21-P032 Regulation of chondrocyte differentiation by posterior Hox genes

S322

MECHANISMS OF DEVELOPMENT

1 2 6 (2 0 0 9) S3 1 4–S 32 8

21-P029

that Sulf1 may be facilitating either the movement or activity of

The new method to induce primordial germ cell using animal cap

Shh during primary neurogenesis. To asses whether Sulf1 is able

and activin treatment

to alter the spatial distribution of Shh, we used a Shh–GFP fusion

Shoko Mori, Hiroki Kuroda

construct to directly visualise the Shh protein. Here we present an

Shizuoka University, Shizuoka, Japan It is well known that activin-treated presumptive ectoderm,

analysis of the effects of Sulf1 on the proliferation and specification of neuronal precursors. doi:10.1016/j.mod.2009.06.895

which is generally called as animal cap, in amphibian differentiates into various types of tissues and organs such as notochord, muscle, heart, and kidney. However, there has been no report that primordial germ cells are induced by activin treatment for animal caps. Anterior or posterior axial mesoderms can be, respectively, induced by middle-low or -high concentrations of activin treat-

21-P031 Insights into signalling mechanisms of Latrophilins Simone Prmel, Tobias Langenhan, Andreas Russ University of Oxford, Oxford, United Kingdom

ments for dissociated animal cap cells. It is reported that sandwich of two different reaggregates from these dissociated

Latrophilins were first described as receptors for the black

animal cap cells has a potent activity of convergent extension.

widow spider toxin. They are highly conserved members of the

Using combination of this assay and microarray gene analyses,

Adhesion G-protein coupled receptor family, an unusual class of

we tried to isolate new regulators of convergent extension phe-

molecules with roles in development and planar cell polarity.

nomenon. As a result, novel candidate molecule was not isolated

For LAT-1, one of the two Latrophilin homologues in Caenorhabdi-

from this assay although only a few convergent extension mark-

tis elegans, our data indicate a role in cell fate specification and

ers were confirmed. Unexpectedly, most genes showing big differ-

morphogenesis. No physiological ligand or signalling pathway

ences of expression levels in this screening were primordial germ

for these receptors with unusual long N-termini and various

cell markers such as germs, Xpat, and DEADSouth RNA helicase.

domains is known so far.

In addition to them, some novel or functionally unreported mol-

Using C. elegans, we have developed the first invivo assay to

ecules were contained in the other high-scored genes of this

investigate the function and possible signalling mechanism of

screening.

LAT-1 on a molecular basis. We were able to make a detailed anal-

This is the first report that activin treatment for animal cap

ysis of different domains in receptor function. We also found that

cells can induce primordial germ cells, suggesting that the sand-

the receptor functions in two different ways, one maintained solely

wich assay that we used in this study is able to be a good strategy

by the N-terminus, while the whole receptor is crucial for the other.

to isolate genes related to primordial germ cell formation.

The N-terminus does not need to be separated from the C-terminus, cleavage at a site in the N-terminus, assumed to be crucial

doi:10.1016/j.mod.2009.06.894

for receptor function, does not seem to be essential. This is in contrast with current literature and challenges the models suggested for possible signalling mechanisms in this receptor class.

21-P030 The role of Sulf1 in Sonic hedgehog mediated neuronal

doi:10.1016/j.mod.2009.06.896

patterning Simon Ramsbottom, Mary Pownall University of York, York, North Yorkshire, United Kingdom Heparan sulphate proteoglycans (HSPGs) are large molecules

21-P032 Regulation of chondrocyte differentiation by posterior Hox genes Stefanie Werner, Andrea Vortkamp

distributed ubiquitously, both at the cell surface and within the

Center for Medical Biotechnology, Department of Developmental Biology,

extracellular matrix. These molecules are known to play essential

University of Duisburg-Essen, 45117 Essen, Germany

roles in developmental cell signalling and the differential sulfation of HSPG chains gives rise to much variability in their binding

In vertebrates, Hox genes of the Hoxa and Hoxd cluster are

specificity. Sulf1, an N-acetylglucosamine O-6 endosulfatase, spe-

necessary for limb development. For establishing the zeugopod

cifically removes sulphate groups from HSPG chains in regions of

Hoxa11 and Hoxd11 are the essential factors. Hoxa11/d11 double

high sulfation, and our lab has previously shown that Sulf1 activ-

mutant mice display a severe forelimb phenotype with dramatic

ity leads to the attenuation both BMP and FGF signalling.

size reduction and delayed chondrocyte differentiation of ulna

The expression profile of Sulf1 within the neural tube is simi-

and radius. Interestingly, almost the same forelimb phenotype

lar to that of Sonic hedgehog (Shh) and work in chick has shown

is observed in Ulnaless mice, in which no Hox gene is disrupted,

that Sulf1 is able to increase the concentration of Shh at the cell

but the complete Hoxd cluster is inverted. Due to this inversion,

surface in a cell autonomous manner [Danesin etal., 2006]. Due to

the posterior Hoxd13 is ectopically expressed in the zeugopod

the fact that HSPG chains are required for the diffusion of Hh

region, where it likely represses specific functions of Hox11

through a field of cells [The etal., 1999], Sulf1 provides a good can-

paralogues.

didate as a regulator of Shh distribution and activity.

