13-P080 Hedgehog signalling in lung development

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MECHANISMS OF DEVELOPMENT

1 2 6 (2 0 0 9) S1 9 5–S 23 8

showing abnormalities of the semicircular canal formation.

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These are consistent with proposals that Fgf10 expression in

Heparan sulphotransferases are regulators of mammalian axon

the developing sensory tissues is required to induce non-sensory

guidance

development. To determine the otic epithelial functions of Fgf3

Christopher D. Conway, David J. Price, John O. Mason,

and Fgf10, we generated double conditional mutants using Tg-

Thomas Pratt

Pax2Cre, and found that as expected, both genes play roles in semicircular canal morphogenesis. Unexpectedly, these genes also appear to have redundant roles in cochlear morphogenesis. We will describe progress in determining the molecular mechanisms by which these FGFs control morphogenesis, as well as a new approach to controlling Fgf3 expression with doxycyline administration that should enable detailed analysis of otic morphogenesis, both in vivo and in culture.

University of Edinburgh, Edinburgh, United Kingdom Heparan sulphate proteoglycans (HSPGs) are extracellular glycoproteins involved in cell–surface interactions and are emerging as regulators of classical signalling pathways. HSPGs exhibit massive structural diversity, conferred partly by extensive post-translational modification of the carbohydrate HS component, including differential sulphation by heparan sulphotransferase (HST) enzymes.

doi:10.1016/j.mod.2009.06.550

The normal functioning of the mammalian central nervous system involves precise interconnections forming between developing neurons. To gain a better understanding of the functional significance of HSPG differential sulphation in connectivity we

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have examined the function of Hs2st and Hs6st1 and the forma-

The canonical and non-canonical Wnt pathways: A complex

tion of major forebrain commissural axon tracts in loss of func-

interplay for the regulation of cardiogenesis

tion mutant mice lacking either Hs2st or Hs6st1. We find that

Tania Papoutsi, Bill Chaudhry, Deborah Henderson

Hs2st and Hs6st1 are expressed at specific locations in the devel-

Newcastle University, Newcastle upon Tyne, United Kingdom Heart disease is one of the leading causes of death in the developed world. Understanding of how naı¨ve cells differentiate into cardiomyocytes is important for preventing congenital heart malformations and repairing the failing adult heart. The Wnt pathway has been extensively studied but its precise temporal role during cardiomyocyte commitment remains unclear. We have used mouse ES cells to investigate the complex roles of the Wnt pathway in early cardiogenesis. These pluripotent cells spontaneously differentiate into diverse cell lineages and model early embryonic development. Specifically, mESC can differentiate through the cardiac lineage and achieve cardiomyocyte fate. Interrogation of the roles played by different parts of the Wnt network has been carried out by inhibition of key pro-

oping brain and that axons make particular mistakes in each mutant. Slit proteins are axon guidance ligands and axon guidance defects in Slit1 and Slit2 loss of function mutants closely resemble those seen in our Hs2st and Hs6st1 mutants suggesting that these HST enzymes are essential regulators of Slit1 and Slit2 function. In vitro experiments show that retinal axons lacking Hs6st1 function are defective in their ability to respond to Slit2. In conclusion, we find that two HST enzymes are deployed in the developing brain so as to regulate specific aspects of axon guidance by regulating the ability of navigating axons to respond to guidance cues. Our findings support the hypothesis that modification to HSPG structure is an important mechanism for regulating signalling events required for the correct wiring of the brain. doi:10.1016/j.mod.2009.06.552

teins during differentiation. Our data indicate that inhibition of RhoKinase and Rac do not significantly impair cardiac fate. In contrast,

inhibition

of

c-Jun-N-terminal-Kinase

completely

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inhibited beating. Similarly, hyper-activation of canonical Wnt

Hedgehog signalling in lung development

signalling throughout mESC differentiation, blocked spontane-

Uda Ho, Elaine Costelloe, Brandon Wainwright

ous contraction. To further investigate these findings we inhibited the noncanonical Wnt/PCP branch in zebrafish embryos before and after gastrulation. Fish embryos treated with a chemical inhibitor for JNK were abnormal and poorly developed. They formed smaller hearts with incomplete looping and bradycardia, compared to their age-matched and stage-matched controls. These data highlight the complexity of Wnt signalling in early cardiogenesis and supports the idea that whilst canonical signalling suppresses cardiogenesis at many time intervals during development, different branches of non-canonical Wnt signalling play essential roles in the regulation of cardiac induction and cardiac morphology.

Institute for Molecular Bioscience, Brisbane, Australia Activation of the Hedgehog (Hh) pathway has been reported in small cell lung cancer progression, but the role of its receptor, Patched1 (Ptc1), is poorly understood. We believed that tumorigenesis is caused by the dysregulation of developmental pathways that maintain normal tissue homeostasis; hence it is imperative that we understand these events in development in order to understand their roles in cancer. This project aims to study the effects of Hh signalling during lung development by crossing the Ptc1 conditional knockout mouse with the Dermo1Cre mouse (Dermo1-Cre+/ ; Ptc1lox/lox), thus activating Hh signalling in the lung mesenchyme. Dermo1-Cre+/ ; Ptc1lox/lox embryos die at E12.0 and showed defects in lung lobe formation. Due to

doi:10.1016/j.mod.2009.06.551

early embryonic lethality, lungs from Dermo1-Cre+/ ; Ptc1lox/lox embryos were isolated at E11.5 for explants cultures and showed

MECHANISMS OF DEVELOPMENT

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failure of lung branching and mesenchymal cell proliferation

