11-P010 Zebrafish and medaka: Model organisms for a comparative developmental approach of brain asymmetry

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MECHANISMS OF DEVELOPMENT

1 2 6 (2 0 0 9) S1 8 4–S 18 8

duplication after the radiation of the protochordate lineages. In

4

developmental terms, the data presented here suggest that devel-

Santiago, Chile

opmental gene loss was highly significant as well.

5

doi:10.1016/j.mod.2009.06.443

Institute of Nutrition and Food Technology, University of Chile, Center for Mathematical Modeling, University of Chile, Santiago, Chile Asymmetry is a conserved and fundamental feature of the

brain, however, little is known of the genetic mechanisms that establish asymmetry during development. Here we used zebrafish to identify novel genes involved in asymmetric brain morphogen11-P007

esis using a reverse genetic approach. We isolated differential

Mechanisms of left–right asymmetric signal generation around

expressed sequences enriched in either left (L) or right (R) adult

the node

brain tissue using subtractive hybridisation technology. These

Aiko Kawasumi1, Naomi Iwai2, Jose´ Anto´nio Belo3,

sequences were employed as probes to hybridise commercial zeb-

Tetsuya Nakamura1, Hidetaka Shiratori1, Hiroshi Hamada1

rafish genetic libraries from which 132 differential clones were

1

Osaka University, Osaka, Japan

identified and amplified by PCR to construct a differential and

2

University of Texas, Texas, United States

3

University of Algarve, Faro, Portugal Despite recent progress in understanding of how left–right (L–

R) asymmetry is generated during vertebrate development, many important questions still unanswered. One such question concerns the mechanism by which the signal responsible for the generation of L–R asymmetry is transferred from the node to the lateral plate. This signal, whose identity remains unknown, is generated in the node, and its arrival in the left lateral plate

non-redundant custom microarray. Of these clones, 41% corresponded to genes of well-known function and 15% were unknown. Asymmetric expression is currently being tested by in situ hybridisation in embryos and adult brain tissue. We have already found clone P5A3 as a novel gene with asymmetric expression in the epithalamic habenular region from larval to adult stages. P5A3 loss and gain of function analyses indicates a role in asymmetric habenular morphogenesis. doi:10.1016/j.mod.2009.06.445

induces the asymmetric expression of Nodal. Although it is known that L–R asymmetry-breaking event in the mouse embryo is the leftward fluid flow (nodal flow) on the node, it is unclear

11-P009

what kinds of molecules and signals become asymmetric around

The roles of Tbx5 and Tbx4 in limb bud initiation and symmetry

the node due to nodal flow.

Fatima Sulaiman, Malcolm Logan

We found that the transcriptional regulatory element, Asymmetric Node Enhancer (ANE), which is located within the 7.5-kb upstream region of human LEFTY-1, could direct left side specific enhancement of reporter gene expression in the node at 1-somite stage, shortly after the beginning of nodal flow. To identify the molecules and signals responsible for the asymmetric activity of ANE, we explored ANE activities in several types of L–R mutant embryos, in which asymmetric Nodal expression in LPM is abnormal. There was a good correlation between the laterality of ANE activity and that of Nodal expression in the LPM. Our results suggest that ANE responds to the L–R asymmetric signals responsible for L–R axis determination. In addition, we want to show how these players interact with each other leading to generate L–R asymmetric signal in the node. doi:10.1016/j.mod.2009.06.444

National Institute for Medical Research, London, United Kingdom Tbx5 and Tbx4 are two closely related transcription factors that are expressed in the forelimb and hindlimb, respectively. Both genes are thought to play equivalent roles in the initiation of the forelimb bud and the hindlimb bud. Deletion of Tbx5 from the presumptive forelimb area leads to a failure of the limb bud to form. However, following deletion of Tbx4 in the presumptive hindlimb area, a hindlimb bud still forms, although it fails to grow. I am studying this phenotype in order to elucidate what factors may be compensating for the absence of Tbx4, and am consequently exploring this. Haploinsufficiency of TBX5 in humans is associated with a congenital disorder known as Holt Oram Syndrome. Clinical characteristics of HOS include heart defects and upper limb abnormalities. A striking feature of the limb defects is left sided asymmetry. Using mouse models in which we have disrupted activity of Tbx5, we have recapitulated the left sided bias of limb asymmetry. We are currently using this mouse strain and others to uncover the

11-P008

origins of asymmetry seen in this limb phenotype.

Identification of genes with asymmetric expression in the zebrafish brain

doi:10.1016/j.mod.2009.06.446

Alicia Colombo1, Marina Mione2, Iskra Signore1, Felipe Olivares4, Ramo´n Pe´rez1, Mauricio Gonzalez4, Andre´s Aravena5, Stephen Wilson3, Miguel Concha1 1

Laboratory of Experimental Ontogeny - LEO, ICBM, University of Chile,

11-P010 Zebrafish and medaka: Model organisms for a comparative

Santiago, Chile

developmental approach of brain asymmetry

2

Instituto FIRC di Oncologia Molecolare, Milan, Italy

Iskra A. Signore1, Ne´stor Guerrero1, Felipe Fredes1, Felix Loosli2,

3

Department of Cell and Developmental Biology, UCL, London, United

Alicia Colombo1, Joachim Wittbrodt3, Miguel Concha1

Kingdom

MECHANISMS OF DEVELOPMENT

1

Laboratory of Experimental Ontogeny - LEO, ICBM, University of Chile,

Santiago, Chile 2

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node, turning and heart looping occur normally suggesting that these events do not require Nodal at the node.

