13-P049 FGF signalling is regulating bone development in zebrafish

MECHANISMS OF DEVELOPMENT

1 2 6 ( 2 0 0 9 ) S 1 9 5 –S 2 3 8

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The neural progenitors tend to differentiate into neurons,

CNEs enhance GLI3-specific expression in evolutionary ancient

when they were dissociated in vitro. This suggests that the cell–

stylopod and zeugopod or modern skeletal structures of the auto-

cell adhesion is required for maintaining the undifferentiated

pod, respectively. Limb specificity is also found in chicken but has

state of the neural stem cells. In fact when we disrupted the junc-

not been detected in zebrafish embryos. Even though fish, birds,

tional complex in the developing neural tube specifically, the neu-

and mammals share an ancient repertoire of gene regulatory ele-

roepithelial cells differentiated into neurons concomitant with

ments within GLI3, the functions of individual enhancers from

down-regulation of Notch signaling.

this catalog have diverged significantly. During evolution, ancient

Notch signaling has long been known to influence cell fate

broad-range regulatory elements within GLI3 attained higher

regulation in the developing nervous system. However, it is not

specificity, essential for patterning of more specialized structures,

known precisely where Notch interacts with Delta along the elon-

by abolishing the ancient potential for redundant expression

gated lateral surface of the neuroepithelia. Given the size of these

control.

transmembrane proteins, we hypothesize that the Notch signaling requires a close and stable distance between the neighboring

doi:10.1016/j.mod.2009.06.521

cells for efficient interaction between Notch and Delta. The adherens junction would provide such environment between the neuroepithelia. Our data suggest that Notch protein is cleaved in the vicinity of the adherens junction, as DAPT treatment, an inhibitor of gamma-secretase that cleaves Notch upon its activation, resulted in accumulation of Notch protein at the adherens junctions in the cultured neural progenitor cells. We also examined the presomitic mesoderm (PSM), and our results obtained

13-P049 FGF signalling is regulating bone development in zebrafish Katharina Felber, Phil Elks, Peter Croucher, Henry Roehl University of Sheffield, Sheffield, United Kingdom

from the PSM were consistent with those from the neural tissue. These results suggest that the adherens junction facilitates

Osteoblasts are specialized cells that are responsible for the

efficient interaction of Notch and Delta to maintain the undiffer-

deposition of bone. Molecular and genetic analysis performed in

entiated state of the neural stem cells. We also suggest that this

mice have identified two transcription factors, RUNX2 and OSX,

may be a general feature of Notch signaling under dynamic cellu-

that act sequentially to direct mammalian osteoblast differentia-

lar environment.

tion. RUNX2 is required during the early stages of commitment and acts in part to activate osx transcription. OSX and RUNX2

doi:10.1016/j.mod.2009.06.520

then act to regulate transcription of bone matrix proteins like osteonectin and col1a2. We have investigated the expression of these genes during zebrafish cranial osteoblastogenesis and find that during the formation of a given bone, the mammalian

13-P048

expression sequence is conserved.

Expression patterning in CNS and limb from fish to mammals by GLI3-intronic enhancers Amir Ali

Abbasi1,2, Zissis Paparidis1, Sajid Malik Malik1,2,

Christian Weirich1, Karl-Heinz Grzeschik1

Moreover we have analysed the roles of FGF and WNT signalling pathways in the regulation of osteoblast differentiation in zebrafish using pharmaceuticals as well as a heat shock (hs) promoter to drive misexpression. Inhibition of WNT signalling with

1

Philipps-Universita¨t, Marburg, Germany

the hsDKK line leads to a downregulation of osx expression. Fur-

2

Quaid-i-Azam University, Islamabad, Pakistan

thermore inhibition of the FGF pathway with the hsdnFGFR1 line or the potent small molecule SU5402 results in a strong and rapid

The zinc-finger transcription factor GLI3 is an important mediator of Sonic hedgehog signaling and thus crucial for patterning of many aspects of the vertebrate body plan. GLI3 expression must be subject to a tight spatiotemporal regulation. Twelve potential enhancers of GLI3 expression identified as conserved non-coding sequence elements (CNEs) distributed exclusively across introns of the gene were studied. Both, signatures highly conserved from humans to fish carrying predicted transcription factor binding sites and less conserved flanking sequences are

downregulation of osx transcription while having a mild effect on runx2b expression. On the other hand, overactivation of the FGF pathway with hsFGF3 results in a slight upregulation of osx and runx2b. Interestingly continuous heat shocks of hsFGF3 embryos from 48 h post-fertilization until 5 days post-fertilization leads to a loss of ossification instead of the expected upregulation. We are currently investigating why ossification is lost in hsFGF3 embryos and how FGF and WNT signalling interact to regulate osx expression.

required to achieve strong activation of reporter gene expression. The regulatory potential of GLI3-intronic CNEs as activators and/

doi:10.1016/j.mod.2009.06.522

or repressors observed in transfected human cell cultures is upheld in transgenic zebrafish, chicken and mouse transgenic embryos. During development of the model organisms, these human enhancers autonomously control expression. Their combined action generates a GLI3-specific pattern of transcriptional activity. In the mouse, three of these elements govern central nervous system specific gene expression, each targeting a subset of endogenous Gli3 transcription sites. In limb buds, two different

13-P050 Transcriptional regulation by the FGF signaling pathway in trophoblast stem cells Kenjiro Adachi, Hitoshi Niwa RIKEN CDB, Kobe, Japan