09-P021 Atonal regulates Rfx during chordotonal organ development in the Drosophila

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MECHANISMS OF DEVELOPMENT

1 2 6 (2 0 0 9) S1 5 1–S 18 1

newborns display a premature fusion of the bones in the cranial

09-P020

base. The anterior cranial base shows an abnormal differentia-

Nkcc1/Slc12a2 is required for the regulation of endolymph in the

tion of chondrocytes in the mutant, with reduced proliferation

otic vesicle and volume of the swim bladder in the zebrafish larva

and increased terminal differentiation. The main factors

Leila Abbas, Tanya Whitfield

responsible to restrict Six2 function to the anterior portion of the cranial base are the tissue-specific transcription of the gene and the presence of compensatory effects from other Six family members, in particular Six1 and Six4. Gain of function experiments indicate that Six2 has a general effect on endochondral bone formation and can affect cartilage development and growth in other body areas. It is likely that Six2 acts by controlling general regulators of chondrocyte differentiation. Our data suggest that Six2 acts to control locally the level of the IGF-axis.

MRC Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom Endolymph is the specialised extracellular fluid present inside the inner ear. In mammals, disrupted endolymph homeostasis can result in collapse or distension of the endolymphatic compartment in the cochlea, and concomitant hearing loss. The zebrafish little ears (lte) mutant larva shows a collapse of the otic vesicle, apparently due to loss of endolymphatic fluid in the ear over a 12- to 18-h period, together with an over-inflation of the swim bladder. Mutant larvae display signs of abnormal vestibular

doi:10.1016/j.mod.2009.06.348

function by circling and swimming upside down. The two alleles of lte are homozygous lethal: mutant larvae fail to thrive beyond 6 days post fertilisation. Patterning of the otic vesicle is apparently normal. However, expression of several genes thought to play a

09-P019 The role of the non-canonical Wnt signalling pathway in branching morphogenesis Laura L. Yates1, Carsten Schnatwinkel3, Jennifer N. Murdoch1, Debora Bogani1, Caroline J. Formstone2, Stuart Townsend1, 1

3

1

Andy Greenfield , Lee A. Niswander , Charlotte H. Dean

role in endolymph production is downregulated, including the sodium–potassium–chloride cotransporter nkcc1(slc12a2) and several Na+/K+-ATPase channel subunit genes. We have cloned the lte mutations and show that they correspond to lesions in nkcc1. Each allele has a point mutation that disrupts splicing, leading to frame shifts in the coding region, and predicting the

1

generation of truncated products. Endolymph collapse in the

Kingdom

lte/nkcc1 mutant shows distinct parallels to that seen in mouse

2

Nkcc1 mutants, validating the zebrafish as a model for the study

United Kingdom

of endolymph disorders. The collapse in ear volume can be ame-

3

Howard Hughes Medical Institute, Dept. of Pediatrics, Section of

liorated in the to27d allele by injection of a morpholino to block

Developmental Biology, University of Colorado Denver School of Medi-

splicing at the new site, forcing use of the original site. This

cine, Aurora, CO, United States

exemplifies the use of morpholinos as potential therapeutic

MRC Harwell Science and Innovation Campus, Oxfordshire, United MRC Centre for Developmental Neurobiology, Kings College London,

agents for genetic disease. The development of organs such as the lung occurs by branching morphogenesis. This developmental process has evolved to

doi:10.1016/j.mod.2009.06.350

provide maximum surface area in minimal space. Currently, the mechanisms underlying this process are poorly understood. It is known however, in addition to reciprocal signalling between epithelial and mesenchymal cells, changes in the cytoskeletal architecture, cell–cell adhesion and cell polarity are essential for the formation of new branches. Mouse mutants of three non-canonical Wnt pathway genes: Vangl2, Ceslr1 and Scribble, display disrupted branching morpho-

09-P021 Atonal regulates Rfx during chordotonal organ development in the Drosophila Lina Ma, Andrew Jarman University of Edinburgh, Edinburgh, United Kingdom

genesis in several branched organs. In the lung we observed cytoskeletal defects and explant cultures revealed mutant endoderm

One major type of neuron in the Drosophila peripheral nervous

was unable to respond to the chemoattractant FGF-10 despite an

system is type I neuron. Both chordotonal organs (Ch) and exter-

intact FGF signalling pathway.

nal sense organs (Es) have this type of neuron with a specialised

Analyses of Vangl2, Celsr1 and Scribble protein expression in

ciliary dendrite. As we know, the proneural gene, Atonal (Ato) ini-

lung epithelium revealed differences in the spatial expression

tiates the genesis of Ch organs, while Scute initiates Es organ

patterns of each protein suggesting that they have independent

development. Although the role of Ato in sense organ precursor

roles in branching morphogenesis. We find that Celsr1 constrains

(SOP) specification has been well characterised, how it regulates

the distal bud during bifurcation whereas Scribble maintains the

the subsequent events and leads to the neuron differentiation is

lumen of the bud epithelium.

still elusive. In order to reveal the gene regulation network in

This data shows a novel and important role for the non-

which Ato participates, a microarray analysis was conducted on

canonical Wnt signalling pathway in vertebrate branching

Ato-expressing cells in our lab, which gives rise to a large number

morphogenesis.

of potential Ato target genes. Rfx is in a high rank within this list. Rfx encodes a conserved transcription factor and is essential in

doi:10.1016/j.mod.2009.06.349

the formation of dendrites of type I neuron. To test the hypothesis that Rfx is a direct target of Ato, the enhancer of Rfx was located

