Title

What can molecular labelling do for developmental biology?

Master subtitle

Mariana R. P. Alves

mariana.alves@embl.de
Twitter @Mariana_RPAlves

Summer School 2019

17/07/2019
 Eukaryotic gene expression

Different Genes
(Units of DNA)

Different proteins

Different cells

Kahn Academy
2 17/07/2019
 Body plan – From head to tail

Different cells form

structures during
development and make
up a diverse adult
organism

3 17/07/2019
 Body plan – From head to tail

The same genes in both

human and flies are
responsible for the
formation of the different
body parts

4 17/07/2019
 Body plan – From head to tail

Hox genes

University of Utah
5 17/07/2019
 Body plan – From head to tail

Hox genes
By Fluorescence
Microscopy

https://www.thermofisher.com/

University of Utah
6 17/07/2019 http://science.sciencemag.org/content/313/5795/1918.full?rss=1
 Body plan – From head to tail

Hox factor Ubx

Specification of thoracic
segments Pb
Dfd

Scr

A1

Wildtype Ultrabithorax (Ubx -)

Drosophila melanogaster
Pearson et al. (2005)

7 17/07/2019
 Eukaryotic gene expression

Transcription

Kahn Academy
8 17/07/2019
 Eukaryotic gene expression

9 17/07/2019
 Discovering gene regulation through Fluorescence Microscopy

Embryo (fluorescent antibody

against Ubx protein)

Ubx regulates svb

gene
(fluorescent antibody
against DIG-RNA
svb = shavenbaby probe)

Larva

10 17/07/2019 Crocker et al., 2015

 Discovering gene regulation through Fluorescence Microscopy

Embryo
Deletion of Hox
factor removes
Ubx regulates svb tricomes
gene

Larva

11 17/07/2019 Crocker et al., 2015

 Discovering gene regulation through Fluorescence Microscopy

Embryo
Overexpression
makes ectopical
Ubx regulates svb tricomes
gene

Larva

12 17/07/2019 Crocker et al., 2015

 Discovering mechanisms through Fluorescence Microscopy

Ubx regulates svb

shavenbaby (svb)
gene Ultrabithorax (Ubx)

13 17/07/2019 Tsai, Muthusamy, Alves et al., 2017

 Looking at proteins in live embryos!

TF-HaloTag 50 msec

Ubx binds DNA in

short and frequent
intervals

A. Tsai

14 17/07/2019 Tsai, Muthusamy, Alves et al., 2017

 Imaging single Ubx molecules in live embryos

Ubx binds DNA in

short and frequent
intervals

A. Tsai

15 17/07/2019 Tsai, Muthusamy, Alves et al., 2017

 Imaging single Ubx molecules in live embryos

HaloTag Ubx

Fusing Ubx with

HaloTag
JF635
Synthesized in the Lavis Lab
λabs/λem
635 nm/652 nm

A. Tsai

16 17/07/2019
Tsai, Muthusamy, Alves et al., 2017, Grimm et al. (2017)
 JF635 into embryos expressing HaloTag-Ubx

Injection site of
HaloTag Ligand-JF635

Fusing Ubx with

HaloTag

A. Tsai

17 17/07/2019
Tsai, Muthusamy, Alves et al., 2017
 Watching RNA formation in live embryos

MS2 system

18 17/07/2019
Garcia et al., 2013
 In our lab

19 17/07/2019
 In our lab

We try to see what we

usually can’t

We try to understand
how the DNA is
organized in space

20 17/07/2019 Crocker et al., 2015

 Observing different parts of the svb gene

Overlapping patterns

A1

21 17/07/2019 Crocker et al., 2015

 In our lab

We try to see what we

usually can’t

We try to understand
how the DNA is
organized in space
(and time during the
different stages of
development).
22 17/07/2019 Adapted from Tsai, Muthusamy, Alves et al., 2017
 In our lab

Fluorescence in situ hybridization (mRNA)

What about the DNA? control

What about when it is

not active?

Co-localization of
‘hubs’ where RNA is
formed

23 17/07/2019 Adapted from Tsai, Muthusamy, Alves et al., 2017

 To know more about it you can read our latest paper

24 17/07/2019 Tsai & Alves et al., 2019

 An example from mammalian cells

Mouse embryo
development

25 17/07/2019 Radzisheuskaya & Silva, 2014

 An example from mammalian cells

What makes these

cells different?

Kahn Academy

26 17/07/2019 Radzisheuskaya & Silva, 2014

 An example from mammalian cells

What makes these

cells different?

Kahn Academy

27 17/07/2019 Radzisheuskaya & Silva, 2014

 An example from mammalian cells

In different cell types

same gene moves
with different
properties

Live imaging of cells

28 17/07/2019 Gu et al., 2018; Radzisheuskaya & Silva, 2014

 An example from mammalian cells

In different cell types

same gene moves
with different
properties

Live imaging of cells

29 17/07/2019 Gu et al., 2018

 An example from mammalian cells

In different cell types

same gene moves
with different
properties

Live imaging of cells

30 17/07/2019 Gu et al., 2018

 In summary

Labelling molecules can teach us many details about how genes are
expressed in different cell types which form throughout development

Advances in Microscopy allows us to look even closer to molecules, and do

it in live embryos, for long periods of time

31 17/07/2019
 Take Home Message

It’s a very exciting time

The possibilities of discoveries are infinite
We need more scientists like you to help us understand these images

32 17/07/2019
 Questions?

About this biology, about our lab, about our papers, about being a PhD at EMBL, or other?

Don’t hesitate to write me an email or message me on twitter!

33 17/07/2019