have dual functions, being activators of FGF signalling, by interact-

ing with and phosphorylation by FGF receptors, and regulators of

cell adhesion. These data suggest that leucine rich repeat trans-

membrane proteins are novel modulators of receptor signalling

and/or cell adhesion during embryonic muscle development.

doi:10.1016/j.mod.2009.06.333

09-P004

Two populations of endochondral osteoblasts with differential

sensitivity to Hedgehog signaling

Chrissy Hammond, Stefan Schulte-Merker

Hubrecht Institute of Developmental Biology and Stem Cell Research &

University Medical Centre, Utrecht, The Netherlands

Hedgehog (Hh) signaling has been implicated in the develop-

ment of osteoblasts and osteoclasts whose balanced activities

are critical for proper bone formation. Since many mouse mutants

in the Hh pathway are embryonic lethal, questions on the exact

effects of Hh signaling on osteogenesis remain. Using zebrafish

larvae mutant in Indian Hedgehog, patched1, patched2 and sup-

pressor of fused (dre), we show that there are two populations of

perichondral/endochondral osteoblasts with differential sensitiv-

ity to Hh signaling. One, formed outside the cartilage structure,

requires low levels of Hh signaling, but still fails to differentiate

in Indian Hedgehog mutants. The other derives from transdiffer-

entiating chondrocytes and requires higher levels of Hh signaling

to form. This latter population develops significantly earlier in

mutants with increased Hh signaling, leading to premature endo-

chondral ossification, and also fails to differentiate in Indian

Hedgehog mutants, resulting in severely delayed endochondral

ossification. By contrast the number of dermal bone osteoblasts

is unaltered in mutants with increased and decreased levels of

Hh. Additionally, we demonstrate that the timing of first osteo-

clast activity positively correlates to Hh levels in both endochon-

dral and dermal bone. Our findings, based on invivo imaging of

osteoblasts, offer a possible explanation for some recently

reported controversial findings regarding Hh requirements in

osteogenesis, and can be integrated in a model that explains

how modulated levels of Hh signaling exert a differential effect

on specific subpopulations of differentiating osteoblasts.

doi:10.1016/j.mod.2009.06.334

09-P005

The Wilms tumour protein is required for kidney function in

adult mice

Ralph Sierig1, Dagmar Kruspe1, Jrgen Kastner2, Claudia Lck1,

Ralph Witzgall2, Christoph Englert1

1Leibniz Institute for Age Research – Fritz Lipmann Institute (FLI), Jena,

Germany2Institute for Molecular and Cellular Anatomy, University of Regens-

burg, Regensburg, Germany

The Wilms tumour suppressor gene Wt1 encodes a zinc finger

transcription factor and was initially identified as a gene inacti-

vated in 15–20% of Wilms tumour, a pediatric kidney cancer.

Besides its tumour suppressor function, Wt1 has been shown to

be an important regulator in the development of several organs

including heart, kidneys and genital system. Mice having no func-

tional Wt1 copy do not form kidneys and die, most probably due

to heart failure, at mid-gestation. In the adult kidney Wt1 expres-

sion is restricted to a single cell population, the podocytes. This

particular cell type forms the major part of the renal filtration bar-

rier. Podocyte-associated defects, as observed in focal glomerulo-

sclerosis, lead to severe kidney malfunction.

Using a conditional Wt1 knock out mouse model we are inves-

tigating Wt1 function in kidney and podocyte development and

maintenance. Mice with early and podocyte-specific Wt1 inacti-

vation die within 24 h after birth and show no signs of urine pro-

duction (anuria). Podocyte restricted Wt1 inactivation in adult

mice results in severe proteinuria, which is accompanied by a sig-

nificant reduction in the number of foot-processes formed by the

podocytes. In most cases this impairment of the kidneys’ filtra-

tion capacity does not affect viability of the animals. In some

cases, however, mice display a continuous and massive protein-

uria and eventually die after 4–6 months.

Our data suggest that Wt1 is not only essential for the devel-

opment of the renal filtration apparatus, but also for its mainte-

nance and functional integrity.

doi:10.1016/j.mod.2009.06.335

09-P006

Epithelial–mesenchymal interactions promote hair follicle neo-

genesis and a new stem cell niche in adult corneal epithelium

James Waters1, Gavin Richardson1, Danielle Dhouailly2,

Colin Jahoda1

1Durham University, Durham, United Kingdom2Universite Joseph Fourier, Grenoble, Isere, France

Previous recombinations of epithelium from the rabbit central

cornea with embryonic mouse dermis containing dermal conden-

sations has shown that dedifferentiation of corneal epithelium is

followed by hair follicle and skin differentiation. Our work aimed

to establish whether adult hair follicle dermal papillae (DP) can

induce trans-differentiation of the central cornea into hair folli-

cles, and to determine the role the underlying stroma plays in

these epithelial–mesenchymal interactions. Methods: Epithelium

from the central cornea of adult rabbit eyes was separated from

the underlying stroma, and hair follicle DP and non-hairy skin

were dissected from vibrissae and footpad of PVG rats, respec-

tively. Skin dermis or corneal stroma were then covered with cor-

neal epithelium with or without DP inserted in between. The

recombined tissue samples were then implanted beneath the kid-

ney capsule of athymic mice. Results: DP inserted between cornea

stroma and epithelium elicited expression changes in the epithe-

lium but no new hair follicles. By contrast, DP supported by foot-

pad dermis induced new follicle formation from cornea

epithelium. Intriguingly, footpad stroma combined with cornea

epithelium alone created new localised limbal niches in which

cells expressed limbal stem cell markers including ABCG2 and

K15. Conclusions: New hair follicles can be induced from central

corneal epithelial cells by adult DP. The underlying stroma is

important for this process since corneal stroma is not able to sup-

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port follicle neogenesis but non-hairy skin dermis can. In long-

term recombinations, the stem cell deficient central cornea cre-

ates new stem cell compartments/niches.