Institute of Toxicology and Genetics, Eggenstein-Leopoldshafen,

Germany 3

1 2 6 ( 2 0 0 9 ) S 1 8 4 –S 1 8 8

doi:10.1016/j.mod.2009.06.448

EMBL, Heidelberg, Germany Comparison between related species is a successful approach

to uncover conserved and divergent principles of development. Here we studied the pattern of epithalamic asymmetry in zebrafish and medaka, two related teleost species with 115–200 mil-

11-P012 Left/right axis specification in the cilia mutant talpid3 Jamie Pinkham, Lynn McTeir, Megan Davey

lion-year of independent evolution. We found that these

The Roslin Institute and R(D)SVS, University of Edinburgh, Edinburgh,

species share a strikingly conserved overall pattern of asymme-

United Kingdom

try in the parapineal–habenular–interpeduncular system. Nodal signalling precedes the left-sided asymmetric positioning and

Talpid3 chicken embryos lack cilia (Yin et al., 2009) and have

connectivity of the parapineal organ, the enlargement of neuro-

severe defects associated with a loss of cilia and corresponding

pil in the left habenula compared to the right habenula and the

loss of Hedgehog signalling such as polydactyly and neural tube

segregation of left–right habenular efferents along the dorso-

and brain patterning defects. However, talpid3 embryos do not

ventral axis of the interpeduncular nucleus. Despite the overall

have heart looping defects suggesting L/R axis specification is

conservation of asymmetry, we observed heterotopic changes

normal despite the requirement for Hedgehog signalling in L/R

in the topology of parapineal efferent connectivity, heterochronic

specification. We have investigated the possibility that cilia may

shifts in the timing of developmental events underlying the

still be present in early talpid3 embryos by examining presence

establishment of asymmetry, and divergent degrees of canalisa-

of cilia in wt and talpid3 embryos at stage 4HH using immunohis-

tion of embryo laterality. We are currently testing hypotheses of

tochemistry. We have also examined the expression of Hedgehog

inter-species variation that link the topology of parapineal effer-

responsive genes and other genes involved in L/R specification in

ent connectivity to the underlying organisation of domains

talpid3 embryos in order to clarify the role of Hedgehog signalling

within the left habenula, and are also expanding our develop-

in L/R specification in the developing chicken.

mental time comparison to a broader ontogenic context.

Supported by the BBSRC.

Together, these findings highlight the usefulness of zebrafish and medaka as comparative tools to study the developmental mechanisms of epithalamic asymmetry in vertebrates.

doi:10.1016/j.mod.2009.06.447

doi:10.1016/j.mod.2009.06.449

11-P013 Gene expression in WT and Pkd2 mutant mouse embryos Petra

Pennekamp, Stefan Feldner, Gerrit Randau, Nadia Koo,

Peter Wieacker, Bernd Dworniczak 11-P011 The role of Nodal in embryo turning and heart looping Kerry-lyn Riley, Dominic Norris MRC Harwell, Didcot, Oxfordshire, United Kingdom

Institute of Human Genetics, UKM, Muenster, Germany The development of an invariant left–right (LR) asymmetry of the visceral organs is a fundamental feature of vertebrate embryogenesis. Failure to establish the normal organ position

Although mammals appear bilaterally symmetrical externally,

(situs solitus) may result in a mirror-image reversal (situs inver-

the placement and patterning of the visceral organs demon-

sus), in left or right isomerism or in heterotaxia (situs ambigu-

strates left–right asymmetry. The signalling molecule Nodal is

ous). While the complete situs inversus does not have adverse

thought to play a definitive role in specifying the left side of

impact on the organisms, heterotaxia frequently is accompanied

the embryo, where it is expressed in the both node and left lat-

by fatal malformations and complex cardiac and cardiovascular

eral plate mesoderm (LPM). It is assumed that left sided expres-

defects. Increasing evidence based on research on genetically

sion of Nodal controls the direction of embryo turning and

modified animals including our Pkd2 knockout mouse suggest,

heart looping. To determine whether this is the case, mutants

that the disturbance of proper LR axis development and thus mis-

expressing varying levels of Nodal have been analysed. The iv

alignment of the developing heart tube is one of the major causes

mutant shows randomised LPM expression of Nodal (either left

for the development of congenital heart disease (CHD) resulting

or right sided) and has a 50/50 chance of a reversed left–right

in early prenatal death in most cases and it is very likely that

axis. Mutants at the Nodal locus can give embryos lacking Nodal

the same mechanisms are responsible in large part for early abor-

from the node, and LPM or show very low LPM Nodal expression.

tions in the first trimester of pregnancy in humans.

In the majority of mutants analysed, both turning and heart

To identify and to characterize novel genes and mechanisms

looping occur in the same direction, indicating that these events

which influence LR axis development in vertebrates and thus might

are coordinated, even in the absence of LPM Nodal expression.

be involved in the development of congenital cardiac defects we

This suggests that other, Nodal independent, asymmetric signals

performed a gene expression analysis of right and left body halves

must be involved. In embryos lacking Nodal expression in the

of 2–3 somite stage male WT and Pkd2 mutant mouse embryos