Gene Expression Patterns 28 (2018) 77–86

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Gene Expression Patterns

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Astacin gene family of metalloproteinases in planarians: Structural T

organization and tissue distribution
Maria Emilia Isolania, Renata Batistonia, Chiara Ippolitob, Anna Maria Bianuccic, Silvia Marraccia,
Leonardo Rossib,∗
a
Department of Biology, University of Pisa, S.S.12 Abetone e Brennero 4, 56127 Pisa, Italy
b
Department of Clinical and Experimental Medicine, University of Pisa, Via Volta 4, 56126 Pisa, Italy
c
Istituto Nazionale per la Scienza e Tecnologia dei Materiali, Via Giusti, 9, 50121 Florence, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: Planarian flatworms possess extraordinary regenerative capability and body plasticity, which rely on a com-
Astacin posite population of stem cells, the neoblasts. Despite impressive advances have been recently achieved in the
Extracellular matrix knowledge of neoblast biology, few is still known about factors that are released by differentiated tissues and
Metallopeptidase influence the neoblast fate. Extracellular matrix (ECM) is a fundamental component of the stem cell niche and its
Planarian
remodeling affects stem cell fate. Here we provide the characterization of the astacin gene family of metallo-
proteinases in planarians, good candidate enzymes for generating dynamicity in the ECM. Ten and eighteen
astacin isoforms were identified in the planarian species Schmidtea mediterranea and Dugesia japonica, respec-
tively. Besides the already characterized Smedolloid, in Schmidtea mediterranea are present eight astacins with a
minimal structure (a signal peptide, an activation domain and a Zn-binding catalytic domain), that are colo-
calized in large cells organized in a peculiar, not yet morphologically characterized, two-ring-shaped structure
located in the middle of the body. A single astacin, characterized by a ShK toxin domain in its C-terminal region,
has been found to be produced in gastrodermal cells.

Section 1 inhibition. However, their primary regulatory mechanism is the zy-

Astacins are secreted and membrane-bound metzincin metallo-
peptidases widespread among different animal phyla and involved in
various physiological processes, including digestion, extracellular ma-
trix (ECM) remodeling, morphogenesis, hatching, tissue remodeling
and differentiation (Gomis-Rüth et al., 2012 and references therein). All
members of this family are characterized by two key elements: the 18-
amino acid zinc-binding motif (HEXXHXXGFXHEXXRXDR), and the
methionine-turn (Met-turn) sequence SXMHY (Gomis-Rüth, 2003; Bond
and Beynon, 1995). The N-terminal hydrophobic signal peptide - that
directs the proteins into the endoplasmic reticulum during biosynthesis
- followed by an activation domain, is another remarkable common
feature of astacins. While minimal astacins do not possess any C-
terminal domain in addition to the protease domain, other members of
the family contain one or more copies of different non-catalytic do-
mains, promoting protein-protein and substrate interactions (Wermter
et al. 2007; Hintze et al. 2006; Garrigue-Antar et al., 2001; Sieron et al.,
2000). Astacins are temporally and spatially regulated through mod-
ulation of gene expression, compartmentalization, allostery or
mogenic latency, provided by the presence of the activation domain
(Bond and Beynon, 1995; Nguyen et al., 1994). This segment physically
blocks the access of substrates to the active site when the enzymatic
activity is not required and it is removed during enzyme maturation
(Khan and James, 1998).
Expression and function of astacins have been studied during em-
bryogenesis of different animals, being BMP-1 and its Drosophila
homologues, Tolloid and Tolloid-like, the best characterized members
(Ge and Greenspan, 2006). BMP-1/Tolloid members perform multiple
functions, including activation of the TGF-β signaling pathway and
formation/remodeling of the ECM during development and metamor-
phosis (Reddi, 1996; Hopkins et al., 2007). BMP-1/Tolloid also plays
crucial roles during regeneration. In Hydra two Tolloid-like proteinases,
HMP-1 and HMP-2, are implicated in head regeneration and transdif-
ferentiation of tentacle battery cells and in foot morphogenesis, re-
spectively (Yan et al., 2000a,b). A BMP-1/Tolloid homolog is involved
in morphogenetic movements leading to folding of the luminal epi-
thelium and gut looping during visceral regeneration in the sea cu-
cumber (Mashanov et al., 2012). A Tolloid-like gene (Smedolloid-1) has

∗
Corresponding author.
E-mail address: leoros@biomed.unipi.it (L. Rossi).

https://doi.org/10.1016/j.gep.2018.03.003
Received 10 January 2018; Received in revised form 2 March 2018; Accepted 12 March 2018
Available online 13 March 2018
1567-133X/ © 2018 Elsevier B.V. All rights reserved.
M.E. Isolani et al. Gene Expression Patterns 28 (2018) 77–86

Table 1
S.mediterranea astacin sequences and Blastx analysis versus Homo sapiens non redundant protein sequences.

