1. Trishala Kalra
u5547625
Summer Research Internship Report
Supervisor: Maja Adamska
Evolutionary Origin of Neurons: Investigation of Expression of “Neuronal” Genes in Sycon
ciliatum (Calcispongia)
Summary: Cross cells seen positively staining with dlg1 and dlg2 embryos, positive staining
around the rim of the osculum in the young syconoid with NaVa and RIMS1 and also positive
staining in the sclerocytes in the young syconoid with RIMS1 has been observed with our results.
No cross cells were observed with gpcr9 and pre and post inversion staining were opposite to what
was expected as per the heatmap for all genes.
Key Words: Sycon ciliatum, NaVa, Dlg1, Dlg2, gpcr9, RIM2, Neurexin 3β, Neurexin 2β
Introduction: Dlg1 is an 866 amino acids long peptide and Dlg2 is an 889 amino acids long
peptide where both encode for a L27 and L27-1 domain, PDZ domain, SH3 domain, P-loop
containing nucleoside triphosphate hydrolase, guanylate kinase/L-type calcium channel β subunit,
guanylate kinase-like domain in order to bind proteins (InterPro 2016). Dlg1 has addition SH3
domains and dlg2 is an isoform (InterPro 2016). Dlg1 is also involved in structural organization
of cell adhesion molecules targeting the epithelial cells and ion channel in synapses and their
downstream signaling pathways (Thomas et al, 1997).
Gpcr9: 976 amino acids long peptide which belongs to the GPCR protein family 3 with
conservative C-terminal, has a periplasmic binding protein-like I domain, receptor for ligand
binding domain in order to form an integral component of the membrane and perform G-protein
coupled receptor activity in the signaling pathway (InterPro 2016).
NaVa: 2782 amino acids long peptide with a sodium channel protein 60E, ion transport
domain, voltage dependent channel with four helix bundle domain, voltage dependent L-type
calcium channel which is a component of the membrane and allows transmembrane transport for
calcium ion transport (InterPro 2016; Barzilai et al., 2012).
Neurexin 2α is 948 amino acids long peptide, Neurexin 2β is 2941 amino acids long peptide
and Neurexin 3β is 1156 amino acids long peptide which all include a concanavalin A-like
lectin/glucanase domain, Laminin domain, EGF-like domain which allows all to bind to proteins
(InterPro 2016). Neurexin 2β also has an EGF-like calcium-binding domain which allows it to
bind to calcium ions for signaling pathways (InterPro 2016). Each of these are synaptic adhesion
proteins and are transcribed by two promoters, where the α form is shorter and the β forms are
longer (Rozic-Kotliroff & Zisapel, 2007).
RIMS1: Regulating synaptic membrane exocytosis protein 4 isoform X3 is a 865 amino
acids long peptide which codes for a Zinc finger, RING/FYVE/PHD-type domain, Rab-binding
domain and C2 domain which together help in Rab GTPase binding and intracellular protein
binding and transport (InterPro 2016). The role being in the functional interaction of the active
zone proteins such as RIM1 in synaptic vesicle priming and thus stimulate exocytosis (Betz et al.,
2001).
The aim of this project was to identify the cells where the expression of 8 neuronal genes
takes place using WMISH and Sycon ciliatum. This will tell us more about any homology between

2. Trishala Kalra
u5547625
Summer Research Internship Report
Supervisor: Maja Adamska
the cnidarians and the bilaterians and see what sponges can tell us about the evolution of animals.
The key question is that marine sponges do not have neurons yet they express “neuronal” genes
and it is thought that the cross cells are the sensory cells in sponges since they do respond to water
movement and to capture food particles.
