NCKX5 is an ion exchanger expressed mostly in pigment cells; however, the functional role for this protein in melanogenesis is not clear. A variant allele of SLC24A5, the gene encoding NCKX5, has been shown to correlate with lighter skin pigmentation in humans, indicating a key role for SLC24A5 in determining human skin colour. SLC24A5 expression has been found to be elevated in melanoma. Knockdown analyses have shown SLC24A5 to be important for pigmentation, but to date the function of this ion exchanger in melanogenesis has not been fully established. Our data suggest NCKX5 may have an alternative activity that is key to its role in the regulation of pigmentation. Here Xenopus laevis is employed as an in vivo model system to further investigate the function of NCKX5 in pigmentation. SLC24A5 is expressed in the melanophores as they differentiate from the neural crest and develop in the RPE of the eye. Morpholino knockdown and rescue experiments were designed to elucidate key residues and regions of the NCKX5 protein. Unilateral morpholino injection at the 2 cell stage resulted in a reduction of pigmentation in the eye and epidermis of one lateral side of the tadpole. Xenopus and human SLC24A5 can rescue the morpholino effects. Further rescue experiments including the use of ion exchange inactive SLC24A5 constructs raise the possibility that full ion exchanger function of NCKX5 may not be required for rescue of pigmentation.

Formation of the vertebrate nervous system requires coordinated cell-cell interactions, intracellular signalling events, gene transcription, and morphogenetic cell movements. Wnt signalling has been involved in regulating a wide variety of biological processes such as embryonic patterning, cell proliferation, cell polarity, motility, and the specification of cell fate. Wnt ligands associate with their receptors, members of the frizzled family (Fz). In Xenopus, five members of the frizzled family are expressed in the early nervous system. We have investigated the role of Xenopus frizzled-10 (Fz10) in neural development. We show that Fz10 is expressed in the dorsal neural ectoderm and neural folds in the region where primary sensory neurons develop. Fz10 mediates canonical Wnt signalling and interacts with Wnt1 and Wnt8 but not Wnt3a as shown in synergy assays. We find that Fz10 is required for the late stages of sensory neuron differentiation. Overexpression of Fz10 in Xenopus leads to an increase in the number of sensory neurons. Loss of Fz10 function using morpholinos inhibits the development of sensory neurons in Xenopus at later stages of neurogenesis and this can be rescued by co-injection of modified Fz10B and beta-catenin. In mouse P19 cells induced by retinoic acid to undergo neural differentiation, overexpression of Xenopus Fz10 leads to an increase in the number of neurons generated while siRNA knockdown of endogenous mouse Fz10 inhibits neurogenesis. Thus we propose Fz10 mediates Wnt1 signalling to determine sensory neural differentiation in Xenopus in vivo and in mouse cell culture.

Macrophages are essential in development, repair and pathology of a variety of tissues via their roles in tissue remodelling, wound healing and inflammation. These biological functions are also associated with a number of human diseases, for example tumour associated macrophages have well defined functions in cancer progression. Xenopus embryonic macrophages arise from a haematopoietic stem cell population by direct differentiation and act as the main mechanism of host defence, before lymphoid cells and a circulatory system have developed. This function is conserved in mouse and human development. Macrophages express a number of matrix metalloproteinases (MMPs), which are central to their function. MMPs are a large family of zinc-dependent endoproteases with multiple roles in extracellular matrix remodelling and the modulation of signalling pathways. We have previously shown MMP-7 to be expressed by Xenopus embryonic macrophages. Here we investigate the role of MMP-7 and two other MMPs (MMP-18 and MMP-9) that are also expressed in the migrating macrophages. Using morpholino (MO) mediated knockdown of each of the MMPs we demonstrate that they are necessary for normal macrophage migration in vivo. The loss-of-function effect can be rescued using the specific MMPs, altered to be resistant to morpholinos but not by overexpression of the other MMPs. Double and triple morpholino knockdowns further suggest that these MMPs act combinatorily to promote embryonic macrophage migration. Thus, our results imply that these three MMPs have distinct functions, which together are crucial to mediate macrophage migration in the developing embryo. This demonstrates conclusively that MMPs are required for normal macrophage cell migration in the whole organism.

Klhl31 is a member of the Kelch-like family in vertebrates, which are characterized by an amino-terminal broad complex tram-track, bric-a-brac/poxvirus and zinc finger (BTB/POZ) domain, carboxy-terminal Kelch repeats and a central linker region (Back domain). In developing somites Klhl31 is highly expressed in the myotome downstream of myogenic regulators (MRF), and it remains expressed in differentiated skeletal muscle. In vivo gain- and loss-of-function approaches in chick embryos reveal a role of Klhl31 in skeletal myogenesis. Targeted mis-expression of Klhl31 led to a reduced size of dermomyotome and myotome as indicated by detection of relevant myogenic markers, Pax3, Myf5, myogenin and myosin heavy chain (MF20). The knock-down of Klhl31 in developing somites, using antisense morpholinos (MO), led to an expansion of Pax3, Myf5, MyoD and myogenin expression domains and an increase in the number of mitotic cells in the dermomyotome and myotome. The mechanism underlying this phenotype was examined using complementary approaches, which show that Klhl31 interferes with β-catenin dependent Wnt signaling. Klhl31 reduced the Wnt-mediated activation of a luciferase reporter in cultured cells. Furthermore, Klhl31 attenuated secondary axis formation in Xenopus embryos in response to Wnt1 or β-catenin. Klhl31 mis-expression in the developing neural tube affected its dorso-ventral patterning and led to reduced dermomyotome and myotome size. Co-transfection of a Wnt3a expression vector with Klhl31 in somites or in the neural tube rescued the phenotype and restored the size of dermomyotome and myotome. Thus, Klhl31 is a novel modulator of canonical Wnt signaling, important for vertebrate myogenesis. We propose that Klhl31 acts in the myotome to support cell cycle withdrawal and differentiation.