Morphological analysis of Hoxa11//d11/ and Ulnaless mice

Over expression of Sulf1 changes the spatial distribution of

revealed a significant delay in chondrocyte differentiation of ulna

molecular markers of specific neuronal precursors, indicating

and radius. At E14.5 hypertrophic chondrocytes are not detect-

1 2 6 ( 2 0 0 9 ) S 3 1 4 –S 3 2 8

MECHANISMS OF DEVELOPMENT

S323

able. In situ hybridization on E14.5 and E16.5 forelimbs confirmed

space [Hou etal., 2007]. The data suggested that xHtrA1 through

delayed chondrocyte differentiation since Indian hedgehog (Ihh)

cleaving proteoglycans release cell-surface bound FGF ligands

is not expressed in the ulna of Ulnaless or Hoxa11//d11/ mice.

and stimulate long-range FGF signaling during establishment of

Fibroblast growth factor 3 (Fgfr3), which is expressed at low levels

the embryonic body plan [Gallagher, 2007]. If not tightly con-

in distal cells and at high levels in columnar chondrocytes, shows

trolled, the proteolytic activity of xHtrA1 would lead to an unlim-

low expression in the Ulnaless and Hoxa11//d11/ zeugopod

ited amplification and propagation of FGF signals. We have

resembling the expression level in distal chondrocytes. However,

isolated a full-length cDNA clone encoding a secreted serine pro-

the Unique cartilage matrix-associated protein (UCMA), a specific

tease inhibitor (xSPI) that may act as a negative regulator of

marker for distal chondrocytes, is only expressed in the outer-

xHtrA1/FGF signals. xSPI shows distinct expression in the early

most cell layers in ulna and radius of both mutant mouse lines.

embryo and promotes anterior development in mRNA-injected

These results indicate that chondrocyte differentiation is

Xenopus embryos. xSPI mRNA induced enlargement of head struc-

arrested at an early step in Ulnaless and Hoxa11/d11 double

tures, suppression of mesoderm and reduction of neuronal differ-

mutant mice, before the differentiation of distal and columnar

entiation. These effects are reminiscent of those caused by

chondrocytes takes place.

knockdown of xHtrA1 or inhibition of FGF signaling [Hou etal., 2007]. In contrast, downregulation of xSPI by specific morpholino

doi:10.1016/j.mod.2009.06.897

oligonucleotides caused microcephaly, a phenotype that is also induced by misexpression of xHtrA1, FGFs or components of the FGF-MAPK pathway. Moreover, xSPI mRNA prevented xHtrA1

21-P033

from inducing ectopic tail-like structures, mesoderm induction

The role of the transcription factor Sox21 in the developing chick

and stimulation of neuronal differentiation. Preliminary results

inner ear

indicate that xSPI and xHtrA1 directly bind to each other.

Stephen Freeman, Nicolas Daudet

Together, the data suggest that xSPI may add another layer to the regulation of the FGF pathway in the extracellular space and

UCL Ear Institute, London, United Kingdom In the developing central nervous system, the balanced activity of different SoxB family transcription factors plays an impor-

via suppression of xHtrA1 proteolytic activity restrict growth factor signalling in the developing embryo. doi:10.1016/j.mod.2009.06.899

tant role in the regulation of neuronal proliferation and differentiation. The SoxB1 gene Sox2 has been shown to inhibit neurogenesis, maintaining cells in a proliferative neuronal precursor state in the chicken neural tube. Conversely the SoxB2 family member Sox21 promotes neurogenesis by antagonizing Sox2 activity [Sandberg, 2005. Nat. Neurosci. 8 (8) 2005, 995– 1001]. A similar interplay may regulate the embryonic production of hair cells in the sensory epithelia of the inner ear. In fact, the Sox2 gene is expressed in progenitor cells that give rise to sensory hair cells, and a previous study has reported expression of Sox21 in the chicken inner ear at HH stage 30 [E6-6.5; Uchikawa, 1999. Mech. Dev. 84 (1–2), 103–120]. Here we analysed in detail the expression pattern of Sox21 during the development of the chicken inner ear, and found that it is first detected in the prosensory domains at E5. Sox21 expression becomes gradually restricted to the hair cell layer as development of the inner ear progresses. We also obtained preliminary data showing that overexpression of Sox21 can down-regulate the expression of the progenitor cell marker Prox-1. These findings suggest that Sox21 could play a role in the specification of inner ear sensory cells. doi:10.1016/j.mod.2009.06.898

21-P035 Drosophila neurotrophic factor DmMANF Mari Palgi1, Riitta Lindstrm1, Vassileios Stratoulias1, Mart Saarma1, Tapio Heino2,1 1

Institute of Biotechnology, University of Helsinki, Helsinki, Finland

2

Department of Biological and Environmental Sciences, University of

Helsinki, Helsinki, Finland In vertebrates, neurotrophic factors have prominent roles in survival, differentiation and maintenance of neurons. Despite extensive searches no neurotrophic factors have been found in invertebrates. On the other hand, cell ablation studies in fruit fly, Drosophila melanogaster, suggest trophic and non-cell autonomous interactions in its neuronal development. DmMANF is a Drosophila homolog of recently discovered vertebrate neurotrophic factors MANF and CDNF. DmMANF mutants die as early second instar larvae and our rescue experiments confirm DmMANF as a ortholog of the human MANF/CDNF genes. We have shown that during embryogenesis DmMANF is expressed in glia and that it is essential for the maintenance of dopamine positive neurites. The abolishment of both maternal and zygotic DmMANF leads to earlier lethality and degeneration of axonal

21-P034 Extracellular regulation of FGF signaling in the early Xenopus embryo Tan Hooi Min, Dobromir Iliev, Edgar M. Pera Lund University, Lund, Sweden

bundles in the embryonic central nervous system and subsequent nonapoptotic cell death. We show results from several rescue and RNAi experiments that aim to find out the critical tissues for survival. We also present preliminary results of a RNAi based screen to identify genes interacting with DmMANF. Finally the expression pattern of DmMANF in the adult Drosophila brain is presented.

Fibroblast growth factors (FGFs) play an important role in development and homeostasis. We previously presented the xHtrA1 as positive regulator of FGF signaling in the extracellular

doi:10.1016/j.mod.2009.06.900