Moreno MM et al. (2021), Anaplastic lymphoma kinase (alk), a neuroblasto...

XB-ART-58121

Gene Expr Patterns 2021 Jun 19;40:119183. doi: 10.1016/j.gep.2021.119183.

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Anaplastic lymphoma kinase (alk), a neuroblastoma associated gene, is expressed in neural crest domains during embryonic development of Xenopus.

Moreno MM , Barrell WB , Godwin A , Guille M .

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Neuroblastoma is a neural crest-derived paediatric cancer that is the most common and deadly solid extracranial tumour of childhood. It arises when neural crest cells fail to follow their differentiation program to give rise to cells of the sympathoadrenal lineage. These undifferentiated cells can proliferate and migrate, forming tumours mostly found associated with the adrenal glands. Activating mutations in the kinase domain of anaplastic lymphoma kinase (ALK) are linked to high-risk cases, where extensive therapy is ineffective. However, the role of ALK in embryonic development, downstream signal transduction and in metastatic transformation of the neural crest is poorly understood. Here, we demonstrate high conservation of the ALK protein sequences among vertebrates. We then examine alk mRNA expression in the frog models Xenopus laevis and Xenopus tropicalis. Using in situ hybridisation of Xenopus embryos, we show that alk is expressed in neural crest domains throughout development, suggesting a possible role in neuroblastoma initiation. Lastly, RT-qPCR analyses show high levels of alk expression at tadpole stages. Collectively, these data may begin to elucidate how alk functions in neural crest cells and how its deregulation can result in tumorigenesis.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: alk ids ltk sox10 twist1

???displayArticle.disOnts??? anaplastic large cell lymphoma [+]

