The Temporal and Spatial Expression of Ptf1-a, Ascl1/2,

NeuroD1/2/4/6, and Mist1 in Strongylocentrotus purpuatus Stages of

Development
Abstract
Strongylocentrotus purpuratus, the purple sea urchin, is an echinoderm whose
development shares similarities with that of chordates. As such, they share many orthologs
critical to embryonic development with humans. The purpose of this study is the characterize
unknown S. purpuratus genes using a basic local search alignment tool (BLAST), phylogenetic
analysis, whole mount in situ hybridization (WMISH) and revere transcription PCR (RT-PCR).
Probes for WMISH were synthesized using DNA cloning and plasmid isolation. Using these
techniques, the genes were determined to be Pancreas transcription factor 1-a (Ptf1-a),
Achaetescute complex like-1/2 (Ascl1/2), Neuronal differentiation factor 1/2/4/6 (NeuroD1/2/4/6)
and Mist1; all are transcription factors part of the basic-helix-loop-helix protein family that have
been known to support pancreatic development in chordates. Ptf1-a was expressed at 72 hours
after fertilization, and possibly at 48 hours according to RT-PCR and WMISH. Ascl1/2 and Mist1
did not appear in any stages of embryonic development. Neurod1/2/4/6 was also expressed after
72 hours of embryonic development. This suggests that Ptf1-a and NeuroD1/2/4/6 play
regulatory roles within the first 72 hours of sea urchin development. Mist1 and Ascl1/2, known
transcription factors in other organisms, might be expressed later on. The characterization of
these genes gave us insight to their specific functions during S. purpuratus embryonic
development.

Results:
DNA Sequencing Analysis Results

Figure 1: Phylogenetic analysis to predict gene identity. The phylogenetic trees generated from
MEGA software after ClustalW alignment, the orthologs and paralogs protein sequences were
obtained through NCBI after researching their identities through Pubmed. The trees include
organisms: Saccroglossus kowalevskii, Homo sapiens, Gallus gallus, Maylandia zebra,
Oreochromis niloticus, Xenopus laevis, Cynoglossus semilaevis, and Rattus norvegicus, Macaca
mulatta, Xenopus tropicalis, Bos Taurus, Macaca fascicularis, Pan troglodytes, Pongo abelii,
Strongylocentortus purpuratus, Bubalus bubalis, Galeopterus variergatus, Danio rerio,
Heterocephalus glaber, Anolis carolinensis, Drosophila melanogaster, Accoglossus kowalevskii,
Latimeria chalumnae, Chrysemy picta bellii, Canis lupus familiaris. The amino acid sequences
were aligned using ClustalW and a tree was constructed based on the similarities and a bootstrap
value of 500. (A) The tree confirms that the predicted identity of the first gene as Ptf1-a because
it clustered in the same clade with known Ptf1-a orthologs. (B) The tree reveals that gene 22554
could be Ascl1/2 because it shares a common ancestor with both Ascl1 and Ascl2. (C) The tree
shows that gene shares a common ancestor with NeuroD1, NeuroD2, NeuroD4 and NeuroD6,
while remaining independent from other orthologs, so the assumed identity if NeuroD1/2/4/6.
(D) The tree supports the conclusion that the gene is part of the Mist1 family because it clusters
with orthologs of Mist1 and remains independent of related BHLH genes NeuroD1 and Atoh1.

