05-P014 Histone deacetylase-mediated control of forebrain neurogenesis: Analyzing the role of specific HDACs through RNA interference

S116

MECHANISMS OF DEVELOPMENT

1 2 6 (2 0 0 9) S1 1 3–S 11 9

commitment and cell cycle exit in multipotent neural progeni-

Prrxl1 isoforms. Transcriptional autoregulation of Prrxl1 is in line

tors, contribute to the specification of neuronal subtype identi-

with its highly restricted expression pattern.

ties, and promote neuronal migration and differentiation. Little is known, however, on the molecular mechanisms underlying

doi:10.1016/j.mod.2009.06.217

the gene regulatory functions of proneural factors. The identity of their target genes, and how do they co-ordinately regulate the neurogenic program is not known. These issues cannot be addressed in the mouse embryo,

05-P013

where the developing nervous system contains a mixture of cells

Analysis of chromatin organization of Hox gene cluster by using

at different developmental stages and with different fates. Neural

3C-based technique

stem cell lines derived from embryonic stem cells or primary neu-

Hyehyun Min, Ji-Yeon Lee, Myoung Hee Kim

ral progenitors represent an excellent model for studying neurogenesis in culture. We have engineered inducible versions of Mash1 and Ngn2, by fusing them to a modified version of the

Department of Anatomy, Embryology Laboratory, Brain Korea 21 project Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea

ligand binding domain of the estrogen receptor (ERT2). These quimeric proteins can only induce proneural target genes when acti-

Spatially and temporally programmed expression of the Hox

vated by the ERT2 ligand tamoxifen. When applied to the NS5 cell

genes along the antero-posterior (A-P) anterior–posterior (A–P)

line, this inducible system allows for the efficient generation of

axis is essential for correct pattern formation during embryonic

Mash1/Ngn2 induced neuronal progenitors that differentiate into

development. However, the precise mechanisms controlling the

mature neurons in a synchronized manner.

spatio-temporal expression of Hox genes during development

We are using a combination of locations analyses and expres-

are not fully understood. Accumulating evidence emphasize

sion profiling in order to characterize the transcription program

the importance of intra- and inter-chromosomal interaction

activated by Mash1/Ngn2 in differentiating NS5 cells and to gain

(’’DNA (‘‘DNA looping’’) between regulatory elements and their

novel insights into the molecular mechanisms underlying the

target genes as a beneath mechanism for gene regulation in

function of proneural factors.

the third dimension. Recently, chromosome conformation capture (3C) and its derivative techniques have been developed

doi:10.1016/j.mod.2009.06.216

and opened a new way to study chromosomal folding in the nucleus. To explore genome-wide chromosomal interactions with high resolution, we are now using circular 3C method,

05-P012 Evidence for autoregulation of the homeodomain transcription factor Prrxl1 Filipe Almeida Monteiro1, Carlos Reguenga1, Francois Guillemot2, Diogo Sampaio Castro2, Deolinda Lima1

termed 4C, which allows identification of regions throughout the genome that are physically close to a single locus of interest. In this study, we focused our analysis on mapping of chromatin interactions specific to upstream enhancer sequences of Hoxc8 and Hoxa7, well known regulatory elements specifying expression boundaries, during invitro induction of their expression in

1

Laboratory of Molecular Cell Biology, Faculty of Medicine of Porto and

F9 murine embryonic cells. Here we will present the dynamic

Morphophysiology of the somatosensory System, Instituto de Biologia

pattern of interactions linked with the active transcription sta-

Celular e Molecular, University of Porto, Porto, Portugal

tus of the Hox genes.

2

Division of Molecular Neurobiology, National Institute for Medical

Research, Mill Hill, London, United Kingdom

This work was supported by Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MEST). (grant (MEST) (Grant code: 7-2009-0034)7-2009-0034).

Prrxl1 is a paired-like homeodomain (HD) transcription factor involved in the development/maintenance of both peripheral and

doi:10.1016/j.mod.2009.06.218

spinal cord dorsal horn nociceptive neurons, dedicated to the processing of pain input. Contrary to the broad knowledge on the defects displayed at the cellular level by the absence of Prrxl1, very little is known about the transcriptional network downstream this

05-P014

factor. Dragon, a member of the repulsive guidance molecule-

Histone deacetylase-mediated control of forebrain neurogenesis:

related family that is co-expressed with Prrxl1 in embryonic DRG

Analyzing the role of specific HDACs through RNA interference

and spinal cord, is so far the only well established Prrxl1 target

Kathrin Weissmueller, Kerry Lee Tucker

gene. Here we show, by the use of in silico analysis, that the tran-

University of Heidelberg, Heidelberg, Germany

scription factor binding sites on Prrxl1 proximal promoter present a conserved HD putative binding site, suggesting the existence of

Histone deacetylases (HDACs) are a family of chromatin-mod-

an auto-regulatory mechanism controlling Prrxl1 expression. In

ifying enzymes that remove acetyl groups from the lysine tails of

vitro DNA pull-down assays using ND7/23 neuronal cell line over-

core histones, leading to modifications of gene transcription.

expressing Prrxl1 show that Prrxl1 interacts with its own promoter.