Planmine ID Genebank ID Best human blastx hit

Smed-ast-1 dd_Smed_v6_85_0_1 HF952107 PREDICTED: astacin-like metalloendopeptidase isoform X2 [Homo sapiens]

E-value: 1e-19; identity: 29%
Smed-ast-2 dd_Smed_v6_51_1_1 HF952108 PREDICTED: astacin-like metalloendopeptidase isoform X4 [Homo sapiens]
E-value: 2e-16; identity: 30%
Smed-ast-3 dd_Smed_v6_345_0_1 HF952109 tolloid-like protein 1 isoform 2 precursor [Homo sapiens]
E-value: 7e-19; identity: 28%
Smed-ast-4 dd_Smed_v6_76_0_1 HF952110 Select seq gb|AAH13871.1| TLL2 protein [Homo sapiens]
E-value: 7e-19; identity: 32%
Smed-ast-5 dd_Smed_v6_497_0_1 HF952111 meprin A subunit beta isoform 1 precursor [Homo sapiens]
E-value: 7e-14; identity: 33%
Smed-ast-6 dd_Smed_v6_51_1_2 HF952112 PREDICTED: astacin-like metalloendopeptidase isoform X4 [Homo sapiens]
E-value: 1e-18; identity: 30%
Smed-ast-7 dd_Smed_v6_961_0_1 HF952113 PREDICTED: astacin-like metalloendopeptidase isoform X1 [Homo sapiens]
E-value: 1e-18; identity: 28%
Smed-ast-8 dd_Smed_v6_254_0_1 HF952114 PREDICTED: meprin A subunit alpha isoform X1 [Homo sapiens]
E-value: 2e-22; identity: 30%
Smed-ast-9 dd_Smed_v6_12688_0_1 PREDICTED: astacin-like metalloendopeptidase isoform X4 [Homo sapiens]
E-value: 3e-20; identity: 32%
Smedolloid-1 dd_Smed_v6_5652_0_1 tolloid-like protein 1 isoform 1 precursor [Homo sapiens]
E-value: 0.0; identity: 45%

Table 2
Percentages of identity between S. mediterranea astacin putative proteins. Font size increases with the increase in identity percentage.

Smed-ast-1 Smed-ast-2 Smed-ast-3 Smed-ast-4 Smed-ast-5 Smed-ast-6 Smed-ast-7 Smed-ast-8 Smedolloid-1

Smed-ast-1 – 82% 60% 34% 50% 77% 38% 26% 27%

Smed-ast-2 82% – 58% 33% 52% 75% 36% 28% 27%
Smed-ast-3 60% 58% – 35% 49% 62% 36% 24% 25%
Smed-ast-4 34% 33% 35% – 34% 39% 76% 31% 29%
Smed-ast-5 50% 52% 49% 34% – 52% 33% 25% 27%
Smed-ast-6 77% 75% 62% 39% 52% – 38% 27% 24%
Smed-ast-7 38% 36% 36% 76% 33% 38% – 29% 30%
Smed-ast-8 26% 28% 24% 31% 25% 27% 29% – 31%
Smedolloid-1 27% 27% 25% 29% 27% 24% 30% 31% –

