Activated in most cancers, the MAPK signaling pathway is among the most attractive targets for novel anti-cancer therapies 
. Like MAPK signaling pathways, most of the Int
pathways - Wnt, Fgf, and Notch - are conserved regulators of development that are frequently activated to promote oncogenesis. We provide evidence that, like other Int
gene products, Int6
is required for vertebrate development (), in part by providing a novel layer of MAPK signal transduction regulation (). With the wide range of cellular activities attributed to INT6, the mechanistic detail of this control remains to be understood; our early investigations indicate reduction of MEK1 in INT6
-siRNA treated mammalian cells is not dependent on the proteasome (M.G. & C.J.N., unpublished data
), making direct MEK1 regulation by INT6-dependent translation a possibility.
Recently, importance of RAS, RAF and MEK in human disease has been extended beyond cancer by the discovery that human germ-line mutations in these genes cause the LEOPARD-Noonan family of syndromes 
. Detailed immunohistochemical studies in mice have identified highly regulated, specific domains of discrete and dynamic ERK phosphorylation throughout development, including the pharyngeal arches and limb buds 
. In the first Int6 protein expression studies in a whole developing animal, we show that Int6 has regionally overlapping domains of protein expression with phospho-Erk, primarily in the craniofacial region (, Figure S4
). Lending biological significance to these observations, we show that phenotypic characteristics are shared between the loss of Int6 and inhibition of Mek activity (). In addition, partial loss of Int6 causes embryos to be highly sensitive to a mildly compromising dose of Mek inhibition, revealing an in vivo
interaction between Int6 protein expression and developmental Mek-Erk signaling (). As early over-expression of Ras-Raf-Mek signaling causes morphologic defects, we are currently generating transgenic lines that allow temporal control of Mek signaling, which will be a valuable tool for deeper genetic dissection of the Int6-Mek-Erk relationship in vivo
. It will be critical in future studies to establish if Int6 is capable of controlling both Mek1 and Mek2; our initial MO studies indicate that MEK2 may have a specific role in melanocyte migration (C.A. & E.E.P, unpublished data
), raising the possibility that the pigment cell migration defects observed in the int6
morphants also reflect altered MEK signaling.
FGF signaling is crucial for skeletal development, exemplified by the mutations that disrupt FGF signaling in human genetic skeletal abnormality syndromes 
. In the developing mouse embryo, most phospho-ERK domains overlap with FGF signaling domains 
. FGF signaling molecules are candidates for upstream activation of the Int6-moderated Mek-Erk signaling that shapes the craniofacial skeleton in vertebrates 
, and candidate downstream targets of Int6-Mek-Erk signaling include the chondrocyte differentiation transcription factor Sox9, which requires Mek activity for transcriptional activity 
. We also note that erk2
, but not erk1
, is specifically expressed in the pharyngeal arches in two-day old zebrafish embryos 
, possibly suggesting that Int6-Mek modulation in the developing craniofacial region may specifically signal through targets of Erk2.
Relating the Int6 modulation of Mek-Erk signaling to cancer development is a new angle for future investigation. One possibility is that in MMTV induced mammary tumors, the truncated Int-6 protein may act as an oncogene by altering MEK-ERK signaling. We propose that the diverse cellular locations of Int6, combined with the temporal expression and localization of Mek1/2 and Erk1/2, may result in fine-tuning of Mek-Erk signaling pathways in specific tissues during development, and may have important implications for the role of INT6 in tumorigenesis.