doi:10.1016/j.mod.2009.06.336

09-P007

The conserved avian Z-linked gene, DMRT1, is required for testis

determination in the chicken embryo

Craig Smith, Kelly Roeszler, Thomas Ohnesorg, Peter Farlie,

Andrew Sinclair

Murdoch Childrens Research Institute, Melbourne, Australia

Sex in birds is chromosomally based, as in mammals, but the

sex chromosomes are different and the mechanism of avian sex

determination is a long-standing mystery. In the chicken and other

birds, the homogametic sex is male (ZZ) and the heterogametic sex

is female (ZW). Two hypotheses have been proposed for the mech-

anism of avian sex determination. The W (female) chromosome

may carry a dominant-acting ovary determinant. Alternatively,

the dosage of a Z-linked gene may mediate sex determination,

two doses being required for male development (ZZ). A strong can-

didate avian sex-determinant under the dosage hypothesis is the

conserved Z-linked gene, DMRT1. DMRT1 encodes a transcription

factor related to male sex regulators in the fly and worm. Here,

we used a novel RNA interference approach to deliver microRNA

directed against DMRT1 into chicken embryos. Knockdown of

DMRT1 in ovo leads to feminisation of the embryonic gonads in

genetically male (ZZ) embryos. Affected males show partial sex

reversal, characterised by a feminised left gonad and a right testis.

The feminised gonad shows reduced DMRT1 protein expression,

disorganised testis cords and a decline in the testicular marker,

SOX9. The ovarian marker, Aromatase, is ectopically activated.

Germ cells also show a female pattern of distribution in the femin-

ised male gonads. This is the first functional test of any candidate

avian sex-determinant. Our results indicate that DMRT1 plays a

key role in chicken testis determination. The data support the Z

dosage hypothesis for bird sex determination, with DMRT1 repre-

senting the elusive avian sex determinant.

doi:10.1016/j.mod.2009.06.337

09-P008 – Withdrawn

09-P009

A signal amplifying regulatory network in lung mesenchyme

regulates mesenchymal and epithelial development

Yongjun Yin, Andrew White, Sung-ho Huh, Matthew Hilton,

Fanxin Long, David Ornitz

Washington University, St. Louis, MO, United States

Lung mesenchyme is a critical determinant of the shape and

size of the lung, the extent and patterning of epithelial branching,

the formation of the pulmonary vasculature and eventually the for-

mation of the interstitial mesenchymal components of the adult

lung. Signaling pathways in lung mesenchyme are not only neces-

sary for regulating mesenchymal growth and differentiation, but

also for regulating epithelial development. Fibroblast Growth Fac-

tor 9 (FGF9) is expressed in lung mesothelium and epithelium

and controls lung development through activation of mesenchy-

mal FGF receptors. Mice lacking FGF9 have decreased lung mesen-

chyme and impaired epithelial branching and die of respiratory

insufficiency in the perinatal period. Here, we identify a mesenchy-

mal signal amplifying regulatory network in developing lung mes-

enchyme through conditional inactivation of FGF9, mesenchymal

FGF receptors and mesenchymal b-Catenin. We show that during

the pseudoglandular stage of lung development this feed forward

regulatory network operates between two molecularly distinct

mesenchymal cell types and where it functions to integrate extrin-

sic FGF9 and Wnt7b signals. Interestingly, both extrinsic input sig-

nals are required to maintain the mesenchymal responsiveness to

FGF9, thus providing a mechanism to limit the activity of this net-

work. This signaling network functions to directly control mesen-

chymal growth and differentiation and to control output signals

(BMP pathway) that regulate lung epithelial growth.

doi:10.1016/j.mod.2009.06.339

09-P010

A nonsense mutation in the TFIIH p52 gene causes abnormal

rRNA processing in the zebrafish intestinal mutant, sycorax

Elizabeth Christie1, Andrew Trotter1, Yeliz Rifat1,

Andrew Badrock1, Adam Parslow1, Heather Verkade1,�,

Elke Ober2,�, Holly Field2, Didier Stainier2, Ross Hannan3,

Graham Lieschke4, Joan Heath1

1Ludwig Institute for Cancer Research, Parkville, Vic., Australia2Department of Biochemistry and Biophysics, University of California,

San Francisco, CA, United States3Research Division, Peter MacCallum Cancer Centre, East Melbourne,

Vic., Australia4Walter and Eliza Hall Institute of Medical Research, Parkville, Vic.,

Australia

sycoraxs845 is a zebrafish mutant identified in the Liverplus ENU-

mutagenesis screen. It exhibits striking defects in the morphol-

ogy of the intestinal epithelium, which is dysplastic, disorganised

and relatively unfolded compared to wild type. Apoptotic cells are

also observed in the intestinal lumen. The causative mutation in

sycorax is a premature stop codon in the general transcription

factor IIH polypeptide 4 (gtf2h4) gene. This gene encodes a com-

ponent of the TFIIH complex which is involved in RNA polymer-

ase I- and II-mediated transcription, nucleotide excision repair

(NER) and cell cycle regulation. TFIIH has been implicated specif-

ically in transcription elongation by RNA polymerase I and previ-

ous studies have linked this process with rRNA processing. To

examine ribosomal gene transcription and processing in sycorax,

� Present address: School of Biological Sciences, Monash Univer-sity, Clayton, Vic., Australia.� Present address: National Institute for Medical Research, MillHill, London, United Kingdom.

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