also been characterized during planarian regeneration and its func-
tional ablation causes the loss of some differentiated tissues at the re-
generative midline (Reddien et al., 2007).
Planarians are free-living Platyhelminthes (Collins, 2017). A popu-
lation of adult stem cells called neoblasts (Aboobaker, 2011), which
includes pluripotent stem cells (Wagner et al., 2011; van Wolfswinkel
et al., 2014), accounts for the astonishing regenerative capabilities,
high body plasticity and continuous cell turnover of these organisms.
Following amputation, neoblasts activate a biphasic proliferative pro-
gram and accumulate to form a regenerative blastema in which novel
tissues and organs are quickly reformed. Following this early epi-
morphic regeneration, morphallatic rearrangements of the entire body
are needed to restore the appropriate body proportion (Elliott and
Sánchez Alvarado, 2013; Rossi et al., 2008). Despite sustained efforts to
identify micro-environmental cues responsible for neoblast fate control,
no conclusive evidence has been provided in demonstrating the ex-
istence of a “neoblast-niche” analogous to that described for adult stem
cells in other organisms. Thus, the molecular mechanisms by which
stem cells respond to differentiated tissue requests are still almost un-
known. ECM remodeling undoubtedly serves a dynamic micro-
environment for stem cell niche and previous work demonstrates that
some matrix metalloproteinases (MMPs) play key roles in planarians,
generating the microenvironment in which cells can migrate and ex-
press their proliferation/differentiation program (Isolani et al., 2013;
Dingwall and King, 2016). With the aim to identify additional candi-
dates for ECM remodeling that might be important for neoblast fate
determination, here we provide the identification and characterization
of a family of astacin-related genes in planarians.
Section 2
2.1. Structural organization of the astacin-like gene family in planarians
By performing a survey of S. mediterranea genome, published tran-
scripts and available transcriptome databases, we managed to identify
ten genes encoding putative astacins that we named Smed-ast-1, Smed-
ast-2, Smed-ast-3, Smed-ast-4, Smed-ast-5, Smed-ast-6, Smed-ast-7, Smed-
ast-8 Smed-ast-9, and Smedolloid-1 (Reddien et al., 2007) (Tables 1 and
2). The analysis of genomic organization revealed that some astacin
genes are amplified. Indeed, at least two copies for Smed-ast-2 and
Smed-ast-5 and 3 copies for Smed-ast-4 and Smed-ast-6 were detected in
the same genome contig. Moreover, the locus coding for Smed-ast-2,
Smed-ast-3, and Smed-ast-6, as well as those coding for Smed-ast-4 and
Smed-ast-9 are relatively close, being included in the same genomic
contig (Supplementary Fig. S1 A).
Based on the genomic organization of introns and exons the pla-
narian astacin genes can be subdivided in three groups. The first group
is represented by Smed-ast-1 and Smed-ast-2, each with four exons and
three introns. The length and position of the introns and exons appear
well conserved between the two genes. The second group is represented
by Smed-ast-3, Smed-ast-4 Smed-ast-6, Smed-ast-7 and Smed-ast-9 with
five exons and four introns, again preserved in length and position.
Finally, the third group is represented by Smed-ast-5 and Smed-ast-8,
with six and seven exons, respectively. Both Smed-ast-5 and Smed-ast-8
are characterized by the presence of a long intron, of about 1 Kb, in
position three and five, respectively (Supplementary Fig. S1 B).
Smed-ast-1, Smed-ast-2, Smed-ast-3, Smed-ast-4, Smed-ast-5, Smed-ast-
6, Smed-ast-7 and Smed-ast-9 encode astacins with a minimal structure,
including a signal peptide, an activation domain and a Zn-binding

78
M.E. Isolani et al. Gene Expression Patterns 28 (2018) 77–86

Fig. 1. Structural organization of astacin gene family. (A) schematic structural organization of S. mediterranea astacins. (B) multiple alignment of the zinc-binding motif and Met-Turn
sequence of S. mediterranea astacins. Substitutions with respect to the consensus sequence are indicated in red. (C) Cladogram clustering S. mediterranea and D. japonica putative astacins.
D. japonica genes are named as: Dj+(gene bank ID for the transcriptome contig).

catalytic domain (Fig. 1A). Conversely, both Smed-ast-8 and Sme-
dolloid-1 possess additional domains in the C-terminal region. Smed-
ast-8 has a single ShK toxin domain (Möhrlen et al., 2006), while
Smedolloid-1 has five CUB domains intercalated with two EGF-like
domains (Sarras, 1996) (Fig. 1A). In Smed-ast-4, Smed-ast-7, Smed-ast-
9 and Smedolloid-1 the zinc-binding motif shows the typical consensus
sequence. Conversely, the second glutamate of the consensus domain is
substituted by a threonine in Smed-ast-1, Smed-ast-2, Smed-ast-3,
Smed-ast-5 and Smed-ast-6 (Fig. 1B). Smed-ast-8 shows a substitution
in both the 9th and 15th position of the consensus motif (Fig. 1B). The
Met-turn sequence is completely conserved in Smed-ast-1, Smed-ast-2,
Smed-ast-3, Smed-ast-5, Smed-ast-6, Smed-ast-8 and Smedolloid while
an aminoacidic substitution is present in Smed-ast-4, Smed-ast-7 and
Smed-ast-9 (Fig. 1B). In order to investigate whether S. mediterranea
astacin isoforms are conserved in the closely related species Dugesia
japonica, we used the S. mediterranea sequences to query D. japonica

