Kyoto
Takatsuki

Osaka

https://www.brh.co.jp/en/https://www.brh.co.jp/en/Our institute has tied with
Graduate School of Science, Osaka
University for more than 20 years.
Laboratory of Biohistory

Search bybrh spider

Morphological diversity in animalsⒸHiroki Oda

Present

Morphological diversity in animalsⒸHiroki Oda

Present

Past

Depth of timeMorphological diversity in animalsⒸHiroki Oda

Present

How can we know the mechanisms behind the diversity?

Past

Depth of timeMorphological diversity in animalsⒸHiroki Oda

Present

How can we know the mechanisms behind the diversity?

Pastwithmutations

Depth of time

Genetic information(genome)Morphological diversity in animals

PresentⒸHiroki Oda

Model species

How can we know the mechanisms behind the diversity?

Past
withmutations

Depth of time

Genetic information(genome)Morphological diversity in animals

PresentⒸHiroki Oda

Model species

Arthropods
How can we know the mechanisms behind the diversity?

Past
with
mutations

Depth of time

Genetic information(genome)Morphological diversity in animals

PresentⒸHiroki Oda

Model species

Arthropods
How can we know the mechanisms behind the diversity?

Past
with
mutations

Depth of time

Genetic information(genome)

Morphological diversity in animals
Model speciesⒸHiroki Oda

PresentArthropods
How can we know the mechanisms behind the diversity?
What happened
to the genome? Past
with
mutations

Depth of time

Genetic information(genome)Morphological diversity in animals
Model speciesⒸHiroki Oda

Present

Arthropods
How can we know the mechanisms behind the diversity?
What happened
to the genome? Past
with
mutations

Depth of time

Genetic information(genome)

The common house spider
Parasteatoda tepidariorumⒸHiroki Oda

The spider genome has been already sequenced!

The common house spider
Parasteatoda tepidariorum

Oda and Akiyama-Oda (2020) EvoDevo 11 CC BY 4.0

The common house spider
Parasteatoda tepidariorum

Oda and Akiyama-Oda (2020) EvoDevo 11 CC BY 4.0

Parasteatoda tepidariorum

Oda and Akiyama-Oda (2020) EvoDevo 11 CC BY 4.0

The common house spider
Parasteatoda tepidariorum

200-300 eggs in an egg sacⒸHiroki Oda

The common house spider Parasteatoda tepidariorum
Oda and Akiyama-Oda (2020) EvoDevo 11 CC BY 4.0
St 2

1st axis formation

St 3Embryogenesis(Germ disc formation)
St 4Egg laying

St 51dLifecycle> ~60d2dAxis growth &segmentation
St 8100 μmEggs

Hatching eggs
Female(Germ band formation)

St 7Male2mm

2nd axis formation

St 93d50 μm

Adults6d

Postembryos

Juveniles 1st instar

7d
100 μm10d

Arthropod phylotypic stage

Shared characteristics of arthropod body structures
formed in early embryos

Body Axes
anterior-posterior/dorsal-ventral

Repetitive Unitscalled segments

Live embryo of the spider
Parasteatoda tepidariorum
Spherical
symmetry

Radialsymmetry

Bilateralsymmetry

Akiyama-Oda and Oda (2006) Development 133 CC-BY

Three experiments that can affect body axes formation
1.

2.

3.

Three experiments that can affect body axes formation
1. Laser ablation of cells in a specific small region of spider embryo

2.

3.

Kill cells in a target region of spider embryo
Laser ablation system+

Applied laser kill cells
Oda and Akiyama-Oda (2020) EvoDevo 11 CC BY 4.0

If you kill the entire cumulus cells at the center of the disc,the formation of the body axes is delayed
Normal development

Affected development

Oda et al. (2020) Development Genes and Evolution 230 CC BY 4.0

Three experiments that can affect body axes formation
1. Laser ablation of cells in a key small region of spider embryo

Delayed development of the body axes
2.