Figure 2: Sequence alignment to find conserved domains among orthologs and paralogs.
The alignment of the sequences using Mega 6.0s ClustalW alignment, the orthologs and
paralogs protein sequences were obtained through NCBI after researching their identities through
Pubmed. The trees include organisms: Saccroglossus kowalevskii, Homo sapiens, Gallus gallus,
Maylandia zebra, Oreochromis niloticus, Xenopus laevis, Cynoglossus semilaevis, and Rattus
norvegicus, Macaca mulatta, Xenopus tropicalis, Bos Taurus, Macaca fascicularis, Pan
troglodytes, Pongo abelii, Strongylocentortus purpuratus, Bubalus bubalis, Galeopterus
variergatus, Danio rerio, Heterocephalus glaber, Anolis carolinensis, Drosophila melanogaster,
Accoglossus kowalevskii, Latimeria chalumnae, Chrysemy picta bellii, Canis lupus familiaris.
(A) Shows a conserved sequence of 30 residues among all orthologs and paralogs of Ptf1-a, the
sequence was found to be a BHLH binding domain. (B) The alignment shows the conserved
BHLH binding domain of 24 residues that is present in Ascl 1/2 and its orthologs and paralogs.
(C) The alignment also highlights the presence of a BHLH binding domain of 32 residues
conserved in its paralogs and orthologs of NeuroD1/2/4/6. (D) The alignment also highlights the
presence of a conserved BHLH transcriptional binding domain in Mist1 that has 23 residues.
4

Gene 2677 BLASTn results yielded a search result with an E-value of 0.00, a query cover
of 100% and a max and total score of 1397. The BLASTx yielded several matches for pancreas
transcription factor 1-a (Ptf1-a) with the E-value of 4x10-165, a query cover of 99%, an identity of
100%, and the presence of three white lines in the mapview BLAST reveal four exons within the
gene. Gene 22554 BLASTx resulted in an uncharacterized loci on the S. purpuratus genome.
The highest scores of this gene were identical with an E-value of 1x10-26, a 91% identity value
and a 12% query cover. BLASTx revealed gene Achaete-scute complex-like 1 (Ascl-1) with a
max score of 98.2, an E-value of 2x10-2, a 79% identity value, and a 25% query cover. Blasting
the sequence against the entire sea urchin genome indicated two possible exons and no
overlapping nucleotides in the sequence. Gene 24918 BLASTn analysis yielded an E-value of
0.0, a query cover of 100% and an identity of 99% for an uncharacterized sequence from the S.
pururatus loci. BLASTx gave result of Neuronal differentiation-1 (Neurod1) with a 96% query
cover, an E-value an identity of 99%. Based on the BLAST against the sea urchin genome, we
concluded that there was only one exon in this gene. Gene 19444 blast results yielded results for
a basic helix-loop-helix family protein a-15 (Bhlha15), an alias for Mist1. The most similar
sequences had an E-value of 4x10-22, an identity of 79%, and a query cover of 20%. BLASTx
confirmed this result by generating more orthologs of Mist1. The highest result had an E-value of
6x10-16, a query cover of 23% and a 71% identical sequence. The mapview BLAST against the
sea urchin genome confirmed that there were no other exons present in the gene.
When designing a phylogenetic tree for Ptf, orthologs from the hemichordate
Saccoglossus kowalevskii (XP_006825561.1) and several Chordate species were selected
including Homo sapiens (NP_835455.1), Danio rerio (NM_207641.2), Rattus Norvegicus
(NP_446416.1), Mus musculus (NP_061279.2) and Xenopus lavevis (NP_001167491.1, Figure
1A). Protein sequence for Gatae was included as a control to root all trees because its sequence
is distantly related to the genes that we are studying. The ten genes, excluding Gatae for
alignment purposes contained, contained a highly conserved basic helix-loop-helix binding
(BHLH) domain containing 30 residues found in previous papers (Figure 2A, Beres et al., 2006).
The phylogenetic tree was consistent with the hypothesis that Gene 2677 was an ortholog of the
gene Ptf1-a. The chordates Ptf1-a grouped in the same clade that shared a recent common
ancestor to S. purpuratus and the acorn worm, an echinoderm and hemichordate respectively.
The alignment parallels that of the known animal phylogeny; the Ptf1-a orthologs were in a
distinct clade from its paralogs, Nato in Homo sapiens (NP_001158470.1), Ferd3l in H. sapiens
(AAI01139.1) and M. musculus (EDL36872.1).
The phylogenetic tree for Ascl included Hemichordate Saccroglossus kowalevskii
(XP_006818759.1), Chordates like Homo sapiens (NP_004307.2, NP_005161.1, NP_065697.1,
NP_982260.2, NP_001257530.1), Maylandia zebra (XP_004548511.1), Oreochromis niloticus
(XP_005475946.1), Cynoglossus semilaevis (XP_008313626.1), and Mus musculus
(NP_032579.2, NP_032580.2, NP_064435.1, AC_000032.1, NC_000067.6) found in Figure 1B.
Gatae was used as a control. The chordates sharing the same orthologs fell within the same
clade, but S. kowalevskii and S. purpuratus remained a separate clade and shared a recent
common ancestor with both Ascl1 and Ascl2, which suggests that when the Chordates began to
5

