Cell death of melanophores in zebrafish trpm7 mutant embryos depends on melanin synthesis.

Transient receptor potential melastatin 7 (TRPM7) is a broadly expressed, non-selective cation channel. Studies in cultured cells implicate TRPM7 in regulation of cell growth, spreading, and survival. However, zebrafish trpm7 homozygous mutants display death of melanophores and temporary paralysis, but no gross morphological defects during embryonic stages. This phenotype implies that melanophores are unusually sensitive to decreases in Trpm7 levels, a hypothesis we investigate here. We find that pharmacological inhibition of caspases does not rescue melanophore viability in trpm7 mutants, implying that melanophores die by a mechanism other than apoptosis. Consistent with this possibility, ultrastructural analysis of dying melanophores in trpm7 mutants reveals abnormal melanosomes and evidence of a ruptured plasma membrane, indicating that cell death occurs by necrosis. Interestingly, inhibition of melanin synthesis largely prevents melanophore cell death in trpm7 mutants. These results suggest that melanophores require Trpm7 in order to detoxify intermediates of melanin synthesis. We find that unlike TRPM1, TRPM7 is expressed in human melanoma cell lines, indicating that these cells may also be sensitized to reduction of TRPM7 levels.

Defective skeletogenesis with kidney stone formation in dwarf zebrafish mutant for trpm7.

Development of the adult form requires coordinated growth and patterning of multiple traits in response to local gene activity as well as to global endocrine and physiological effectors. An excellent example of such coordination is the skeleton. Skeletal development depends on the differentiation and morphogenesis of multiple cell types to generate elements with distinct forms and functions throughout the body. We show that zebrafish touchtone/nutria mutants exhibit severe growth retardation and gross alterations in skeletal development in addition to embryonic melanophore and touch-response defects. These alterations include accelerated endochondral ossification but delayed intramembranous ossification, as well as skeletal deformities. We show that the touchtone/nutria phenotype results from mutations in trpm7, which encodes a transient receptor potential (TRP) family member that functions as both a cation channel and kinase. We find trpm7 expression in the mesonephric kidney and show that mutants develop kidney stones, indicating renal dysfunction. These results identify a requirement for trpm7 in growth and skeletogenesis and highlight the potential of forward genetic approaches to uncover physiological mechanisms contributing to the development of adult form.