3.

Three experiments that can affect body axes formation
1. Laser ablation of cells in a key small region of spider embryo

Delayed development of the body axes2.

Formation of twinned embryos
3.

Formation of twinned embryos

Three experiments that can affect body axes formation
1. Laser ablation of cells in a key small region of spider embryo

Delayed development of the body axes2.

Formation of twinned embryos
3.

Formation of twinned embryos

Jumping spider
Hasarius adansoniⒸHiroki Oda

Jumping spider
Hasarius adansoniⒸHiroki Oda

Jumping spider’s eggs

1 mm

ⒸHiroki Oda

House spider’s eggs

Embryogenesis of the jumping spider
Hasarius adansoni

Oda et al. (2020) Development Genes and Evolution 230 CC BY 4.0

The cumulus was graftedto the opposite side of embryo

Oda et al. (2020) Development Genes and Evolution 230 CC BY 4.0

The cumulus was graftedto the opposite side of embryo
Partially twinned embryos were formed

Oda et al. (2020) Development Genes and Evolution 230 CC BY 4.0

Three experiments that can affect body axes formation
1. Laser ablation of cells in a key small region of spider embryo

Delayed development of the body axes
2. Transplantation of the key cells to the opposite side of embryo

Formation of twinned embryos
3.

Formation of twinned embryos

Laser ablation of a large region that
separates the left- and right-side fields
Laser ablation system+Oda and Akiyama-Oda (2020) EvoDevo 11 CC BY 4.0

Laser ablation of a large region that
separates the left- and right-side fields
Partially twinned embryos were formed

By Iwasaki

Oda et al. (2020) Development Genes and Evolution 230 CC BY 4.0

Each separated embryonic field self-regulates
to recover the bilateral symmetry
dorsalSingleembryonic field

posterioranterior
ventraldorsalEach separated embryonic field self-regulatesto recover the bilateral symmetry
dorsalRight-side
fieldSeparated
embryonic fields

posterioranterior
ventralLeft-side
field

dorsalEach separated embryonic field self-regulates
to recover the bilateral symmetry
dorsalanterior

ventraldorsalposteriordorsalventraldorsalanterior

Three experiments that can affect body axes formation
1. Laser ablation of cells in a key small region of spider embryo

Organizer

Delayed development of the body axes
2. Transplantation of the key cells to the opposite side of embryo

Formation of twinned embryosOrganizer

3. Laser ablation of cells in a large region to separate the left and right

Formation of twinned embryos Self-regulation

Identification of genes involved inthe formation of the body axes in the spider

Parental RNA interference, pRNAi, is a powerful technique in the spider
Mating0Day after 1st injection
1020

dsRNA injection
Inject ~1−2 μl of dsRNA
solution (1−2 μg/μl) fortarget gene multiple times

Egg laying

RNAi effect+++

Oda and Akiyama-Oda (2020) EvoDevo 11 CC BY 4.0++++++

You can see the full version of this video at https://www.brh2.jp/

Parental RNA interference, pRNAi, is a powerful technique in the spider
Mating0Day after 1st injection
1020dsRNA injection
Inject ~1−2 μl of dsRNA
solution (1−2 μg/μl) fortarget gene multiple times

Egg laying

RNAi effect+++

+++

+++

Oda and Akiyama-Oda (2020) EvoDevo 11 CC BY 4.0

pRNAi for hedgehog

pRNAi for gfp (control)

Akiyama-Oda and Oda (2010) Development 137

Easy to analyze gene functions

Parental RNAi that affects the body axes
sognormalgfp

Delta

Akiyama-Oda and Oda (2006) Development 133
Oda et al. (2007) Development 134

gfp

hedgehog

patchedAkiyama-Oda and Oda (2010) Development 137

Morphogens operate
inside the cell
outside the cells
vsThe centerof germ disc

Posterior

Anterior
Bicoid（transcription factor）

Hedgehog（secreted protein）

Syncytial

Cell-based

Akiyama-Oda and Oda (2010) Development 137
Kanayama et al. (2010) Arthrop. Struct. Devlop. 39

