ALKALs are in vivo ligands for ALK family receptor tyrosine kinases in the neural crest and derived cells

Abstract

Mutations in anaplastic lymphoma kinase (ALK) are implicated in somatic and familial neuroblastoma, a pediatric tumor of neural crest-derived tissues. Recently, biochemical analyses have identified secreted small ALKAL proteins (FAM150, AUG) as potential ligands for human ALK and the related leukocyte tyrosine kinase (LTK). In the zebrafish Danio rerio, DrLtk, which is similar to human ALK in sequence and domain structure, controls the development of iridophores, neural crest-derived pigment cells. Hence, the zebrafish system allows studying Alk/Ltk and Alkals involvement in neural crest regulation in vivo. Using zebrafish pigment pattern formation, Drosophila eye patterning, and cell culture-based assays, we show that zebrafish Alkals potently activate zebrafish Ltk and human ALK driving downstream signaling events. Overexpression of the three DrAlkals cause ectopic iridophore development, whereas loss-of-function alleles lead to spatially distinct patterns of iridophore loss in zebrafish larvae and adults. alkal loss-of-function triple mutants completely lack iridophores and are larval lethal as is the case for ltk null mutants. Our results provide in vivo evidence of (i) activation of ALK/LTK family receptors by ALKALs and (ii) an involvement of these ligand-receptor complexes in neural crest development.

Keywords: ALK; ALKAL; FAM150; Ltk; iridophore.

Fig. 1.

Ectopic expression of ALKALs and overactivation of DrLtk leads to supernumerary iridophores in D. rerio. (A) Domain structure of the human and zebrafish ALK/LTK RTK family. GR, glycine rich; L, LDLa; M, MAM; PTK, kinase domain; TM, transmembrane domain. The activating F993I (DrLtk) and F1174L (HsALK) mutations within the kinase domain are indicated. (B) ltk loss-of-function shady mutants (ltk^j9s1) lack iridophores, while gain-of-function moonstone (ltk^mne) exhibit increased numbers of iridophores. (C) Alignment of ALKAL proteins from different species. Underlined, the FAM150 domain; red asterisks, conserved Cys. Note high conservation of the C-terminal half of the FAM150 domain.

Fig. 2.

Expression of DrAlkals is sufficient for ectopic production of iridophores. (A) Schematic of ALKAL-expression constructs. Amp^R, ampicillin resistance; Tol2, medaka Tol2 transposase recognition sequence. (B) alkal expression in 72 hpf albino larvae. Weak alkal1 signal can be observed in the head, swim bladder (B1), notochord (B2; green arrowheads), and iridophores in dorsal and ventral stripes (B2; blue arrowheads). alkal2a mRNA is detected in the head (B3), swim bladder (B4), notochord (B5; green arrowheads), and iridophore stripes (B5; blue arrowheads). alkal2b mRNA is detected in swim bladder, a row of cells at the ventral aspect of the head (B7; white arrowhead), bilateral clusters in the head (B6; red arrowheads), behind the eyes (B6; black arrowheads), notochord (B8; green arrowheads), and iridophore stripes (B8; blue arrowheads). ltk expression is visible in swim bladder (B9), eyes (B10), notochord (green arrowheads), and iridophores (blue arrowheads). Negative controls, corresponding sense probes. (C) Supernumerary iridophores were observed in 5 dpf larvae upon ectopic expression of indicated DrAlkals in F₀-injected fish. ltk^mne larvae are used as positive controls. (D) Distribution of phenotypes of C. Fisher exact probability test showed significant differences (P < 0.001) in phenotype distribution in the following comparisons: any Alkal overexpressing fish against uninjected control and Akal2a overexpressing fish against Alkal1 or Alkal2b overexpressing fish. No significant difference was found between Alkal1 and Alkal2b overexpressing fish (P = 0.2). (E) Mosaic overexpression of alkal2a produces patches of supernumerary iridophores in adults.

Fig. 3.

Human and zebrafish ALKALs activate DrLtk and HsALK. (A) Ectopic expression of either HsALK or DrLtk in combination with HsALKALs, but not alone, leads to a disorganization of the ommatidial pattern in Drosophila eye. (B) Expression of DrAlkals and DrLtk together, but not alone, results in rough eye phenotypes. Temperatures at which flies were grown are indicated. (C) Neurite outgrowth in PC12 cells expressing either pcDNA3 vector control, DrAlkals or DrLtk alone, combinations of DrLtk and DrAlkals, or DrLtk^mne in the presence or absence of the ALK tyrosine kinase inhibitor lorlatinib as indicated. (D) Quantification of neurite outgrowth in PC12 cells as indicated in C. Error bars: SD. (E) Immunoblot of whole-cell lysates from PC12 cells expressing DrLtk and DrAlkals alone or in combination as indicated. (F) Immunoblot of whole-cell lysates from PC12 cells expressing wild-type DrLtk and DrLtk^mne in the presence or absence of lorlatinib as indicated. (G and H) Activation of endogenous HsALK by ALKAL-containing medium in neuroblastoma cell lines: IMR-32 (G) and NB1 (H). Antibodies against ALK and ERK were used as loading controls. α-myc, total DrLtk protein; HA antibodies, ALKAL proteins; pALK-Y1278, phosphorylated HsALK or DrLtk. pERK1/2 indicates activation of downstream signaling. Agonist mAb46 ALK was employed as a positive control.

Fig. 4.

DrAlkals affect iridophore development via DrLtk. (A) Ectopic expression of DrAlkal2a does not rescue loss of iridophores in 4 dpf transheterozygous ltk knockout zebrafish larvae (2 bp insertion/22 bp insertion, n = 9/9), whereas in heterozygous or wild-type siblings and ltk^j9s1 larvae, it leads to overproduction of iridophores (n = 29/29 and 34/35). Overexpression of DrAlkal2a in ltk^mne mutant slightly enhances the phenotype. (B) Treatment with lorlatinib results in diminished iridophore numbers in 4 dpf larvae. (C) Mosaic overexpression of DrAlkal2a produces patches of supernumerary iridophores in wild-type and ltk^mne adults. Overexpression of DrAlkal2a in ltk^mne mutants enhances production of supernumerary iridophores compared with uninjected ltk^mne. (D) Zebrafish alkal mutants display defects in iridophore development. alkal1^ko mutants have reduced iridophores in eyes and operculum, and removal of both alkal1and alkal2b results in a complete loss of eye iridophores. alkal2a^ko mutants show the strongest phenotype, with reduced numbers of iridophores in the trunk, especially anteriorly. This phenotype displays increased penetrance in alkal2a;alkal2b double mutants. (E) Triple mutant for all alkal genes is embryonic lethal and displays total loss of iridophores, similar to ltk transheterozygous knockout.