distinguish between Ascl1 and Ascl2, the hemichordate and echinoderm retained the same protein
that is a combination of the two. Paralogs Ascl3 and Ascl 4 remained separated from the gene
studied. It is suggested that this gene is Ascl 1/2. The alignment of the sequences revealed a
highly conserved sequence in the genome of 24 residues (Johnson et al. 1990): a BHLH binding
domain according to Krapp et al. (Figure 2B, Krapp et. al, 2006).
The NeuroD genome was aligned with six other paralogs: NeuroD2, NeuroD3, NeuroD4,
NeuroG, Neuro G2, and the control Gatae from different species for a total of twenty-two
organisms (Figure 1C). The organisms include: Macaca mulatta (NM_001265801.1), Homo
sapiens (NM_002500.4, NM_006160.3), Xenopus tropicalis (NM_001097399.2), Bos Taurus
(NM_001144105.1), Rattus norvegicus (NM_001109237.1), Macaca fascicularis
(NM_001284857.1), Pan troglodytes (XM_009432275.1), Pongo abelii (XM_009251872.1),
Strongylocentortus purpuratus (NM_001005725.1), Bubalus bubalis (XM_006046985.1),
Galeopterus variergatus (XM_008567126.1), Danio rerio (NM_130978.1, NM_131041.1), and
Heterocephalus glaber (XM_004836210.1). Paralogs NeuroG1 and NeuroG2 were organized
into distinct clades from SpNeuroD1. Curiously, SpNeuroD1 shared a most recent common
ancestor with paralogs NeuroD1, NeuroD4, NeuroD2 and NeuroD6, which suggest the gene is an
older form NeuroD that Chordate species have since branched from. This suggests that the
identity is NeuroD1/2/4/6. The alignment of these sequences highlighted a highly conserved
BHLH binding domain of 32 residues to activate transcription (Figure 2C, Bouard et. al).
Sequences from Saccroglossus kowalevskii (XM_006822677.1), Homo sapiens
(NM_002500.4, NM_005172.1, AB032369.1), Gallus gallus (NM_204920.1), Rattus norvegicus
(AF049874.1), Danio rerio (DQ995273.1), Mus musculus (AF091858.1, NM_007500.4,
AB021220.1), Anolis carolinensis (XM_008120891.1, XM_008111198.1), Drosophila
melanogaster (M_001273745.1), Accoglossus kowalevskii, Latimeria chalumnae
(XM_005998714.1, XM_006010356.1), Chrysemys picta (XM_005306556.1), Chrysemys picta
bellii (XM_005306556.1),and Canis lupus familiaris (EU371515.1) were used to construct a
phylogenetic tree for Mist1 (Figure 1D). Mist1 does not have any immediate paralogs, so genes
from the basic-helix-loop family families such as NeuroD1 and Atoh1 were included in the tree
to confirm the sea urchin genes identity. The alignment of these thirteen genomic sequences
yielded a highly conserved BHLH binding domain containing 23 residues (Lemercier et al.,
1998). MIST, another genome was also a possible paralog of MIST1 because of the similarity in
names, but from the phylogenetic analysis, it is of a very distant relation and will not be
considered a paralog. For the purpose of comparison, it was also included in the tree. NeuroD1
and Atoh1 orthologs clustered within the same clade. Overall, Mist1 orthologs grouped in the
same clade, S. purpuratus grouped with S. kowalevskii, the hemichordate, confirming its identity.
The genes will be identified in this paper as Ptf1-a, Ascl1/2, NeuroD1/2/4/6 and Mist1
from here on.

