Proper maternal nutrition is critical for early embryonic development. The Dutch Famine Study examined the consequences of nutrient deprivation on developmental outcome during severe food shortages near the end of the Second World War and clearly demonstrated that inadequate nutrient availability during human gestation increases the likelihood of developmental anomalies 
. From these initial observations arose the well-recognized link between maternal folate supplementation and the suppression of neural tube defects 
. Despite overwhelming epidemiologic data indicating the benefits of folate and other nutrient supplementation we do not fully understand the genetics of predisposition to these abnormal developmental phenotypes when faced with suboptimal nutrient levels. There are several large difficulties in the study of these processes in mammals that have prevented faster progress. The first is that the genetics of mammals has been cumbersome. The second, and more important, is that development of placental animals occurs in utero
making rapid detection of developmental phenotypes difficult. Finally, controlling the level of nutrient available to the developing embryo cannot be done with precision as it depends both on the genetics of the mother and the embryo as well as maternal nutrition.
Copper is an essential nutrient which when absent results in severe developmental abnormalities. This is most clearly illustrated by Menkes disease (OMIM #309400), a rare X-linked disorder of copper metabolism. Patients with Menkes disease have an array of symptoms including seizures, neurodegeneration, hypopigmentation, and lax skin which result from decreased copper incorporation into critical enzymes such as dopamine-β-hydroxylase and lysyl oxidase 
. This usually fatal disease is caused by mutations in a copper transporter, ATP7A
(NM_000052), which resides in the secretory pathway and is responsible for transport of copper into this compartment. The Menkes gene product is also responsible for placental copper transport. While patients complete in utero
development apparently normally, it is clear from biochemical studies at birth that there are significant defects that arise from gestational copper deficiency 
In order to study the effects of developmental copper deprivation our lab has previously created a zebrafish model of severe copper deficiency 
. High doses of the cell permeable copper chelator neocuproine cause embryonic zebrafish to exhibit a Menkes-like phenotype with neurodegeneration, hypopigmentation, and connective tissue defects. Isolation and cloning of the mutant calamity
, which shared these same characteristics, revealed a loss-of-function mutation in the zebrafish orthologue of ATP7A
(NM_001042720). In this current study we expand this model to study the effects of induced genetic alterations on the developmental response to mild
copper deprivation. We describe two mutants sensitive to nutritional copper deficiency that illustrate the potential power of this approach to overcome the limitations of studying gene-nutrient interactions in vertebrate organisms and that define combinations of loss-of-function mutations of known ion homeostatic pathways that result in aberrant development.