79
M.E. Isolani et al.
transcriptome databases. We managed to identify 18 D. japonica as-
tacin-like genes that we multialigned with S. mediterranea astacin se-
quences. Phylogenetic tree analysis (Fig. 1C) allowed us to group as-
tacins in five clusters: cluster I including Smed-ast-1, -2, -3, -5, -6 and
their D. japonica homologues and further divisible in cluster IA in-
cluding Smed-ast-1, -2, -3, -6 and their D. japonica homologues and
cluster IB including Smed-ast-5 and its D. japonica homologues; cluster
II including Smed-ast-4, -9 and -7, as well as their D. japonica homo-
logues, cluster III and IV including respectively Smed-ast-8 and Sme-
dolloid-1 together with the corresponding D. japonica homologues.
Multiple alignments of the active and catalytic domains of S. medi-
terranea and D. japonica putative minimal astacins reveal high con-
servation at the amino acid level (Supplementary Fig. S2).
2.2. Expression of astacin genes in intact planarians
Colorimetric whole mount in situ hybridizations (WISH) in intact S.
mediterranea show that most astacin genes (Smed-ast-1 to Smed-ast-7
and Smed-ast-9) are strongly expressed in cells that are confined to two
ring-shaped areas localized dorsally and ventrally around the pharynx
in the central region of the body (Fig. 2 and Supplementary Fig. S3 A
and B).
We wondered if astacins confined to these ring-shaped areas are co-
expressed in the same cells or in different cells. To this aim we co-
localized Smed-ast-1 with the other astacin transcripts expressed in the
two-ring-shaped area. As shown in Fig. 3A and Supplementary Fig. S3 C
and D, Smed-ast-1 almost completely colocalizes with Smed-ast-2, Smed-
ast-3, Smed-ast-4, Smed-ast-5, Smed-ast-6, Smed-ast-7 and Smed-ast-9
with the exception of rare cells that were found positive for only one of
the analyzed genes (Fig. 3A and Supplementary Fig. S4). For example,
in Smed-ast-1 and Smed-ast-2 hybridized specimens very rare (ast-1+/
ast-2-) and (ast-1-/ast-2+) cells can be detected scoring the entire an-
imal. Further experiments are necessary to find out if these rare cells
belong to a different lineage or to the same lineage but are in a less
differentiated state. Confocal analysis at high magnification shows that
the positive cells are large cells (30–50 μm in diameter) slightly poly-
gonal or bottle-shaped and with a low nucleus/cytoplasmic ratio
(Fig. 3B and C). As a two-ring shaped expression pattern has also been
demonstrated for the cysteine-rich neurotrophic factor TCEN49 (Bueno
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Gene Expression Patterns 28 (2018) 77–86
Fig. 2. Expression of Smed-ast-1 to Smed-ast-6 genes in
intact S. mediterranea. (A) Representative images of col-
orimetric in situ hybridization for Smed-ast-1 to Smed-ast-6
genes. Scale bar corresponds to 200 μm. Anterior is at the
top (B) Lateral view of transcript distribution of Smed-ast-1.
This expression pattern is shown as representative example
of the typical two-ring-shaped structure. Anterior is on the
left. Scale bar corresponds to 200 μm. (C) A transverse
section of an intact S. mediterranea hybridized with Smed-
ast-2 probe is shown as example of localization of tran-
scripts of minimal astacins. Scale bar corresponds to
50 μm. g: gut. Dorsal side is at the top.
et al., 2002; Iglesias et al., 2008), we wondered if astacin-positive cells
of the two-ring-shaped structure are also positive for TCEN49 tran-
scripts. To this aim double fluorescent in situ hybridization was per-
formed using TCEN49 and smed-ast-1 labelled probes. As shown in
Fig. 4 all TCEN49-positive cells also express smed-ast-1 transcripts. The
expression pattern of Smed-ast-1, Smed-ast-2, Smed-ast-3, Smed-ast-4,
Smed-ast-5 and Smed-ast-6 appeared conserved in the related species D.
japonica (Supplementay Fig. S4), as demonstrated by in situ hy-
bridization with S. mediterranea probes.
Smedolloid-1 transcripts are mainly localized on the dorsal side of
the body, as previously demonstrated (Reddien et al., 2007) and its
expression appears conserved in the related species D. japonica, as de-
monstrated by in situ hybridization with S. mediterranea probe
(Supplementary Fig. S5).
Smed-ast-8 is mostly expressed in some gastrodermal cells
(Fig. 5A–C) that are also positive for the goblet cell marker smed-ra-
punzel (Reuter et al., 2015) (Fig. 5F). To exclude a certain degree of
colocalization between Smed-ast-8 and the astacin genes expressed in
the two-ring-shaped area we performed colocalization experiments
with Smed-ast-8 and Smed-ast-1 probes. As shown in Fig. 5 D, E, astacin-
positive cells of the two-ring-shaped structure are differently localized
with respect to Smed-ast-8-positive cells. The expression pattern of
Smed-ast-8 appeared conserved in the related species D. japonica, as
