ShroomB and Zipper

Candidate Gene in Neural

Defects
Emaya Anand, Mishika Mehta, Sydney Lloyd, Instructor Christina
Grogan, Dr. Hildebrand
Neural tube formation through apical constriction is
fundamental to embryonic development.

Mammalian Neural Tube

Plessis, Volpe, 2018.
Failure to close the neural tube during development results
in defects of the brain and spinal cord  

Neural Tube Defects

Botto, et al., 1999.

Folic acid deficiencies lead to an increased prevalence of
neural tube disorders

Center for Disease Control, 2017

Individuals in rural areas have less access to prenatal screening,
leading to a higher incidence rate of pregnancy complications

Hung, Henning-Smith, Casey, Kozhimannil, 2017

Shroom protein is necessary for normal constriction of the
neural tube

The difference in development between the

control and the disrupted shroom gene 
Haigo, et. al, 2003.
Shroom is responsible for cellular constriction in
epithelial cells

F & G: image of cells

comparing the control
and Shrm induced 
Hildebrand et al.,  2005
Shroom colocalizes and works with actin and myosin to
constrict the neuroepithelial cell

Martin and Goldstein, 2014

Hildebrand and Soriano, 1999
Who else does Shroom work with to drive
apical constriction in epithelial cells?
Neural tube and wing disc formation perform similar functions
during development 

Neural Tube Wing Disc

Development  Formation

Wolpert et al., 2011

Phenotypic changes from Shroom overexpression in the wing allows
identification of candidate proteins impacting the Shroom pathway

Wildtype and Overexpressed ShroomA Overexpressed ShroomA

overexpressed ShroomA, with candidate loss-of- with candidate loss-of-
respectively function function

Hildebrand et al, 2021

ShroomA and ShroomB exhibit different
binding sites in the Shroom pathway
Actin-Binding Rok-binding

Rok-binding

Hildebrand et al, 2021

What is the ShroomB over-expression
phenotype of the wing? 
 apical
apical

basal basal
 apical apical

basal basal
ShroomB over-expression leads to smaller wings and a
change in phenotypical expression in male Drosophila flies
Shroom B over-expression leads to smaller wings on
average in male Drosophila flies

P>0.05
Zipper (non-muscular myosin II heavy chain) is important in
the actin/myosin network in neural tube development

Zipper encodes myosin heavy chain in NMII for

actin binding Actin and myosin in neural tube Rolo, 2016
Betapudi, 2014
Zipper heavy chain pulls actin for apical constriction in mammalian epithelial cell
development. 

Myosin Heavy Chain

Myosin complex representation

Myosin complex binding to actin

Takeya, 2014
Muscle Contraction: Actin and Myosin Bonding, 2013
Zipper proteins are represented in both mammals and Drosophil
flies. 

Vicente-Manzanares, 2009
Kirchner et. al, 2007
Zipper also constricts epithelial cells in wing
disc development of Drosophila flies.

Drosophila wing disc epithelial cells

Apical and basal representation

of wing disc cells 
Duda, 2019
Nakajima, 2021
 apical apical

basal basal
Methods
Zip1, Zip2, and Zip02957 are loss of function mutations of the
zipper protein

**Zip1 is not
mapped but
Zip02957 is known to
be loss of
function

Zip2

FlyBase Consortium, 2021

Genetic cross for candidate alleles
Drosophila Lifecycle Timeline at 25°C
Day -7 = (Collection of virgin females)
Day 0 = addition of males
Day 10 Day 14 = Adult F1 Progeny were Collected an
Day 21 = Wings dissected and measured
Pupa
Embryo
Adult flies 

Day 7 (Males
Removed)

Hatching

3rd instar
larva 2nd instar 1st instar Day 1 Oxford University Press, 2017 
Day 4-5 Day 2-3 larva
larva Tauber Lab, n.d.
The reduction of Zipper candidate alleles led to overall larger
wings with ShroomB overexpression
The Zipper protein interacts in the ShroomB pathway
in female Drosophila flies.  

P<0.05
P<0.05
nn=24 P<0.05

n=18 n=20
n=22 n=19
n=15
The Zipper protein interacts in the ShroomB pathway in
male Drosophila flies. 

P<0.05
In conclusion, the data supports our hypothesis with female
flies and partially supports with male flies.

• All three candidate allele mutations

displayed significant increases in
wing area for female flies

• Only the Zip2 candidate allele

mutation led to a significant
increase in wing area for male flies
Future Directions

• Test how the over-expression of zipper along with over-expression of

ShroomB and/or ShroomA affects Drosophila fly wings

• Analyze where exactly zipper localizes in fly wing disc cells (using
fluorescence imaging and intensity) and compare it with the way the
shroom protein localizes  

• Looking at how reducing certain alleles would change the localization

of zipper and Shroom in wildtype epithelial cells
Attributions and Acknowledgments

• Instructor Christina Grogan - for teaching and aiding us throughout this

experiment
• Dr. Hildebrand – providing us the opportunity to contribute to this project
• Dr. Dawn Bisi – maintaining and supplying the flies
• Kylie and Emily (our TAs) - helping us through our experiments in lab
• Lauren, Holly, and Madeline - contributing to wing images. 
• In addition, we each investigated specific alleles [Emaya (Zip1), Sydney (Zip2), and
Mishika(Zip02957)], by conducting individual genetic crosses and investigating the
resulting phenotypes through imaging, measuring, and dissecting. We all
contributed to figures and graphs in this presentation.