19-P019 Retinoic acid signaling in vertebrate appendage regeneration

S296

MECHANISMS OF DEVELOPMENT

1 2 6 (2 0 0 9) S2 9 1–S 30 4

Our experiments demonstrated that blastema cells do not

its expression during the growth phase of juvenile fins, indicating

acquire pluripotency since none of the grafts generated progeny that

that the activation of the smp gene is associated with a transcrip-

contributed to all limb tissues. Although we saw some relaxation of

tional program that is different from that used for the developmen-

lineage boundaries towards developmentally related lineages, the

tal outgrowth of larval and juvenile fins. Morpholino knockdown of

final fate of GFP+ blastema cells strongly reflected their origin.

smp significantly reduced regenerative outgrowth by decreasing cell proliferation as measured by BrdU incorporation and histone-

doi:10.1016/j.mod.2009.06.803

3 phosphorylation. In addition, smp knockdown increased the expression of msxb, msxc, and shh, suggesting that loss of smp de-repressed genes associated with patterning of the regenerating

19-P016

tissues. In accordance, loss of smp resulted in the formation of

Investigation of the immuno-response and signaling pathway in

ectopic bone. Taken together, these data indicate a requirement

Xenopus embryonic wound healing

for smp in fin regeneration through control of cell proliferation,

Jingjing Li

for the regulation of specific genes and for proper patterning.

Healing Foundation Centre, The University of Manchester, Manchester,

doi:10.1016/j.mod.2009.06.805

United Kingdom Wound healing is a series of complex events to heal and regenerate epidermal and dermal tissues after injury. Adults often heal their wounds with scars, while embryos can accomplish this process perfectly, without scars. Previous studies have provided some comparisons between adult and embryonic wound healings; however, details in the inflammatory phase of

19-P018 Notch signalling in colonic epithelial cells: Potential roles in inflammatory bowel disease and tissue repair David Qualtrough, Phil Rees, Massimo Pignatelli University of Bristol, Bristol, United Kingdom

wound healing, which is believed to be a main reason for scarring, still remain unknown. Based on our microarray results in

Notch signalling is required for stem cell maintenance in the

Xenopus wound healing and regeneration, I have identified some

intestinal epithelium and directs cells toward the absorptive line-

interesting genes that are up-regulated or phosphorylated by

age upon differentiation. Its role in intestinal disease is unclear.

wounding. They may responsible for the cell activation, wound

Inflammatory bowel disease (IBD) defines a set of incurable and

closure and tissue remodeling at the wound site, or the inflam-

debilitating illnesses which afflict around 2.2 million people

matory cell recruitment to the wound. My later research will

across Europe. Acute inflammation in IBD can destroy the epithe-

focus on the specific functions of these candidate genes and their

lial barrier and remission requires both reduced inflammation

signaling pathways, and try to determine whether they attribute

and adequate repair of the mucosa.

to the scarless wound healing in embryos. doi:10.1016/j.mod.2009.06.804

Tissue repair is a two-stage process consisting of a period of ‘restitution’ where epithelium at the edge of a wound flattens and migrates across the denuded area to reconstitute the epithelial barrier. Following restitution, regeneration involves rapid cell proliferation and differentiation as well as reconstruction of the

19-P017 Simplet controls cell proliferation and gene transcription during

three-dimensional tissue structure. Using immunohistochemistry we have demonstrated an up-

zebrafish caudal fin regeneration

regulation of Notch-1 expression in IBD compared with normal

Caghan Kizil1, Georg Otto1, Robert Geisler1, Christiane Nu¨ssleinVolhard1, Christopher Antos1,2

tissue. Foci of Notch expression was observed in flattened cells

1

Max-Planck Institut fu¨r Entwicklungsbiologie, Tu¨bingen, Germany

2

Center for Regenerative Therapies Dresden/Technische Universita¨t

Dresden, Dresden, Germany

undergoing restitution and in areas of regeneration. These data suggest an altered role for Notch in achieving remission in IBD. Furthermore, we have investigated the effects of 5-amino salicylate (widely used to treat IBD), and the dietary factor sodium butyrate on Notch expression and activity in vitro. Butyrate leads to rapid and sustained down regulation of Notch, whereas low

Epimorphic regeneration of new tissue requires initiating and

doses of 5-ASA stimulate Notch expression and activity. These

controlling extensive cell proliferation after partial loss of an organ

drug treatments also produce converse effects on cell motility,

or appendage. It is unclear what the molecular determinants are

which is vital for wound closure.

that coordinate cell proliferation with patterning of the tissues during tissue regeneration. We found that fam53b/simplet (smp) regu-

Taken together these data imply a vital role for Notch signalling in IBD and intestinal wound repair.

lates both cell proliferation and the transcription of specific genes that are associated with tissue patterning. In situ hybridization

doi:10.1016/j.mod.2009.06.806

and quantitative RT-PCR experiments showed that amputation of zebrafish hearts and fins resulted in expression of the smp gene. In regenerating adult fins, we observed smp expression in the distal mesenchyme which later expanded to the basal layers of the distal

19-P019

epidermis and distal tip epithelium. While we observed significant

Retinoic acid signaling in vertebrate appendage regeneration

up-regulation of the gene in regenerating tissues, we did not detect

Nicola Blum, Gerrit Begemann

MECHANISMS OF DEVELOPMENT

1 2 6 ( 2 0 0 9 ) S 2 9 1 –S 3 0 4

S297

Zoology and Evolutionary Biology, University of Konstanz, Konstanz,

for survival during homeostasis, with RNAi worms eventually lys-

Germany

ing, possibly due to a build up of excess ECM materials.