demonstrated by in situ hybridization with S. mediterranea probe
(Supplementary Fig. S5).
2.3. Morphogenesis of the two-ring-shaped structure by visualization of
Smed-ast-2 expression during regeneration
To analyze how astacin-positive cells reform the two-ring-shaped
structure, we monitored the expression of Smed-ast-2, as a re-
presentative marker, during the regeneration process. We produced two
groups of regenerating animals. In the first group, we cut the animals
through the pharynx (and consequently through the two-ring-shaped
structure). In the second group, we cut the animals under the head, in
way to produce anterior fragments devoid of the two-ring-shaped
structure and posterior fragments including the whole structure.
Animals from both groups were sacrificed at 1, 3, 6, 8 or 10 days after
the cut. As shown in Fig. 6A when the cut was performed through the
M.E. Isolani et al.
two-ring-shaped structure, Smed-ast-2-positive cells initially did not
change in distribution. In the posterior fragments including the whole
structure (Fig. 6B), Smed-ast-2 expression pattern remained unaltered
during the entire regeneration process. The analysis of the anterior
fragments, devoid of the two-ring-shaped structure, revealed that novel
Smed-ast-2-positive cells were produced late during the regeneration
process and not in the blastema region. Indeed no Smed-ast-2-positive
cells were found 1 day after transection and only a few positive cells
were detectable in the stump of only about 50% of hybridized animals
(6 out of 15) 3 days after transection (Fig. 6B). From this time point, the
number of Smed-ast-2-positive cells increased rapidly and 6 days after
transection a large number of cells were distributed in the parenchyma
to embrace the regenerating pharynx. Ten days after cut, when pharynx
regeneration was completed (Kobayashi et al., 1999), novel Smed-ast-2-
positive cells reformed in the posterior region to complete the two ring-
81
Gene Expression Patterns 28 (2018) 77–86
Fig. 3. Colocalization of Smed-ast-1 to Smed-ast-6 transcripts in
intact S. mediterranea (A) Double fluorescent in situ hybridization
showing examples of Smed-ast-1 (red) transcript distribution with
that of the other astacin members (green) expressed in the two-ring-
shaped structure. Arrows point to rare single positive cells. Scale bars
correspond to 200 μm Anterior is at the top. Images are the result of
the superimposition of confocal optical sections collected through
the animal at an interval of 2.5 μm. (B,C) Two examples of double
positive cells. Scale bars correspond to 10 μm. Red is for Smed-ast-1
and green is for Smed-ast-2 in B and for Smed-ast-3 in C. Blue is for
nuclei. Images are the result of the superimposition of confocal op-
tical sections collected through the central part of the labelled cells
at an interval of 0.35 μm.
shaped structures (Fig. 5B). Similarly, when anterior and posterior
fragments cut through the pharynx regenerated, two novel complete
ring-shaped structures are reformed after that the pharynx is com-
pletely reformed (Fig. 5A). No apparent difference was observed in the
reformation of the dorsal ring with respect to the ventral ring.
Section 3
It is presumable that ECM remodeling is a key issue for highly
plastic tissues. However, due to its complexity, redundancy and dyna-
micity, the understanding of the role of ECM and ECM remodeling in
complex morphogenetic processes is extremely challenging. In this
context, planarians offer the possibility to carry out single and multiple
knockdown assays by RNAi on large number of animals, thus, providing
an in vivo “platform” for combined functional studies on ECM
M.E. Isolani et al.
Fig. 4. Colocalization of Smed-ast-1 and smed-TCEN49 transcripts in intact S. med-
iterranea. A transverse section of an intact S. mediterranea hybridized with Smed-ast-1
(Red) and smed-TCEN49 (green) probes. Nuclei (blue) are stained with Hoechst 33342.
Scale bar corresponds to 20 μm.
remodeling enzymes. Here, to help in these future functional studies,
we provide a detailed characterization of the complex astacin gene
family in planarians.
We found ten genes coding for astacins in available S. mediterranea
transcriptomes. Although a higher number of astacin genes is present in
the related species D. japonica, all the sequences clusterize with a S.
mediterranea gene indicating that the duplication events that generated
the paralogs pre-dated the divergence between the two species. A high
number of astacins is an usual feature of invertebrate organisms. A
general expansion of astacin gene family occurred in fact in nematodes
(Caenorhabditis elegans: 40 genes); 16 genes encoding astacins were
found in Drosophila and 23 genes in sea urchin (Park et al., 2010;
Möhrlen et al., 2003). Only two astacins were found in the blood fluke,
