03-P090 Visualising plant growth and shape in 3D using optical projection tomography

MECHANISMS OF DEVELOPMENT

1 2 6 ( 2 0 0 9 ) S 6 7 –S 1 0 6

S93

ferent time points during forelimb development and that its activ-

We have explored the use of optical projection tomography

ity is tightly regulated. We are exploring the potential post-tran-

(OPT) as a method for capturing 3D morphology and gene activity

scriptional and/or post-translational mechanisms that regulate

at a variety of developmental stages and scales from plant spec-

Tbx5 protein activity during forelimb development.

imens, in collaboration with the Medical Research Council, James Sharpe and Bioptonics. OPT can be conveniently applied to a

doi:10.1016/j.mod.2009.06.141

wide variety of plant material including seedlings, leaves, flowers, roots, seeds, embryos and meristems. At the highest resolution large individual cells can be seen in the context of the

03-P089

surrounding plant structure. 3D domains of gene expression

A simple leaf with compound gene expression: Indeterminate

can be visualised using either marker genes such as b-glucuron-

leaves co-express ARP and KNOX genes

idase, or more directly by whole-mount in situ hybridization. To

Kanae Nishii , Michael Moeller , Catherine Kidner ,

interactively analyse and quantify 3D OPT data we are develop-

Alberto Spada3, Raffaella Mantegazza3, Hao-Chun Hsu1,

ing software using haptics to accurately place points on volumes

Toshiyuki Nagata4, Chun-Neng Wang1

in 3D space. These tools will enable us to create 3D statistical

1

2

2

shape models to analyse phenotypic variation in Arabidopsis

1

National Taiwan University, Taipei, Taiwan

2

leaves. For naturally semi-transparent structures, such as roots,

Royal Botanic Garden Edinburgh, Edinburgh, United Kingdom

3

live 3D imaging using OPT is possible. 3D gene expression pat-

Milan University, Milan, Italy

4

terns in living transgenic plants expressing fluorescent GFP

Hosei University, Tokyo, Japan

markers can also be visualised by OPT. We are using GFP marked trichomes to track leaf growth in 4D, by obtaining OPT time-

The rosulate acaulescent Streptocarpus rexii produces unequal

course data for Arabidopsis plants growing in the OPT device.

cotyledons (anisocotyly), the larger of which (macrocotyledon)

Computer vision techniques are being developed to analyse

grows continuously via a basal meristem (BM), and produces fur-

sequential OPT datasets. The combination of 4D time-course

ther leaves from the groove meristem (GM) on the petiole. On the

data, 3D point-placing, trichome tracking and modelling will

contrary, the caulescent S. glandulosissimus also exhibits anisoco-

allow us to understand mechanisms controlling growth and

tyly, but develops opposite decussate pairs of simple leaves from

shape from earliest stages of leaf growth to maturity.

a shoot apical meristem (SAM). In Arabidopsis ARP genes are expressed in leaf primordia, and negatively regulate KNOX1 (BP1) genes that are expressed in the

doi:10.1016/j.mod.2009.06.143

SAM. Another KNOX1 gene, STM, is also expressed it the SAM and negatively regulates ARP. To characterize the underlying genetics of Streptocarpus meristem and leaf development, we com-

03-P091

pared the expression patterns of KNOX1 (STM, BP) and ARP homo-

COPII-dependent vesicular traffic is necessary for normal eye

logs in the two Streptocarpus morphs. Our results showed that

development in zebrafish

KNOX1 and ARP genes are all co-expressed in the GM/SAM, and

Katy Schmidt, Yi Feng, David Stephens

in the BM macrocotyledons, and in the proximal region of foliage leaves in both, acaulescent and caulescent, Streptocarpus.

University of Bristol, Bristol, United Kingdom

Additionally, leaf morphology, venation, cell size measurements, Aniline Blue staining and Histone 4 expression were used

Eye morphogenesis covers lamination, epithelial differentia-

to determine the meristematic phases of leaves. The results show

tion and interdependence of cellular differentiation during devel-

that both acaulescent and caulescent Streptocarpus, possessed an

opment. Retinal pigment epithelium and photoreceptors are

extended basal leaf meristem activity.

known to interrelate during development and function and in

Our results thus indicated that, as no difference in gene

both cases vesicular traffic is of major importance. Here we show

expression between the two morphs was detected, KNOX1 and

that the outer layer of the COPII coat is necessary for eye develop-

ARP genes are not responsible for the gross morphological differ-

ment in zebrafish. The COPII complex coordinates cargo collec-

ences between acaulescent and caulescent Streptocarpus species.

tion, vesicle formation and transport from ER to Golgi and

However, our data suggest that an extended basal meristem activ-

consists of an inner, cargo-binding layer (Sec23/24) and an outer,

ity seems to require the co-expression of these genes.

structural layer (Sec13/31). We have shown previously that depletion of Sec13 leads to concomitant loss of Sec31 - hence the entire

doi:10.1016/j.mod.2009.06.142

outer coat and is necessary for transport of large cargo resulting in chondrogenesis defects in zebrafish (Townley et al., 2008). Sec13 morphant larvae also show microphtalmia with a protrud-

03-P090 Visualising plant growth and shape in 3D using optical projection tomography Karen Lee1, Johann Strasser1,2, Jerome Avondo1,2, Paul Southam1,2, Andrew Bangham1,2, Enrico Coen1

ing lens at five days of development (5dpf). Whereas the lens matches control fish even on an ultrastructural level, the retina and retinal pigment epithelium are grossly disorganised. Electron micrographs show missing outer segments of photoreceptors. This phenotype occurs prior to 3dpf on a cellular level and manifests itself morphologically at 4dpf. In ongoing experiments we

1

John Innes Centre, Norwich, United Kingdom

investigate the identity of cells present in the morphant eye and

2

University of East Anglia, Norwich, United Kingdom

the transport of rhodopsin in photoreceptors, and of collagen in