The most prominent and dramatic example of regeneration in

doi:10.1016/j.mod.2009.06.808

vertebrates is the complete reconstitution of amphibian limbs and fish fins by epimorphic regeneration. Research into the molecular regulation of this fascinating biological problem is of

19-P021

high medical interest due to the potential application in replacing

Analysis of genes involved in post-transcriptional regulation of

old or damaged tissues in humans.

gene expression in planarians

Retinoic acid (RA), a small lipophilic molecule which is synthe-

Labib Rouhana, Kiyokazu Agata

sized from dietary sources of vitamin A, plays important roles during embryonic development by modulating gene expression.

Kyoto University, Kyoto, Japan

It is well-known that treatment with RA during limb- and fin regeneration causes a number of striking morphological effects

Planarians regenerate complete organisms from small body

on the regenerate, but its functional involvement in vertebrate

fragments produced artificially or by transverse fission during

appendage regeneration has not been addressed.

asexual reproduction. The regenerative ability of planarians is

We have now provided first evidence for the requirement of

attributed to a population of stem cells (neoblasts) which com-

RA signaling for fin regeneration. We could show that RA signal-

pose approximately 30% of their body. Neoblasts can be abolished

ing pathway components as well as potential targets are strongly

entirely by X-ray irradiation, which leads to loss of regenerative

expressed after amputation in the regenerating caudal fin of zeb-

ability. Neoblasts contain large ribonucleoprotein (RNP) aggre-

rafish. Inhibition of RA signaling by using a competitive reversible

gates analogous to mRNA processing structures found in the

inhibitor of RA synthesis (DEAB) blocks regeneration of the larval

germ-line and early development of other eukaryotes. Fifty-seven

fin. Moreover, regeneration is also impaired in homozygous

planarian homologues to human RNP-granule components were

aldh1a2 (neckless) mutant larvae. To address the involvement of

cloned, and their expression analyzed by in situ hybridization.

RA signaling in adult fin regeneration, we have generated a trans-

Over 75% of the genes analyzed are preferentially expressed in

genic line which allows the temporally controlled degradation of

neoblasts, the central nervous system, or both. Functional analy-

endogenous RA. Most importantly, inhibition of RA signaling dur-

sis by RNA interference showed that some of these genes are

ing adult fin regeneration blocks the regeneration process.

required for proper regeneration, stem cell maintenance, differ-

Together, our findings demonstrate an essential role for RA

entiation and neuronal function. We are currently investigating subcellular distribution of some of these proteins and their role

signaling in vertebrate appendage regeneration.

in chromatoid body stability. Work supported by a NSF Minority Postdoctoral Research Feldoi:10.1016/j.mod.2009.06.807

lowship (0804021) to L.R., a Grant-in-Aid for Creative Scientific Research (17GS0318) to K.A. and the gCOE program A06 of Kyoto University.

19-P020 Schmidtea mediterranea’s matrix metalloproteases and their

doi:10.1016/j.mod.2009.06.809

importance for regeneration and survival Mohammed Bakkali, Aziz Aboobaker Institute of Genetics, University of Nottingham, Nottingham, United Kingdom

19-P022 The roles of Drosophila melanogaster developmental gene orthologues in Schmidtea mediterranea regeneration

One of the cornerstones of multi-cellularity is the extra-cellular matrix. Its correct organization is key to almost every aspect of the

Suthira Owlarn, Aziz Aboobaker University of Nottingham, Nottingham, United Kingdom

biology of the multi-cellular organism. Among the enzymes regulating its composition and dimensions are the matrix metallopro-

Freshwater planarians have a population of totipotent stem

endo-peptidases

cells called neoblasts which allow them to regenerate in response

biochemically regulate the composition of the extra-cellular

to traumatic injuries and to rescale according to environmental

matrix (ECM) which in turn provide information to cells affecting

conditions. Patterning is essential in both replacement and remod-

processes such as proliferation and migration. In this work, we

elling of tissues to invariably produce fully functional, proportional

identify the MMPs of the planarian Schmidtea mediterranea – a flat-

and seamless replicas of the original animal. Although the mecha-

worm notorious for its regeneration capacity. We use RNAi medi-

nisms underlying these processes are not well understood at the

ated gene knockdowns to analyse the involvement of these MMPs

molecular level, there is increasing evidence in various species that

in the regeneration process. The knockdown phenotypes have

regeneration is comparable to embryogenesis and requires reacti-

been characterized using immuno-stainings and in situ hybridiza-

vation of key developmental genes. We have isolated orthologues

tions. Only one of the four MMPs identified has a detectable effect

of Drosophila melanogaster genes known to encode developmental

on regeneration. These RNAi worms fail to regenerate possibly

transcription regulators, many of which are pivotal for embryonic

due to loss of the migration capacity of this animal’s stem cells

patterning. Using dsRNA-mediated interference to knock down

(called neoblasts). Adequate activity of this MMP is also essential

the expression of these genes, we screened for abnormalities and

teases

(MMPs).

These

zinc-dependant