Schistosoma mansoni genome (genomic contigs: Smp_047460.1
Smp_134430.1), suggesting that loss of astacin genes occurred during
evolution of parasitic flatworms (Park et al., 2010).
82
Gene Expression Patterns 28 (2018) 77–86
Analysis of genomic organization indicates that astacin genes were
object of events of duplication probably resulting in an advantageous
increase in protein synthesis level, but also a source for isoform varia-
tion. Intron/exon organization suggests that two groups of astacin
genes (ast-1/ast-2 and ast-3/ast-4/ast-6/ant-7/ast-9) include sequences
derived by two independent events of duplication.
Planarian astacins are exclusive for post-mitotic differentiated cells.
A structure similar to the two-ring-shaped area has previously been
identified (Isolani et al., 2013) by the expression of two distinct me-
talloproteinases: Smed-mmp1 and Smed-mmp2, suggesting that groups of
cells of this area are specialized in storage and secretion of ECM de-
grading enzymes. Reasons for their specific distribution pattern around
the pharynx region are still to be clarified. At the moment, no detailed
literature is available on the specialized cells forming the two-ring-
shaped structure. Due to their localization and characteristics, the peri-
pharyngeal cyanophilic secretory cells described by Pedersen, (1963)
and Zayas and colleagues (Zayas et al., 2010), are the main candidates
to express the astacins localized in the two-ring-shaped structure. A
possibility is that astacin-positive cells of the two-ring-shaped structure
are secreting cells with long protrusions projecting into the pharynx for
food digestion. This is consistent with the finding of ast-2, ast-5 and ast-
1 in the proteome of planarian regurgitate (Goupil et al., 2016). On the
contrary, some evidence suggests that astacins might also be secreted in
the mesenchyme and act as ECM remodeling enzymes. First, a gland-
like system of cells surrounding the pharynx and projecting into the
pharynx cavity has been described by the use of 2C11 antibody
(Forsthoefel et al., 2014) and lectin staining (Zayas et al., 2010).
However, in both cases this gland system appears organized as a single
ring. Second, we demonstrate that astacin-positive cells also express the
TCEN49 transcripts that code for a secreted trophic factor involved in
the regulation of cell survival in the central body region (Bueno et al.,
2002). According to immunogold labeling on ultrathin section experi-
ments TCEN49 protein is contained in a subset of the cyanophilic se-
cretory cells, highly concentrated within large secretory granules whose
content is shed into the extracellular matrix (Bueno et al., 1996).
During regeneration the expression pattern of the genes encoding
minimal astacins did not change significantly in trunk fragments, while
some labelled cells began to be detected in the stump of head fragments
only after 3 days of regeneration. This suggests that a putative role for
these minimal astacins during regeneration should be played in later
regeneration steps, when body proportion reestablishment is needed.
Interestingly, as also demonstrated for TCEN49 (Bueno et al., 1996), the
reappearance of transcripts encoding minimal astacins occurs con-
comitantly to the major morphological changes of pharynx regenera-
tion. Pharynx regeneration is a complex process that occurs in the
stump region. Its reformation starts early during regeneration, begins
with the accumulation of pharynx precursors 1/2 days after cut and
only around 4/5 days (for head pieces) a true pharynx primordia be-
come observable, protrudes in a novel pharynx cavity and connects its
novel lumen with that of the gut (Kobayashi et al., 1999). ECM re-
modeling enzymes may play a fundamental role in such morphallactic
process that includes tissue degradation and remodeling, as well as cell
migration processes. Although we cannot exclude that pharynx re-
generation plays a driving role for the reformation of the two-ring-
shaped structure, an intriguing hypothesis is the opposite scenario, in
which minimal astacins are required for the ECM remodeling needed
for pharynx reformation. Future functional studies might shed light on
this possibility. A similar function has also been proposed in C. elegans
where a surprisingly large number of astacins have been found ex-
pressed in the marginal cells of the pharynx and supposed to be secreted
towards the basement membrane and involved in processing basement
membrane components of the pharynx (Park et al., 2010).
The ast-8 expression in the digestive system is a feature typically
conserved in genes encoding astacins with a ShToxin domain. For ex-
ample, in Podocoryne carnea, expression of a gene encoding an astacin
with the ShK toxin domain (PMP1) is concentrated to the manubrium,
M.E. Isolani et al. Gene Expression Patterns 28 (2018) 77–86