Basic characteristics of arthropod body structures
formed in early embryos

Body Axes
anterior-posterior/dorsal-ventral

Repetitive Unitscalled segments

Diversity of dynamic processes generating periodic stripes(reconstructed by staining of sibling spider embryos)
headstripe-splitting

thoraxOpisthosoma
oscillations

Hemmi et al. (2018) Dev. Biol. 437, 84-104 (CC BY 4.0)

Opisthosoma
oscillations

Hh signaling plays a role in the formation of the total polarity
in the spider embryo
before its components appear to function as segment polarity genes.

Akiyama-Oda and Oda, Sci. Adv. 6 (2020) CC BY 4.0
Double negative regulation and negative feedback

hhptc

smociXYpRNAi and RNA-seq enables genome-wide gene discovery
Late stage 5Day -2
Day 0Day 8EggControl embryos

RNAhh dsRNA injection, 1sthh dsRNA injection, 2ndhh dsRNA injection, 3rd
hh dsRNA injection, 4thRNA-seq

CompareDay 15Day 20

hh pRNAi embryos

RNARNA-seq

注射後の日数

RNAi effectAkiyama-Oda and Oda, Sci. Adv. 6 (2020) CC BY 4.0

Genome-wide identification of hedgehog signaling targets
normalhh pRNAiptc pRNAi

pRNAi for hh and ptc
Egg laying
Identify differentially expressed genes byRNA sequencing

hh pRNAi embryos
ptc pRNAi embryos
Positivelyregulated by hh

Negativelyregulated by hh

ptc pRNAihh pRNAicilabotdopa

98 genes

msx1 gene

Akiyama-Oda and Oda, Sci. Adv. 6 (2020) CC BY 4.0

Embryo shows dynamic msx1 expression giving rise to spatial periodicity

Akiyama-Oda and Oda, Sci. Adv. 6 (2020) CC BY 4.0

time

ci

Delta

All dynamic processes that generate spatial periodicity are blocked by msx1 knockdown
msx1
pRNAi
NormalAkiyama-Oda and Oda, Sci. Adv. 6 (2020) CC BY 4.0

All dynamic processes that generate spatial periodicity are blocked by msx1 knockdown
msx1pRNAi
Normalmsx1 gene suppression in cell clones（see arrows）
HeadThorax

Opisthosoma

splittingsubdivisionoscillation

msx1 RNAiin cell cloneci

Delta

normalAkiyama-Oda and Oda, Sci. Adv. 6 (2020) CC BY 4.0

What differs between fly and spider?
Insect (fruit fly)

Chelicerate (spider)

Gene expression behaves like waves

Temporal repetition gives riseto spatial repetition
There are different modes of gene
expression dynamics depending onthe body regions
The possible causes of the differences
Nature of the field (syncytium vs cells)
Nature of morphogen/signal
Historical aspects (derived vs ancestral)

Akiyama-Oda and Oda, Sci. Adv. 6 (2020) CC BY 4.0time

Morphological diversity in animals
Model speciesⒸHiroki Oda

PresentArthropods
How can we know the mechanisms behind the diversity?
What happened
to the genome? Past
Many drasticchanges
occurred!

Depth of time

Genetic information(genome)

withmutations

Yasuko Akiyama-Oda,
PhD
Model system development,
Technical development,
Genome studies,
Body axes, SegmentationSawa Iwasaki-Yokozawa,
PhD

Contributors
Please visit our web sites:
Search by

brh spider

Laser ablation,
twinned embryos

Masaki Kanayama,Previous student,
PhD

Natsuki Hemmi,
Previous student,
MS

Splitting-type segmentation,
Microinjection

Reconstruction ofpatterndevelopment