Results and Discussion:
vitellogenesis cleavage preinversion post inversion larvae Young syconoid
Dlg 1
Dlg 2
Gpcr9
NaVa
Neurexi
n 2β
Negative
Neurexi
n 3β

3. Trishala Kalra
u5547625
Summer Research Internship Report
Supervisor: Maja Adamska
RIMS1 Negative
Table 1: Results showing the different stages: vitellogenesis, cleavage, pre and post inversion, larvae and young
syconoid for each of the seven genes: dlg1, dlg2, gpcr9, NaVa, Neurexin 2β, Neurexin 3β, RIMS1.
From table 1, we observe that the heatmap for these were followed however with the
following exceptions. Dlg1- we expected positive post inversion, however we observed it to be
negative. Dlg2- we expected positive post inversion and negative larvae, however we observed the
opposite of those two. In Dlg1 and Dlg2, we observed cross cells being both positive in both the
preinversion stage. Gpcr9- we expected negative oocytes, positive preinversion and positive
larvae, however we observed the opposite for each of those stages. Also in gpcr9, we expected
cross cells but were not able to locate any. NaVa- we observed positive vitellogenesis and
cleavage, negative pre and post inversion and larvae and positive young syconoid as expected.
Neurexin 2β- we expected positive pre-and post-inversion, however we observed the opposite for
both stages. Neurexin 3β- we expected that vitellogenesis would be negative, post inversion and
larvae would be positive, however the opposite was observed for each of these stages. RIMS1-we
expected positive pre and post inversion and negative young syconoid, however the opposite was
observed in each of these stages and in the young syconoid, we observed positive staining on the
sclerocytes. Overall, we also observed positive choanocytes and negative pinacocytes cells.
Staining around the embryo. Similar pattern seen in NaVa preinversion
samples as well RIMS1 Sclerocytes stained in young
sponge
Gpcr9
Neurexin 2β
Table 2: Consistent patterns observed in gpcr9, NaVa, Neurexin 2β and another new pattern in RIMS1.
Table 2 shows us that embryos in NaVa preinversion, gpcr9, Neurexin 2β all have this
consistent pattern where the choanocytes surrounding the outer rim are stained but not the inside
of the embryo. Also positive staining on the sclerocytes in the young sponge with RIMS1.
I also thought that Neurexin 2α and Neurexin 2β may have a regulatory role where one
gene is only expressed where the other gene is not expressed, however Neurexin 2α was lost in the
project and may need to be repeated in the future. Yet none of the other genes showed such a
pattern as the oocytes were always highly stained in each case. Gpcr9 will also need to be repeated

4. Trishala Kalra
u5547625
Summer Research Internship Report
Supervisor: Maja Adamska
due to no cross cells being located as expected. For RIMS1, double ISH will need to be performed
to confirm the existence of genes being expressed in the sclerocytes and compared with other
RIMS gene versions. The oocytes being stained suggests that these genes are being expressed but
it seems as if the expression is being lost as the sponge develops since most of the results are
negative afterwards even though we expect some of them to be positive.
Materials and Methods:
Primer design, Reverse Transcription, PCR, DNA purification
NaVa, Dlg1, Dlg2, gpcr9, RIM2, Neurexin 3-β, Neurexin 2-β and Neurexin 2-α were the potential
candidate genes to investigate their expression during the early and late vitellogenesis, fertilization,
early and late cleavage, early and late pre- and post- inversion and young syconoid stages. For
these, F1, F2 and R1 primers were designed using full length sequences and Sequencher and
checked using Sigma and diluted. The protocol for First-Strand cDNA Synthesis Using
SuperScript II Reverse Transcription from RNA was performed using Oligo (dT) and random
primers and then diluted as old cDNA was also diluted as control. Using F1R1 primers for old and
new cDNA, the protocol for PCR was followed for 40 cycles and ran on a gel. PCR was repeated
using 35 cycles and with F2 primer for NaVa. The protocol for MinElute DNA PCR purification
kit was followed and ran it on a gel.
