa4b can be detected from stage 28 in the cement gland (cg) and weakly in the otic vesicles (ov) (a and b). Expression of anxa4a in the pronephric anlagen (pn) can be first observed at stage 28 (b) and from stage 33 in the developing tubules (pt) (e and h). anxa4b is also in the pronephric tubules (d and g) but in a transitory manner, being lost from this region in later stage embryos (data not shown). anxa4b is expressed strongly in the developing liver (l) from stage 33 (d and g). A combination of the two expression pat‐ terns is detected with Xt anxa4 probe, which shows similar homology to both X. laevis genes. Strong staining in the pronephric tubules (pt) can be observed from stage 33 (f) and in the liver (l) at stage 39 (i). (B) Anxa4 probes from each of the Xenopus orthologs show identical in situ expression patterns on X. tropicalis embryos. Xt anxa4 is first detected in the cement gland (cg) and otic vesicle (ov) at stage 28 (a) and remains expressed in these two organs throughout their development (d and g). From stage 32, expression can be seen in the liver (l) (d). Xt anxa4 remains strongly expressed in this organ at later stages (g). The same expression pattern is detected with both anxa4a (c, f and i) and anxa4b (b, e and h) probes, although as expected the signal is weaker since probes are being used cross‐species, confirming the homology of these genes.
	Figure 5. Comparison of anxa4a and anxa4b spatial expression. (A) Xenopus anxa4 genes are expressed in a common domain, the gall blad‐ der, in stage 41 embryos. In situ hybridization was carried out on X. laevis (a and b) and X. tropicalis (c) embryos. anxa4a (b) is expressed strongly in the pronephric tubules (pt) and also in the gall bladder (gb). anxa4b (a) is detected in the anterior part of the liver (l), in the gall bladder (gb) but not expressed in the pronephros (pt) (white arrow). Xt anxa4 (c‐ventral view) is only expressed in the gall bladder (gb). The cement gland (cg) is indicated to allow orientation of the embryos. (B) anxa4a and anxa4b expression is dif‐ ferent during pronephric development. RT‐PCR was performed with total RNA extracted from dissected kidneys from different stages of X. laevis embryos. anxa4a is expressed in the kidney throughout its formation whereas anxa4b is only expressed in this organ from stage 28. (C) anxa4a and anxa4b display different spatial expression at late tadpole stages. RT‐PCR was performed with total RNA extracted from dissected pronephric kidneys and livers from stages 41 and 44 X. laevis embryos (W.E.). At stage 41, anxa4a is slightly more expressed in the kidney than the liver and anxa4b is more highly expressed in the liver. At stage 44, this difference of expression is more highly pronounced
	Figure 6. The anxa4 MO2 knocks down anxa4a but not Xt anxa4 transla‐ tion. (A) Alignment of the 5� sequences of X. laevis wild type and mutant anxa4 cDNA and its X. tropicalis homolog showing the complementarity of anxa4a MO1 and MO2 in relation to these cDNAs. The ATG is underlined and mismatch residues are indicated in bold. Four nucleotides (in grey) differ between Xt anxa4 cDNA and the MO2 sequence. Two mismatch nucleotides created in anx4a‐MUT cDNA are highlighted in light grey. Two nucle‐ otides (in dark grey) are different between anxa4b and MO2 sequences. (B) anxa4a MO2 does not interfere with Xt anxa4 translation; autora‐ diograph of an SDS‐PAGE analysis gel of in vitro translated 35S‐Methionine radio‐labelled anxa4 proteins. 0.5 mg of anxa4a mRNA was incubated alone (lane 1) or in combination with 10 mg of anxa4a MO1 (lane 2) or MO2 (lane 3). Both MO block the translation of anxa4a mRNA. 0.5 mg of anxa4a‐MUT mRNA was incubated either alone (lane 4) or in combination with 10 mg of anxa4a MO1 (lane 5) or MO2 (lane 6). Translation of the mutant mRNA is severely affected by MO1 but unaffected by MO2. 0.5 mg of Xt anxa4 mRNA was incubated alone (lane 7) or in combination with 10 mg of anxa4a MO1 (lane 8) or MO2 (lane 9). MO2 does not block the translation of Xt anxa4 whereas the addition of MO1 affects its translation. These results demonstrate that Xt anxa4 can be used to rescue MO2.
	Figure 7. Anxa4 function is conserved between X. laevis and tropicalis. (A) Embryos were injected in the V2 blastomere of the 8‐cell stage with 20 ng of anxa4a MO2 alone (a) or with 5 ng of anxa4a‐MUT mRNA (b) or with Xt anxa4 mRNA (c), cultured until stage 41 and subjected to whole‐mount anti‐ body staining with the pronephric tubule specific antibody 3G8. Five nano‐ grams of Xt anxa4 mRNA (d), 20 ng of cMO (e), 20 ng of anxa4a MO1 (f) were also injected as controls. LacZ (2 ng) was used as lineage tracer in all injections. Embryos injected with MO1 and MO2 show similar phenotype, with shortened, enlarged tubules whereas cMO injection does not affect the tubule morphology. Overexpression of Xt anxa4 induces no apparent phenotype; however, as anxa4a‐MUT mRNA, it is able to rescue the MO2 phenotype. (B) Cryostat transverse sections of stage 41 Xenopus pronephroi stained with tubule‐specific antibody 3G8 and counterstained with Hoechst. The slides were inspected under white light (a�c, g�h) to identify 3G8 stained tubules and the LacZ stained injected side (inj) (a, c and g) and under UV illumination to identify Hoechst nuclei staining (d�f, i�j). Embryo injected with anxa4a MO2 displays enlarged pronephric tubule phenotype on the injected side (inj) (a and d) compared to the uninjected side (uninj) (b and e). Embryo co‐injected with anxa4a MO2 and anxa4a‐MUT mRNA shows rescue of the phenotype [compare g and h (injected side) to i and j (uninjected side)]. No differences between the injected and the uninjected sides can be observed in embryo co‐injected with anxa4a MO2 and Xt anxa4 mRNA (c and f).
	Xl anxa4-a st 28
	Xl anxa4-a st 33
	Xl anxa4-a st 39
	Xl anxa4-b st 28
	Xl anxa4-b st 32
	Xl anxa4-b st 39
	XT ANXA4 ST 28
	Xt anxa4 st 33
	Xt anxa4 st 39
	anxa4-a xl st41
	anxa4-b xl st 41
	anxa4 Xtrop st 41

References [+] :

Barnett, Neural induction and patterning by fibroblast growth factor, notochord and somite tissue in Xenopus. 1998, Pubmed, Xenbase