function analysis of FGF proteins in Drosophila

S216

MECHANISMS OF DEVELOPMENT

1 2 6 (2 0 0 9) S1 9 5–S 23 8

may also be important downstream of Wnt4. To facilitate further

13-P072

identification of NFAT activity we have created reporters driven by

Interfering with Wnt signalling alters the periodicity of the seg-

NFAT-responsive elements and generated transgenic mice to

mentation clock

examine activity in vivo.

Sarah Gibb, Kim Dale

The second, complementary approach we are taking is to manipulate the pathway in cells, embryos, and components of the embryonic kidney. Cre-regulated dominant-negative and constitutively-active forms of NFAT have been generated and will be employed on both the wild type and Wnt4-deficient background. In addition to these genetic manipulations, drugs have been used to alter the pathway in cultured kidney rudiments. Treatment 2+

with cyclosporin A (CSA), an inhibitor of calcineurin-NFAT Ca

signalling, decreases nephron formation – a phenotype similar to that in Wnt4 the Wnt4

/

/

embryos. We are now attempting to rescue

phenotype using Ionomycin, an activator of the

2+

Ca /NFAT pathway. By combining the described reporters, lines, and reagents it is hoped that the functions of Wnt-Ca

2+

signalling

in kidney development can be dissected. Finally, we have extended our analyses of NFAT signalling to encompass the whole embryo. Novel sites of NFAT expression have been identified and an evo-devo approach has been applied to reveal insights into this family of transcription factors. doi:10.1016/j.mod.2009.06.543

University of Dundee, Dundee, United Kingdom Segmentation is first apparent in developing vertebrates with the formation of the somites, which are the embryonic precursors of the ribs, vertebrae and certain dermis tissue. Somite formation is a periodic process regulated by a molecular clock which drives cyclic expression of a number of clock genes in the presomitic mesoderm, the tissue which gives rise to the somites. To date the mechanism regulating the period of clock gene oscillations is unknown. Strikingly we find that modifying Wnt signalling changes the period of Notch driven oscillations in both mouse and chick but these oscillations continue. The use of either casein kinase I inhibitor or soluble frizzled receptor protein inhibits Wnt signalling and results in slower oscillations of clock genes. In addition, activation of the Wnt pathway does not accelerate the oscillations but can rescue the slow down of oscillations caused by Wnt inhibition. The effect of Wnt attenuation on oscillation pace is not mediated by FGF signalling and it is not due to an indirect effect on cell proliferation. We propose that the Wnt pathway is a conserved mechanism that is involved in regulating the period of cyclic gene oscillations in the presomitic mesoderm.

13-P071 A temporal gradient of BMP signalling controls neuronal subtype

doi:10.1016/j.mod.2009.06.545

identity in the dorsal neural tube Samuel Tozer, James Briscoe NIMR, MRC, London, United Kingdom

13-P073 Structure/function analysis of FGF proteins in Drosophila

In the spinal cord, BMPs secreted by the roof plate have been

Sarah Payne, Angela Stathopoulos

proposed to act as morphogens to specify the pattern of generation of dorsal interneurons dI1-3. However the way in which BMPs

California Institute of Technology, Pasadena, CA, United States

perform this function remains unclear. Here, using ex vivo assays, we show that the progenitors of distinct neuronal sub-

Drosophila has three FGF ligand genes and two of them, pyra-

types are induced in response to different durations of BMP sig-

mus (pyr) and thisbe (ths), represent an ancient gene duplication,

nalling. Exposure to different concentrations of BMP resulted in

and are present in all Drosophilids. Pyr and Ths are most closely

the induced levels of signalling activity being sustained, even

related to FGF8 in vertebrates and are similarly involved in gastru-

after ligand removal. However, this ‘‘memory effect” could be

lation. The vertebrate FGFs are 18–30 kDa in size, whereas the Dro-

blocked up to 3 h after ligand exposure by the BMP antagonist

sophila FGFs are 79–82 kDa, with no other predicted domains

Noggin. This resulted in the production of dI2 progenitors but

besides the homologous FGF domain. The longer C-terminus of

not the more dorsally located dI1 progenitors. By contrast, pro-

Drosophila FGFs may serve a regulatory purpose where the func-

longed BMP signalling resulted in the generation of predomi-

tion of the FGF domain is activated or attenuated by protein pro-

nately dI1 progenitors. Consistent with these data, the in vivo

cessing. Processing by proteases is a common mechanism for the

specification of dI3, dI2 and dI1 neurons requires progressively

regulation of ligand proteins and the highly dynamic expression

longer periods of BMP signalling. Moreover, a reporter of the

pattern of pyr and ths suggests that such regulation may be

BMP pathway indicated that BMP activity becomes gradually

involved. Using modified constructs of Pyr and Ths we have

restricted to the dorsal most region of the neural plate. Together,

undertaken a structure/function analysis to understand which

these data suggest that a temporal gradient of BMP signalling pat-

part of the proteins are required for function and which part is

terns the dorsal neural tube and determines the progressive spec-

available for processing by proteases. We have evidence that the

ification of dI1, dI2 and dI3 neurons. We propose that BMP

FGF domain alone is not always sufficient to support activity,

antagonists produced by the neural tube and surrounding tissues

and favor a model in which proteases function in a regulatory

are responsible for this profile of signalling activity?

manner to control the activity of FGFs.

doi:10.1016/j.mod.2009.06.544

doi:10.1016/j.mod.2009.06.546