Gene Cloning Results

Figure 3: Gel electrophoresis of PCR amplified products. The gel electrophoresis of the PCR
products on a 1.5% agarose gel against a 2-log ladder. The genes were amplified using gene
specific primers and then placed on the gel for 1 hour at 400V and 100mA. The two log ladder
sizes are indicated in base pairs. The sizes were determined by calculating the 2-log ladder
standard equation. The expected size for gene Ptf1-a, Ascl1/2, NeuroD1/2/4/6, Mist1 and the two
Gatae are 438.8bp, 788.7bp, 857.3bp, 665.8bp, 610.9bp and 657.6bp respectively.

Figure 4: Gel electrophoresis of purified PCR amplified products. The gel electrophoresis of
the purified PCR products on a 1.5% agarose gel against a 2-log ladder. The genes were purified
using a Qiagens MinElute PCR Purification Kit and then placed on the gel for 1 hour at 400V
and 100mA. The two log ladder sizes are indicated in base pairs. The sizes were determined by
calculating the 2-log ladder standard equation. The expected size for gene Ptf1-a, Ascl1/2,
NeuroD1/2/4/6, Mist1 and Gatae are 442.4bp, 704.3bp, 786.9bp, 675.2bp, and 657.6bp
respectively.
The gel electrophoresis of the PCR products with gene specific primers revealed no band
for gene Ptf1-a, a band size of 788.7bp for Ascl1/2, 857.3bp for gene NeuroD1/2/4/6, 665.8bp for
Mist, and 610.9bp for control Gatae (Figure 3A). A second PCR on gene Ptf1-a yielded a
calculated size of 438.8bp, and Gatae with 535.4bp (Figure 3B). After purification, the second
gel electrophoresis yielded sizes of 704.3bp for Ascl1/2, 786.9bp for NeuroD1246, 675.2bp for
Mist1, and 657.6 for Gatae (Figure 4A). Ptf1-a was purified later, its gel electrophoresis size was
442.4bp (Figure 4B). The concentration, determined by band intensities, was 24.5 ng/ul for
Ptf1a, 28.5 ng/ul for Ascl1/2, 24.5 ng/ul for NeuroD1/2/4/6, 20 ng/ul for Mist1, and 62 ng/ul for
Gatae.

Table 1: The Transformation Efficiency and Ligation Efficiency calculated by counting the
number of blue and white colonies per reaction condition.
Transformation Number of
Number of Blue Transformation Ligation
Condition
White Colonies Colonies
efficiency
Efficiency
(colonies/g)
(coloniges/g)
No DNA
0
0
0
1ng pBSK
0
367
6.60x105
5
10ng pBSK
0
3728
7.71x10
5
100ng pBSK
0
6704
1.21x10
Ptf1-a
431
1.68x104
Ascl1/2
818
3.36x104
NeuroD1/2/4/6
1012
4.79x104
Mist1
1720
6.74x104
Gatae
1210
5.38x104
Based on the concentrations, a ligation reaction was set up to insert the genes into cloning
vectors that were then used to transform competent cells. The control transformations using
pBSK vectors were used to calculated the transformation efficiency: 0.001 g of pBSK yielded
6.60x105 cfu/g, 0.01 g of pBSK yielded 6.71x105 cfu/g and 1.0 g of pBSK yielded
1.21x105cfu/g (Table 1). The ligation efficiency was calculated based off the number of white
colonies: Ptf1-a had an efficiency of 1.68x104cfu/g, Ascl1/2 efficiency was 3.36x104cfu/g,
NeuroD1 had a ligation efficiency of 4.79x104cfu/g, Mist1 was 6.74x104cfu/g, and Gatae had
a ligation efficiency of 5.38x104cfu/g (Table 1).