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	Fig. 1. ALK protein is conserved among different organisms. (A) Protein sequence alignment of ALK in different organisms (right). Residue conservation is based on its relative entropy threshold. Red represents highly conserved and blue represents less conserved columns, when there are no gaps. The scale represents the number of amino acids of the consensus alignment. Start and end numbers are the total amino acid count for each sequence. (B) Phylogenetic tree of ALK protein shows similarity between the ALK orthologues (scale bar: 0.5 distance represents 50% difference in sequences) (COBALT). NCBI reference sequence IDs (right). (C) Comparison of ALK and LTK protein domains of Homo sapiens, Xenopus laevis and Xenopus tropicalis orthologues, highlighting MAM (blue), LDLa (yellow), glycine-rich (green) and kinase domains (purple). (D) Amino acid alignment of human and Xenopus ALK kinase domains showing conservation of active site (yellow), ATP binding site (purple) and mutation hotspots (grey arrowheads) (T-Coffee). (E) Schematic of Xenopus alk and ltk mRNA with UTRs (red) and coding sequences (black) showing RT-qPCR primer amplicons (green) and in situ hybridisation antisense probe target (purple) (MAM = meprin, A5 protein, and receptor protein tyrosine phosphatase (MAM) domains; LDLa = low density lipoprotein receptor A domain; Gly-rich = glycine-rich domain; ATP = adenosine triphosphate; UTR = untranslated region).
	Fig. 2. alk expression pattern in Xenopus laevis. mRNA in situ hybridisation for alk and neural crest markers, sox10 and twist, in Xenopus laevis embryos at stages 8, 13, 17, 26, 37 and 42 (Nieuwkoop and Faber, 1994). Animal (A, D and G) and lateral (Aâ€², Dâ€² and Gâ€²) view of stage 8 embryos (alk n = 11, sox10 n = 5, twist n = 4). Dorsal (B, E and H) and anterior (Bâ€², Eâ€² and Hâ€²) view of stage 13 embryos (alk n = 12, sox10 n = 4, twist n = 2). Dorsal (C, F and I) and anterior (Câ€², Fâ€² and Iâ€²) view of stage 17 embryos (alk n = 7, sox10 n = 4, twist n = 4). Lateral view (J, M and P) of stage 26 embryos and zoomed insets (grey rectagles) of embryos heads (Jâ€², Mâ€² and Pâ€²), showing otic vesicles (OV) and neural crest streams (yellow arrowheads) (alk n = 8, sox10 n = 7, twist n = 5). Lateral view (K, N and Q) of stage 37 embryos (alk n = 10, sox10 n = 7, twist n = 5) and stage 42 embryos. Lateral view (L, O and R) of stage 42 embryos (alk n = 11, sox10 n = 5, twist n = 7).
	Fig. 3. alk is expressed in head structures of Xenopus laevis tadpoles. Coronal sections of Xenopus laevis tadpoles at stage 37, after in situ hybridisation, reveal internal structures with alk expression (Nieuwkoop and Faber, 1994). Anterior to posterior (Aâ€“C) 150 Î¼m sections at the levels of the optic cup (A), ear vesicle (B) and somites (C) (r = retina, l = lens, p = pharynx, b = brain, hm = head mesenchyme, cg = cement gland, nt = notochord, s = somite, ev = ear vesicle, eym = endodermal yolk mass).
	Fig. 4. alk expression pattern in Xenopus tropicalis. mRNA in situ hybridisation for alk and neural crest markers, sox10 and twist, in Xenopus tropicalis embryos at stages 8, 13, 17, 26, 37 and 42 (Nieuwkoop and Faber, 1994). Animal (A, D and G) and lateral (Aâ€², Dâ€² and Gâ€²) view of stage 8 embryos (alk n = 13, sox10 n = 8, twist n = 7). Dorsal (B, E and H) and anterior (Bâ€², Eâ€² and Hâ€²) view of stage 13 embryos (alk n = 9, sox10 n = 3, twist n = 5). Dorsal (C, F and I) and anterior (Câ€², Fâ€² and Iâ€²) view of stage 17 embryos (alk n = 5, sox10 n = 4, twist n = 4). Lateral view (J, M and P) of stage 26 embryos and zoomed insets (grey rectagles) of embryos heads (Jâ€², Mâ€² and Pâ€²), showing otic vesicles (OV) and neural crest streams (yellow arrowheads) (alk n = 31, sox10 n = 31, twist n = 21). Lateral view (K, N and Q) of stage 37 embryos (alk n = 6, sox10 n = 5, twist n = 4) and stage 42 embryos. Lateral view (L, O and R) of stage 42 embryos (alk n = 10, sox10 n = 4, twist n = 4).
	Fig. 5. Expression of alk and ltk in Xenopus laevis and Xenopus tropicalis. RT-qPCR of embryos samples at stages 8, 13, 17, 26, 37 and 42 (Nieuwkoop and Faber, 1994). Graphs show the absolute quantification (in Log10 scale) of alk (A and B) and ltk (C and D) levels, relative to the expression of the housekeeping gene, ef1Î±. Xenopus laevis (A and C) and Xenopus tropicalis (B and D) embryos were pooled in sets of 3 embryos for each stage and used for RNA extration, cDNA synthesis and RT-qPCR. Each icon represents one replicate (pool of 3 embryos). When there are less than 4 icons, no product was detected for these replicates. Bars represent the arithmetic mean between replicates of each stage. (A.U. = arbitrary units).
	alk (ALK receptor tyrosine kinase) gene expression in X. tropicalis embryo, NF stage 26, assayed via in situ hybridization, showing head region, lateral view, anterior left, dorsal up.
	alk (ALK receptor tyrosine kinase) gene expression in X. tropicalis embryo, NF stage 37, assayed via in situ hybridization, lateral view, anterior left, dorsal up.
	Figure 2. alk expression pattern in Xenopus laevis.mRNA in situ hybridisation for alk and neural crest markers, sox10 and twist, in Xenopus laevis embryos at stages 8, 13, 17, 26, 37 and 42 (Nieuwkoop & Faber, 1994). Animal (A, D and G) and lateral (A’, D’ and G’) view of stage 8 embryos (alk n=11, sox10 n=5, twist n=4). Dorsal (B, E and H) and anterior (B’, E’ and H’) view of stage 13 embryos (alk n=12, sox10 n=4, twist n=2). Dorsal (C, F and I) and anterior (C’, F’ and I’) view of stage 17 embryos (alk n=7, sox10 n=4, twist n=4). Lateral view (J, M and P) of stage 26 embryos and zoomed insets (grey rectagles) of embryos heads (J’, M’ and P’), showing otic vesicles (OV) and neural crest streams (yellow arrowheads) (alk n= 8, sox10 n=7, twist n=5). Lateral view (K, N and Q) of stage 37 embryos (alk n= 10, sox10 n=7, twist n=5) and stage 42 embryos. Lateral view (L, O and R) of stage 42 embryos (alk n= 11, sox10 n=5, twist n=7).
	Figure 3. alk is expressed in head structures of Xenopus laevis tadpoles.Coronal sections of Xenopus laevis tadpoles at stage 37, after in situ hybridisation, reveal internal structures with alk expression (Nieuwkoop & Faber, 1994). Anterior to posterior (A-C) 150 µm sections at the levels of the optic cup (A), ear vesicle (B) and somites (C) (r=retina, l=lens, p=pharynx, b=brain, hm=head mesenchyme, cg=cement gland, nt=notochord, s=somite, ev=ear vesicle, eym=endodermal yolk mass).
	Figure 4. alk expression pattern in Xenopus tropicalis.mRNA in situ hybridisation for alk and neural crest markers, sox10 and twist, in Xenopus tropicalis embryos at stages 8, 13, 17, 26, 37 and 42 (Nieuwkoop & Faber, 1994). Animal (A, D and G) and lateral (A’, D’ and G’) view of stage 8 embryos (alk n=13, sox10 n=8, twist n=7). Dorsal (B, E and H) and anterior (B’, E’ and H’) view of stage 13 embryos (alk n=9, sox10 n=3, twist n=5). Dorsal (C, F and I) and anterior (C’, F’ and I’) view of stage 17 embryos (alk n=5, sox10 n=4, twist n=4). Lateral view (J, M and P) of stage 26 embryos and zoomed insets (grey rectagles) of embryos heads (J’, M’ and P’), showing otic vesicles (OV) and neural crest streams (yellow arrowheads) (alk n= 31, sox10 n=31, twist n=21). Lateral view (K, N and Q) of stage 37 embryos (alk n= 6, sox10 n=5, twist n=4) and stage 42 embryos. Lateral view (L, O and R) of stage 42 embryos (alk n= 10, sox10 n=4, twist n=4).
	Figure 5. Expression of alk and ltk in Xenopus laevis and Xenopus tropicalis.RT-qPCR of embryos samples at stages 8, 13, 17, 26, 37 and 42 (Nieuwkoop & Faber, 1994). Graphs show the absolute quantification (in Log10 scale) of alk (A and B) and ltk (C and D) levels, relative to the expression of the housekeeping gene, ef1α. Xenopus laevis (A and C) and Xenopus tropicalis (B and D) embryos were pooled in sets of 3 embryos for each stage and used for RNA extration, cDNA synthesis and RT-qPCR. Each icon represents one replicate (pool of 3 embryos). When there are less than 4 icons, no product was detected for these replicates. Bars represent the arithmetic mean between replicates of each stage. (A.U.=arbitrary units).