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after 72 h in culture. It is hypothesised that inappropriate activa-

The canonical Wnt-signalling pathway is active in the extratoes

tion of Hh signalling inhibits Fgf10 expression, thus leading to

(Gli3Xt/Xt) mouse mutant forebrain

impaired lung branching morphogenesis. Fgf10 expression is

Vassiliki Fotaki, David Price, John Mason

reduced in Dermo1-Cre+/ ; Ptc1lox/lox lungs at E11.5 and addition of Fgf10 recombinant protein to Dermo1-Cre+/ ; Ptc1lox/lox lung explants cultures can partially rescued the branching defect,

Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom

which further supports the role of Hh signalling in regulating Fgf10 expression during lung branching morphogenesis. The results obtained in this study have reflected the importance of proper Hh signalling pathway in regulating lung development. Understanding the role of Ptc1 in lung development will provide insight into the Hh pathway’s involvement in lung repair and cancer, which may improve the diagnosis and prognosis for lung disease patients.

The Gli3 transcription factor, a downstream target of the Shh pathway, is essential for mammalian development. Mice homozygous for a Gli3 null mutation (extratoes or Gli3Xt/Xt mice) die at birth showing severe reduction in the size of the forebrain. In extratoes embryos, the dorsal telencephalic midline fails to invaginate and dorsomedial structures such as the cortical hem, a rich source of Wnt molecules, never form. At present it is not known how and to what extent the absence of cortical hem in the Gli3Xt/ Xt

doi:10.1016/j.mod.2009.06.553

mutant affects Wnt signalling in the developing forebrain. To

shed light on this, we crossed Gli3Xt/+ mice to the BAT-gal mouse, a reporter of canonical Wnt signalling, to generate compound Gli3Xt/Xt; BAT+ mutant embryos. Compound mutants were col-

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lected from embryonic day (E) 8.0 to E12.5 and beta-galactosidase

Distinct dynamics of Sonic Hedgehog signaling are required to

staining was used as a read-out of Wnt/beta-catenin signalling.

specify floor plate and ventral neuronal identities in the verte-

At E8.0, beta-galactosidase was detected in the posterior neural

brate spinal cord

plate and its pattern was similar between wild-types and

Vanessa Ribes, Nikolaos Balaskas, Noriaki Sasai, Eric Dessaud,

mutants. By E8.5, beta-galactosidase was expressed in the ante-

Catarina Cruz, James Briscoe

rior neural plate showing a decrease in extratoes compared to wild-types. At E10.5, the abundant beta-galactosidase staining

National Institute for Medical Research, Mill Hill, London, United

found in wild-type dorsal telencephalon was limited to a few iso-

Kingdom

lated positive cells in the extratoes mutant. By E12.5, only a few beta-galactosidase positive patches were detected in the mutant

A few signaling molecules are repeatedly used during develop-

dorsal telencephalon in contrast to the extensive staining

ment, however little is known about how diverse cellular

observed in wild-types. These results indicate Wnt signalling is

responses are generated by an individual signal. To address this

reduced, but not completely abolished, despite the loss of the cor-

issue, we focused on Sonic Hedgehog (Shh) directed pattern for-

tical hem in the Gli3Xt/Xt telencephalon.

mation in the vertebrate neural tube. Increasing concentrations and durations of Shh signaling generate successively more ventral neuronal identities. However this mode of action is not appli-

doi:10.1016/j.mod.2009.06.555

cable to the most ventral cell type, the floor plate (FP). We show here that Shh signaling is required only transiently for its specification. In contrast to the establishment of other ventral identities, this requirement takes place prior to the neural tube

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closure. Shortly after the initiation of somitogenesis, neural pro-

Apical localization of RhoGEF2 and adherens junctions during

genitors lose their ability to generate FP. Concomitantly, Shh sig-

gastrulation in Drosophila melanogaster

naling is progressively downregulated in presumptive FP cells.

Verena Ko¨lsch1,2, Gregorio J. Fernandez-Ballester3, Luis Serrano4,

Importantly, although high levels of Shh exposure are necessary

Maria Leptin2

and sufficient for FP induction at early stages, the later downreg-

1

ulation of signaling is a prerequisite for the elaboration and main-

UCSD, San Diego, CA, United States

2

tenance of FP identity. Maintaining Shh signaling in these cells

Institut fu¨r Genetik, Universita¨t Ko¨ln, Ko¨ln, Germany

3

Instituto de Biologica Molecular y Celular, Universidad Miguel Her-

generates progenitors with a ventral neuronal identity. We further show that the transcription factor FoxA2, a direct Shh target, plays a central role in relaying the early Shh signal and does so independently of Shh signaling. Together the results suggest a clear separation in time and space between the specification of FP and ventral neural progenitors. In addition, they provide new insights into how the timing and duration of morphogen signaling is exploited to extend the patterning capacity of an individual signal.

nandez, Elche, Spain 4

Centre de Regulacio Genomica, Barcelona, Spain Gastrulation in Drosophila is driven by apical constriction of

the mesodermal cells, leading to the formation of the ventral furrow and the invagination of the mesoderm. Apical constriction requires a contractile network and involves the activation of myosin II via a G protein activated pathway involving RhoGEF2, Rho1, and Rok. Whereas RhoGEF2 is absolutely essential for furrow formation, the loss of the G protein alpha subunit Concertina (cta) or

doi:10.1016/j.mod.2009.06.554

the ligand folded gastrulation (fog) only results in inefficient gastrulation.