Figure 5: The gel electrophoresis of the colony PCR products. Three blue colonies streaked
on an agar plate and its contents were boiled and added to a PCR reaction. The products and 2log
ladder were run on a 1.5% agarose gel at 1 hour at 100V and 400mA. The calculated band sizes
are similar to the expected size. The sizes of the band are indicated in base pairs. The sizes were
determined by calculating the 2-log ladder standard equation. (A) The band sizes for Gatae are
both 932.87bp, 671.67bp for Ptf1-a, and 932.872bp for Ascl1/2. (B) The band sizes for
NeuroD1/2/6/4 are 1253.57bp, 1319.43bp, 988.25bp, 1245.57bp, 718.314bp, 1336.42bp and
727.6bp respectively. (C) The calculated band for Mist1 709.167bp.
The purpose of the colony PCR is to verify whether or not the correct genomic sequence
has been cloned, and to determine which bacteria to inoculate for plasmid isolation. It was
determined that the products for Gatae are 932.87bp, 671.67bp for Ptf1-a, and 932.872bp for
9

Ascl1/2. The band sizes for NeuroD1/2/6/4 are 1253.57bp, 1319.43bp, 988.25bp, 1245.57bp,
718.314bp, 1336.42bp, and 727.6bp respectively (Figure 5A-B). The calculated band for Mist1
709.167bp (Figure 5C).

Figure 6: The gel electrophoresis of the restriction digests. The plasmid was isolated from the
bacteria used for cloning using the miniprep kit, and digested using EcoR1. The products and a
2-log ladder were run on a 1% agarose gel for 1hour at 100V and 400mA. The sizes were
determined by calculating the 2-log ladder standard equation. The products indicative of the
plasmid vector at 3000bp and band sizes for Ptf1-a, Ascl/2, NeuroD1/2/4/6, Mist1 and Gatae
with 436.5bp, 514.8bp, 399bp, 392bp and 519.9bp respectively.
The concentration for the isolated plasmids from the cloning bacteria (in the miniprep kit
solution) for genes Ptf1-a, Ascl1/2, NeuroD1/2/4/6, Mist1 and Gatae was 99.0ng/l, 145.0ng/l,
85.0ng/l, 82.0ng/l, 87.0ng/l respectively. The 260/280 ratio for genes Ptf-1a, Ascl1/2,
NeuroD1/2/4/6, Mist1 and Gatae was 1.736, 1.643, 1.737, 1.721, 1.629 respectively. The
concentration values were used to calculate the volume of DNA to add to the EcoR1 restriction
digest which resulted in a 3000bp band (indicating the presence of a vector). As well as the bands
at 436.5bp, 514.8bp, 399bp, 392bp and 519.9bp, which represent the gene insert for of Ptf1-a,
Ascl/2, NeuroD1/2/4/6, Mist1 and Gatae respectively (Figure 6). After using the 4peaks
program, sequencing done by Laragens, to obtain the sequence, the NCBI BLAST tool was used
to determine that Ptfi-1 and Gatae were inserted sense relative to the T7 promoter region, while
Ascl1/2 was inserted antisense to the T7 promoter region. Sequencing for NeuroD1/2/4/6 and
Mist1 failed; the miniprep was borrowed from other groups that already had sequenced the two
genomes with concentrations 252ng/l for Mist1 (Uni), and 128ng/l for NeuroD1/2/4/6 (DH).