1 2 6 ( 2 0 0 9 ) S 1 5 1 –S 1 8 1

MECHANISMS OF DEVELOPMENT

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by reporter gene analysis. Also, site-directed mutagenesis showed

of undifferentiated and differentiated avian stomachs. We iden-

the requirement for two sites which match Ato binding motif

tify Scleraxis, a basic-helix-loop-helix transcription factor, as a

within that enhancer for regulation by Ato in Ch cells. Our results

new marker of stomach mesenchyme and find that expression

also suggest that unlike Ato, Rfx is indirectly activated by Scute.

of Scleraxis defines the presence of two tendons closely associ-

Different regulation mechanisms in Ch and Es cells lead to the

ated to the two visceral smooth muscles. Using targeted gene

striking differences inRfx expression level, which may be respon-

misexpression, we show that FGF signaling is sufficient to induce

sible for their different neuronal morphologies.

Scleraxis expression and to establish two tendon domains adjacent to the smooth muscle structures. We also demonstrate that

doi:10.1016/j.mod.2009.06.351

the tendon organization is perturbed by altering Scleraxis expression or function. Moreover, using primary cells derived from stomach mesenchyme, we find that undifferentiated stomach

09-P022

mesenchyme can give rise to both SMCs and tendon cells. These

An exvivo model of placental development

data show that upon FGF activation, selected stomach mesenchy-

Didem Sarikaya, Loydie Jerome-Majewska

mal cells are primed to express Scleraxis and to differentiate into tendon cells. Our findings identify a new anatomical and func-

McGill University, Montreal, Que., Canada

tional domain in the vertebrate stomach that we characterize as being two intermuscular tendons closely associated with the vis-

The placenta facilitates nutrient and gas exchanges between the

ceral SMC structures. We also demonstrate that coordinated

mother and growing fetus. It does this through highly branched

development of both tendon and smooth muscle domains is

embryonic blood vessels called villi. In the mouse, villi start to form

essential for the correct morphogenesis of the stomach.

when the allantois attaches to the chorion. This attachment is mediated through a thin layer of mesothelium on the chorion, which

doi:10.1016/j.mod.2009.06.353

becomes indistinguishable from the allantoic cells after attachment. Abnormalities in placental development underlie human pathologies such as pre-eclampsia, miscarriages, and intra-uterine growth-retardation. The genetic and structural features of the

09-P024

human placenta are conserved in mice, and the murine placenta

The transcription factor Trps1 interacts with the activator form of

is well characterized invivo. However, there are no systems to study

Gli3 to regulate chondrocyte proliferation and differentiation

and manipulate the placenta exvivo. We hypothesized that co-cul-

Manuela Wuelling1, Frank J. Kaiser2, Diana Braunholz2,

tures of pre-attachment chorions and pre-attachment allantoises

Reinhard Depping3, Andrea Vortkamp1

will mimic early invivo events in placental development, under

1

the correct culture conditions. Chorions and allantoises of wild type mouse embryos were dissected at somite stage 3–6, and were combined and cultured for 12, 24, and 36 h exvivo. We found that under our culture conditions, the allantois attached to the chorion, and the mesothelium was indistinguishable from the allantois. We will present our morphological and molecular characterization of this exvivo model. This model will further our knowledge of early placental development as it allows for invitro manipulation of the allantois and chorions prior to culture. doi:10.1016/j.mod.2009.06.352

Center for Medical Biotechnology, University Duisburg-Essen, Essen,

Germany 2

Institute for Human Genetics, University of Luebeck, Luebeck, Germany

3

Department of Physiology, Center for Structural and Cell Biology in

Medicine, University of Luebeck, Luebeck, Germany Trps1, the gene mutated in human Tricho-Rhino-Phalageal syndrome, represents an atypical member of the GATA-family of transcription factors. Here we show that Trps1 interacts with Indian hedgehog (Ihh)/Gli3 signalling and regulates chondrocyte differentiation and proliferation. We demonstrate that Trps1 specifically binds to the transactivation domain of Gli3 invitro and invivo, whereas the repressor form of Gli3 does not interact with

09-P023 Intermuscular tendons are essential for the development of vertebrate stomach Ludovic Le Guen, Ccile Notarnicola, Pascal de Santa Barbara INSERM, ERI 25, Montpelier, Languedoc-Roussillon, France

Trps1. GST pull-down experiments were used to verify the interaction of the isolated GLI3 activator domain with TRPS1. Through the use of different truncated TRPS1 constructs, a domain of 185 aa, containing three predicted zinc fingers, was shown to be sufficient for the interaction with GLI3. Using different mouse models we find that in distal chondrocytes

University Montpelier I, EA4202, Montpelier, Languedoc-Roussillon,

Trps1 and Gli3 are required to expand distal cells and locate the

France

expression domain of Parathyroid hormone related peptide. In columnar chondrocytes Trps1 and Ihh/Gli3 signaling are required to

Gastrointestinal motility is ensured by the correct coordina-

activate chondrocyte proliferation. The differentiation of columnar

tion of the enteric nervous system and the visceral smooth mus-

and hypertrophic chondrocytes is supported by Trps1 independent

cle cells (SMC), and defective development of SMCs result in gut

of Gli3. Trps1 seems thus to organize chondrocyte differentiation

malformations and intestinal obstructions. In order to identify

interacting with different subsets of co-factors in distinct cell types.

the molecular mechanisms that control the differentiation of the visceral mesenchyme into SMCs in the vertebrate stomach, we developed microarrays to analyze the gene expression profiles

doi:10.1016/j.mod.2009.06.354