They have recently been associated with the control of both

This same interaction was confirmed invivo by chromatin immu-

neuro- and astrogliogenesis. Here, the role of specific HDACs in

noprecipitation (ChIP) performed on mouse dorsal spinal cord tis-

the development of the murine brain is adressed. addressed.

sue at embryonic age 14.5 (E14.5). Gain-of-function experiments in

Pharmacological inhibition of class I and II HDACs with trichosta-

ND7/23 cells reveal differential autoregulatory expression by two

tin A in invitro-differentiating neural precursors derived from

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1 2 6 ( 2 0 0 9 ) S 1 1 3 –S 1 1 9

MECHANISMS OF DEVELOPMENT

embryonic striatum led to a dramatic reduction in neurogenesis

05-P016

(Shaked etal., PLoS ONE 2008). To further investigate this finding,

DNA

several different approaches have been taken. First, the expres-

mediterranea

sion of HDAC-regulated candidate genes responsible for control-

Priscilla Lo, Farah Jaber, Aziz Aboobaker

methylation

in

the

freshwater

planarian

Schmidtea

ling neurogenesis has been examined through quantitative realtime RT-PCR. TSA-based inhibition of HDAC activity resulted in

Institute of Genetics, University of Nottingham, Nottingham, United

upregulation of Bmp2, Stat1, Stat3, Ngn1 and Ngn2 in neural pre-

Kingdom

cursors from both cortex and striatum. In contrast, the expression of Smad7, an inhibitor of BMP2/4 signaling, decreased as a result of HDAC inhibition. This suggested that HDACs control neurogenesis by inhibiting the BMP2/4 signaling pathway, with additional involvement of JAK/STAT signaling and the Ngn transcription factors. Second, in order to ascertain which HDAC family

members

influenced

neurogenesis,

the

expression

of

individual HDAC genes was inhibited via RNA interference. Expression of HDAC 1, 2, 4 and 5 could be effectively downregulated, as seen on the mRNA level. The effect of the knockdown on neurogenesis and astrogliogenesis will be elucidated. Third, the expression patterns of individual HDACs in developing brain tissue will be presented. doi:10.1016/j.mod.2009.06.219

The freshwater planarian Schmidtea mediterranea can regenerate missing body regions following wounding or amputation. Both planarian tissue homeostasis and regeneration depend on a population of pluripotent somatic stem cells called neoblasts; however, the signals required for neoblast differentiation are unknown. Recent studies show that epigenetic mechanisms such as DNA methylation can direct the terminal fate of stem cells during differentiation. DNA methylation is somatically heritable, but it can be altered during development and therefore may play a role in regulating differentiation of planarian neoblasts. In a screen to identify candidate genes involved in DNA methylation, we identified a planarian DNA methyltransferase (Smed-dnmt2) and a planarian methyl-binding domain protein (Smed-mbd2/3). Their expression patterns were characterised by insitu hybridization on intact and regenerating worms. Their functions were ascertained by RNAi and observed during tissue homeostasis and regeneration following amputation. Smed-mbd2/3 was

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expressed in the region containing proliferating neoblasts of

Developmental regulatory targets of the homeobox transcription

regenerating worms and found in the posterior midline of both

factor HESX1

regenerating and non-regenerating worms. Smed-mbd2/3 knock-

Nicoletta Charolidi1, Cynthia, L. Andoniadou1, Massimo Signore1, Richard, G. Jenner2, Juan Pedro Martinez-Barbera1

down led to a delay in regeneration and arrest in the formation of the brain and photoreceptors. Long term Smed-mbd2/3 RNAi

1

affected neoblast self-renewal. Our results suggest that Smed-

2

mbd2/3 may be involved in neoblast fate specification during

Institute of Child Health, UCL, London, United Kingdom Centre for Medical Molecular Virology, MRC/UCL, London, United

Kingdom

differentiation, and further investigation using BrdU staining is required. No specific role is yet defined for Smed-dnmt2. DNA

The homeobox gene Hesx1 codes for a transcriptional repres-

methylation detection assays such as bisulfite sequencing, diges-

sor that is expressed during mouse development in the prospec-

tion of DNA with methylation sensitive restriction enzymes, and

tive forebrain and Rathke’s pouch, the primordium of the

5-aza-2-deoxycytidine treatment will provide further information

anterior pituitary. Work from our laboratory has shown that

about methylation patterns in S. mediterranea genome.

Hesx1 plays a role in the normal patterning of anterior forebrain by preventing the activation of posterior neural markers. In the

doi:10.1016/j.mod.2009.06.221

anterior pituitary, Hesx1 has been shown to regulate normal cell proliferation of pituitary progenitor cells. Several lines of evidence suggest that Hesx1 is required to antagonize the activation of Wnt/beta-catenin signalling. The interaction of HESX1 with several co-repressors and with DNMT1 (DNA Methyltransferase 1) has been proposed to underlie HESX1 molecular activ-

05-P017 Epigenetic regulation of winged eye capable of inducing transformation in Drosophila

ity. To date, however, HESX1 target genes remain essentially

Rumi

unknown. To gain further knowledge into the molecular func-

Yoshiteru Oshima1, Shoichiro Kurata1

tion of HESX1, we have combined microarray gene expression profiling with chromatin immunoprecipitation-on-chip technology for the identification of direct HESX1 genetic targets. For this analysis we have used embryonic stem cells, where Hesx1 is normally transcribed, translated and imported into the nucleus. The role of DNA methylation in normal forebrain and pituitary gland development is being studied by combining molecular with genetic approaches. Data from these analyses are presented here. doi:10.1016/j.mod.2009.06.220

Nakajima1, Tomonori Katsuyama1,2, Jun Terashima1,

1

Grad. School Pharm. Sci, Tohoku Univ., Sendai, Japan

2

ETH Zurich, Basel, Switzerland In Drosophila, Drosophila, the forced activation of Notch signal-

ing induces various ectopic appendages in the eye field in a context-dependent manner. We previously identified winged eye (wge), the overexpression of which induced ectopic wings on the head. WGE localized on polyten chromosomes, and almost all Posterior sex combs (PSC) that one of Polycomb group (PcG) protein were coincident with some WGE binding sites. The analysis of genetic interaction between wge and PcG genes revealed that wge antagonizes PcG genes. Surprisingly, wge also antagonizes tri-