10

Synthesis of RNA Probes Results

Figure 7: The gel electrophoresis of RNA probe template synthesis. (A-C) The gel
electrophoresis of the PCR probe template. The probe template was synthesized in a PCR
reaction using Sp6 and T7 primers. Its products were run on a 1.5% gel at 1 hour along a 2-log
ladder. The sizes were determined by calculating the 2-log ladder standard equation. (A) The first
successful template synthesis was for Mist1 and Gatae with 809.39bp and 831.7bp respectively.
(B) The calculated size for NeuroD1/2/4/6 is 956.92bp. (C) The size of Ptf1-a and Ascl1/2 are
558.15bp and 816.10bp. (D) The gel electrophoresis of the PCR template after purification using
a Qiagens Minelute PCR Purification kit. Its products were run on a 1.5% gel at 1 hour along a
2-log ladder. The sizes for Ptf1-a, Ascl1/2, NeuroD1/2/4/6, Mist1 and Gatae are
519.97bp, 772.82bp, 864.19bp, 749.59bp, 741.86bp respectively. (E) The RNA Gel
electrophoresis of the RNA probes for genes Ptf1-a, Ascl1/2, Mist1,Gatae, and NeuroD1/2/4/6,
along with a RNA ladder, the RNA Century Marker-Plus. The probe was synthesized using either
a T7 or Sp6 RNA polymerase and then precipitated using LiCl. The RNA probe was denatured
using formamide and fomaldehyde and then placed on a 1.5% denaturing gel. The gel was run at
100V for 1 hour. The calculated size of the gel probes are: 608.22bp (Ptf1-a),
891.62bp (Ascl1/2), 979.99bp (Neurod1/2/4/6), 863.98bp (Mist1), and 765.13bp (Gatae).
RNA probe synthesis first required a DNA template to be synthesized using PCR. To
verify that we synthesized the right sized template for the probe a gel electrophoresis was
conducted which yielded band sizes 809.39bp for Mist1, 831.7bp for Gatae, 956.92bp for Gatae,
and 558.15bp and 816.10bp for Ptf1-a and Ascl1/2 respectively (Figure 7A-C). The template was
11

purified using a Qiagen Minielute PCR Purification kit and then run a second time. The sizes for
Ptf1-a, Ascl1/2, NeuroD1/2/4/6, Mist1 and Gatae are 519.97bp, 772.82bp, 864.19bp, 749.59bp,
and 741.86bp respectively (Figure 7D). The RNA probe was then synthesized using either a T7
or Sp6 RNA polymerase depending on the gene orientation relative to the T7 promoter region.
Genes Ptf1-a and Gatae utilized Sp6 polymerase, and Ascl1/2, NeuroD1/2/4/6, Mist1 utilized T7
polymerase. The probes were precipitated and then diluted: the concentrations of the probes for
Ptf1-a, Ascl1/2, NeuroD1/2/4/6, Mist1 and Gatae are 448.0ng/l, 258.0ng/l, 222.0ng/l,
110ng/l and 244ng/l respectively. The 260/280 ratio for Ptf1-a, Ascl1/2, NeuroD1/2/4/6,
Mist1 and Gatae was 1.845, 1.836, 1.708 and 1.772. The RNA gel against a RNA Century
Marker-plus revealed that probe sizes: 608.22bp (2677), 891.62bp (22554), 979.99bp (24918),
863.98bp (24918), and
765.13bp for Gatae
(Figure
7E).
RT-PCR

12

Figure 8: Gel electrophoresis of RT-PCR products. Following fertilization the total RNA was
isolated at different embryonic stages (0h, 24h, 48h and 72h) and reverse transcribed. The DNA
products were then run under PCR using gene specific forward and reverse primers. The gel
electrophoresis of the RT-PCR products against a 2-log ladder and various controls. The products
were run on a 1.5% agarose gel at 100V for 1 hour. 0h-72h are the RT-PCR products, 0h-72h-RT
are the RNA templates that are negative controls used against genomic DNA. The positive
control, Control DNA contains the plasmid from the gene's miniprep. The 2-log ladder sizes
13