Fig. 5. Expression of Smed-ast-8 in intact S. mediterranea. (A) Representative image of colorimetric in situ hybridization for Smed-ast-8. Scale bar corresponds to 200 μm. Anterior is at
the top. (B–C) Smed-ast-8 positive cells are visualized by in situ hybridization on a wax section. Scale bar corresponds to 50 μm in B and 10 μm in C. g: gut; gl: gut lumen; ph: pharynx.
Dorsal is at the top. (D) Representative image of double fluorescent in situ hybridization showing transcript distribution of Smed-ast-1 (green) and Smed-ast-8 (red). Scale bar corresponds
to 200 μm. Anterior is at the top. (E) High magnification of Smed-ast-1- (green) and Smed-ast-8-positive cells. Scale bar corresponds to 10 μm. (F) Transverse section of an intact S.
mediterranea hybridized with Smed-ast-8 (Red) and smed-rapunzel (green) probes. Nuclei (blue) are stained with Hoechst 33342. Scale bar corresponds to 10 μm.

the feeding organ of this organism (Pan et al., 1998). Ast-8 is specifi-
cally expressed in goblet cells, secretory cells that release enzymes
(Garcia-Corrales and Gamo, 1986), as demonstrated by the exclusive
colocalization with the goblet cell marker smed-rapunzel (Reuter et al.,
2015). This strongly suggests that the presence of the ShK toxin domain
in the planarian ast-8 is required for digestion. Conflicting with this
possibility Goupile and coworkers did not found ast-8 in planarian re-
gurgitate (Goupil et al., 2016).
Despite their discovery in late 1960 (Sonneborn et al., 1969), as-
tacins metalloproteinases, especially those with a minimal structure,
remain an understudied family of proteins and only a few members
have been analyzed in details. For example, it is known that minimal
astacins possess broad substrate specificity with possible redundant, yet
unknown, functions (Gomis-Rüth et al., 2012; Möhrlen et al., 2006).
Our results will allow, according to structural data and tissue expression
specificity, to select groups of astacins for co-silencing experiments in
way to preferentially impact in the dynamics of specific ECM compo-
nents in selective body regions or in a particular temporal window
during regeneration.
Section 4
4.1. Animals
A clonal strain of the asexual Schmidtea mediterranea and an asexual
strain (GI) of the planarian Dugesia japonica were used in this study.
Animals were kept in artificial water (for D. japonica: CaCl2 2.5 mM;
MgSO4 0.4 mM; NaHCO3 0.8 mM; KCl 77 μM; for S. mediterranea: NaCl
1.6 mM; CaCl2 1 mM; MgSO4 1 mM; Mg Cl2 0.1 mM; NaHCO3 1.2 mM)
at 18 °C, and starved for at least a week before being used in the ex-
periments. For regeneration studies, animals were cut posterior to the
auricles or across the pharynx.
4.2. Identification and characterization of S. mediterranea and D. japonica
astacin sequences by in silico analysis
Genes encoding astacins of other organisms were used to query all
the S. mediterranea transcriptomes available in Planmine database
(http://planmine.mpi-cbg.de/planmine/begin.do; Brandl et al., 2016).
Several sequences were identified, assembled and used to query, by
discontiguous megablast analysis, S. mediterranea genome database and
D. japonica transcriptome shotgun assembly. Transcripts with sig-
nificant homology were taken. Multialignment and phylogenetic tree
were obtained by using www.Phylogeny.fr site, which combines:

83
M.E. Isolani et al. Gene Expression Patterns 28 (2018) 77–86

Fig. 6. Expression of Smed-ast-2 during regeneration. Smed-ast-2 was used as a representative marker for astacins of the two-ring-shaped structure. (A) Animals were cut through the
pharynx (and thus through the two-ring-shaped structure). Regenerating fragments were sacrificed 1, 3, 6, 8 or 10 days after cut. Blastemal region is outlined with dotted black lines in 3
day regenerating fragments. Red asterisks indicate the new pharynx rudiment in regenerating tail fragments. (B) Animals were cut behind the pharynx, directly under the head, in way to
produce head pieces devoid of the two-ring-shaped structure and tail pieces with a whole two-ring structure. Regenerating fragments were sacrificed 1, 3, 6, 8 or 10 days after cut.
Blastemal region is outlined with dotted black lines in 3 day regenerating fragments. Red asterisks indicate the new pharynx rudiment in regenerating head fragments. Ph: pharynx. Scale
bars correspond to 500 μm.

84
M.E. Isolani et al.
MUSCLE for multialignment; PhyML for phylogeny analysis and
treeDyn for phylogenetic three rendering. The signal peptide was pre-
dicted using SignalP 4.1 on the server http://www.cbs.dtu.dk/services/
SignalP/ (Petersen et al., 2011). All identified genes and predicted
proteins were named based on their structural motifs with the no-
menclature proposed by Reddien et al. (2008). Analysis of genomic
organization and exon/intron boundaries was performed by aligning
the cDNA sequence of each astacin gene with the corresponding
genomic contig.
4.3. In situ hybridizations
DNA templates for S. mediterranea astacins were prepared by RT-
PCR from wild-type planarian cDNA, by using primer pairs including a
T7 promoter adapted reverse primer, as described in Supplementary
Table 1. Purified amplification products were in vitro transcribed in the
presence of DIG-labelling mix (Roche) to obtain digoxigenin (DIG)-la-
belled RNA probes or in the presence of fluorescein-labelling mix
(Roche) to obtain fluorescein-labelled RNA probes. For colorimetric and
double fluorescent in situ hybridization experiments on S. mediterranea,
specimens were treated with N-acetyl-cysteine, fixed and reduced as
described by Pearson et al., (2009). Bleaching, hybridization and signal
detection was performed according to King and Newmark (2013). For
regenerating fragments proteinase K treatment was replaced by HIAR
treatment as described by King and Newmark (2013). Colorimetric in
situ hybridization on D. japonica specimens was performed according to
Rossi et al. (2014) using heterologous probes from S. mediterranea and
reducing the hybridization temperature from 55 °C to 52 °C. Colori-
metric in situ hybridization on paraffin-embedded sections (8 μm
thickness) was performed as described by Kobayashi et al. (1998). For
double fluorescent section in situ hybridization planarians were fixed as
described by Kobayashi and coworkers (Kobayashi et al., 1998), hy-
bridization and signal detection was performed according to King and
Newmark (2013). Images of colorimetric whole mount hybridization
were captured on a Nikon SMZ1500 stereomicroscope with Leica
DFC310FX camera using the Leica LAS AF Software. Images of colori-
metric in situ hybridization on wax sections were taken using Nikon
Eclipse E600 microscope with Digital Sight Camera NIS-Elements.
Specimens from double fluorescent in situ hybridization experiments
were mounted in 80% glycerol, and scanned under the Leica TCS SP8
confocal microscope. At least 10 animals were hybridized for each
probe or probes combination. Adobe Photoshop was used to orient,
scale and adjust images and improve clarity.
Funding
This work was supported by University of Pisa funding (Rating
2015-16) to Leonardo Rossi and to Renata Batistoni.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.gep.2018.03.003.
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