Ligation, Transformation, PCR, DNA purification, transcription assay, RNA purification
The protocol for Ligation using 2x rapid ligation buffer and transformation of JM109 High-
Efficiency Competent cells were performed. 8 white colonies were isolated for each gene into a
LB grid plate and into tubes for PCR, except Neurexin 2-alpha and gpcr9, and ran on a gel. DNA
was purified and then ran on a gel to estimate the volume of template needed for non-radioactive
transcription assay. This was then performed using SP6 polymerase as it is downstream of M13R
which was with our gene of interest. The protocol for RNeasy MinElute RNA purification kit was
followed and ran on a gel where all genes worked except NaVa which was later found to be
degraded. At a later stage, the protocol from ligation onwards was repeated for Neurexin 2-alpha
and gpcr9 like before. Neurexin 2-alpha was now completely lost in this project due to no white
colonies. The NaVa and gpcr9 columns were re-eluted with water as the purification efficiency
was low and this probe dilution was taken into account for WMISH 3. Probes stored at -80ºC.
Whole Mount In-situ Hybridization
WMISH 1 was performed using the ISH Protocol Sycon with specimens collected on 19/5,
26/5, 6/6, 17/6, 23/6 cut from the equator and into pieces, using uncut small sponges and 1: 100
probe dilution. WMISH 2 was performed using 1: 50 probe dilution and late cleavage 10/6/14,
preinversion 17/6/13 and 1/7/13, vik 27/8/13 post inversion, 1/7/13 late post-inversion pigment
and #2 samples cut like before, young sponges and reusing antibodies. For WMISH 3, Dlg1 was
used as a control, NaVa and gpcr9 were investigated using both previously cut samples. All
samples from the WMISH in 75% glycerol were mounted onto slides with double wells and viewed
under DIC on a compound microscope.

5. Trishala Kalra
u5547625
Summer Research Internship Report
Supervisor: Maja Adamska
Tissue Culture, plasmid DNA purification, Sanger Sequencing
Tissue culture was prepared using a colony from each gene grid plates into ampicillin and
LB mix overnight. The protocol for Plasmid DNA purification was followed and then ran on a gel
and its concentration measured using NanoDrop. The procedure was repeated with autoclaved LB
and using more volume of mixture such that the tubes were then diluted to send 15 µl of 100ng/µl
DNA for Sanger sequencing. The orientation and the product purification percentage was
evaluated using Sequencher to make sure we had investigated our genes of interest.
Acknowledgement: I would like to thank my supervisor Dr. Maja Adamska for giving me this
project and guiding me at every step. Also Marcin Adamski for the bioinformatics support and
Ursula Wiedemann for the lab technical support.
References:
Betz, A., Thakur, P., Junge, H.J., Ashery, U., Rhee, J.S., Scheuss, V., Rosenmund, C., Rettig, J.,
Brose, N. (2001). Functional interaction of the active zone proteins Munc13-1 and RIM1 in
synaptic vesicle priming. Neuron, 30(1): 183-196.
Brazilai, M.G., Reitzel, A.M., Kraus, J.E.M., Gordon, D., Technau, U., Gurevitz, M., Moran, Y.
(2012). Convergent Evolution of Sodium Ion Selectivity in Metazoan Neuronal Signaling. Cell
Reports, 2(2): 242-248.
InterPro 2016, InterProScan sequence search. Available from:
<http://www.ebi.ac.uk/interpro/search/sequence-search>. [28th
January 2016].
Thomas, U., Kim, E., Kuhlendahl, S., Koh, Y.H., Gundelfinger, E.D., Sheng, M., Garner, C.C.,
Budnik, V. (1997). Synaptic clustering of the cell adhesion molecule fasciclin II by discs-large and
its role in the regulation of presynaptic structure. Neuron, 19(4): 787-799.
Rozic-Kotliroff, G., Zisapel, N. (2007). Ca (2+)-Dependent Splicing of Neurexin II
alpha. Biochem. Biophys. Res. Commun., 352(1): 226-230.