are indicated in base pairs and any white arrow indicates that there are bands. The sizes of the
bands determined by calculating the 2-log ladder standard curve. (A) Ptf1-a shows expression
during 48h, 72h and the Control DNA lane with a band size at 518.18bp. (B) Ascl1/2 does not
have any expression except in the control DNA (652.144bp). (C) NeuroD1 may be expressed
during the 72h stage and is present in the control DNA (642bp). (D) Mist1 does not have any
expression except in the control DNA with a size of 493bp. (E) As expected, Gatae shows
expression in 24h, 48h, 72h and the control DNA with a size of 568.78bp.
The RNA isolated from the embryos as different growth stages had a concentration of
41ng/l (0h), 212ng/l (24h), 108ng/l (48h), and 388ng/l (72h). The 280/260 ratios for 0h,
24h, 48h and 72h were 1.768, 1.904, 1.598, 1.808 respectively. Due to a low concentration of
RNA, RNA from the SHARPS replaced that of 0hr (175ng/l), 24hr (198ng/l) and 48hr
(224ng/l). After RT-PCR, Ptf1-a showed a band size of 518.18bp at the 48h, 72h and positive
control lane (Figure 8A). Ascl1/2 had no visible bands during any development period aside from
the control DNA with a size of 652.144bp (Figure 8B). NeuroD1/2/4/6 RT-PCR had a band at
72h and control DNA with a size of 642bp (Figure 8C). Mist1 did not have any bands besides the
positive control DNA at 568.78bp (Figure 8D). Gatae showed expression in 24h, 48h, 72h and
the control DNA with a size of 568.78bp (Figure 8E).

14

Figure 9: Stained embryos for WMISH under a light microscope.The embryos were fixated
24h, 48h and 72h after fertilization before being washed, dehydrated, and exposed to the
antisense RNA probe. After one week of hybridization, they were washed and then mounted on a
slide to be viewed under a Zeiss inverted microscope. (A) Ptf1-a appears to be present in the
midgut of the 72h stage of embryo. (B) Ascl1/2 is not present in any of the stages. (C)
NeuroD1/2/4/6 can possibly be stained in the gut area at 72h. (C) Mist1 does not appear to be
present in any of the stages. (D) Gatae is stained at 24h, 48h and 72h in the primary mesenchyme
cells, the foregut and the hindgut, and in the midgut respectively.

15

The results of the whole mount in situ hybridization reveal that Ptf1-a is possibly
expressed in the midgut during the pluteaus larvae stage of sea urchin development (Figure
9A). It is also possible that Neurod1/2/4/6 is expressed in the pluteaus larvae in the gut area
(Figure 9C). Gatae, our control, showed staining in the primary mesenchyme cells during the
blastula stage, in the foregut and hindgut during gastrula stage and was restricted to the midgut
during pluteus larvae stage (Figure 9E). Ascl1/2 and Mist1 did not have any staining.

Conclusion
A combination of RT-PCR, whole-mount in situ hybridization, sequence and phylogenetic
analysis has given light to the function as well as the spatial and temporal expression of the genes
present in sea urchin development. The light staining and RT-PCR places Ptf1-a at 72hrs in the
midgut, the latter also showed evidence of the genes expression at 48hrs. The genes identity
matches with its location: Ptf1-a is involved in gut development acting as a transcriptional
regulator with its basic-helix-loop helix binding domain. RT-PCR suggests the presence of
NeuroD1/2/4/6 at 72 hours of development, staining in the foregut region in 72hr embryos also
might support this conclusion, but due to insufficient probe binding the results are unclear.
NeuroD1/2/4/6 might also be involved in gut cell differentiation or neural cell development
which explains its location. Neither Ascl1/2 nor Mist1 showed presence in the embryonic
staining or the RT-PCR which suggests that their involvement in sea urchin development might
occur at a later time. Their orthologs in chordates suggest that they are transcription factors
involved in gut development. Little research has been done on the overall spatial and temporal
expression, let alone function of these genes in sea urchin embryos. Future experiments might
include silencing of these genes to monitor their effects on sea urchin embryo development.

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http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4106228C.pdf
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