Development 139, 3786-3794 (2012) doi:10.1242/dev.082230
© 2012. Published by The Company of Biologists Ltd
Autonomy in specification of primordial germ cells and their
passive translocation in the sea urchin
Germ line specification is essential for an organism to produce
offspring. Despite its importance in species propagation, the
process appears highly variable among organisms even within the
same phylum, and probably maximizes the diversity of
reproductive niches. Two major germ line specification
mechanisms have been proposed: autonomous and conditional
specification. Autonomous, or inherited, specification includes
several model organisms such as Drosophila (Williamson and
Lehmann, 1996), C. elegans (Kimble and White, 1981) and
zebrafish (Yoon et al., 1997). Primordial germ cells (PGCs) in these
organisms are specified early in development by inherited
cytoplasmic determinants from maternal stores (Gao and Arkov,
2012). By contrast, conditional, or inductive, processes specify
PGCs later in development by intercellular communication; this
form of specification is primarily responsible for PGCs in mouse
(Lawson and Hage, 1994; Tam and Zhou, 1996), axolotl
(Nieuwkoop, 1947), cnidarians, sponges (Extavour and Akam,
2003) and ascidians (Shirae-Kurabayashi, 2006).
Although the mechanisms of germ line specification vary in each
organism, PGCs share many characteristics. For example,
regardless of the mechanism of specification, autonomous or
conditional, conserved genes are important in this process and
include the RNA helicase vasa, the translational repressor nanos,
and the small RNA regulator piwi. These genes and, depending on
the organisms, many others are responsible for PGC specification,
differentiation and/or maintenance in the germ line among
metazoans (for reviews, see Ewen-Campen et al., 2010; Extavour
and Akam, 2003; Raz, 2000). During specification, the PGCs
initially cluster with each other and then later in development
migrate collectively to specific regions where the gonads will form.
E-cadherin appears to be essential for regulating these events, and
E-cadherin-deficient PGCs fail to cluster, migrate or even specify
themselves as PGCs in mouse, zebrafish and Drosophila (Cano et
al., 2000; Okamura et al., 2003; Marthiens et al., 2010; Kardash et
al., 2010; Matsui, 2010; Saga, 2010; Tarbashevich and Raz, 2010).
Echinoderms are a sister group to the Chordates and are an
early branching group in Deuterostomia. Although the molecular
mechanisms of germ line specification in this phylum are not
known, their eggs do not have obvious preformed germ line
components (Ransick et al., 1996; Juliano et al., 2006) and thus
its germ line is considered to be conditionally specified during
late development. Recent studies, however, suggest that small
micromeres (SMics) formed at the fifth cellular division have
PGC features. The SMics are located at the vegetal tier in
association with endomesodermal precursors, divide more slowly
than their adjacent cells (Tanaka and Dan, 1990), express germ
line-related genes such vasa, nanos and piwi (Juliano et al.,
2006), and are involved in germ cell formation in the adult
(Yajima and Wessel, 2011a). SMics are not necessary for larval
development and contribute only to subregions of the coelomic
pouches (Pehrson and Cohen, 1986), the precursor of the adult
rudiment (see Fig. 7B). After early larval stages, the left
coelomic pouch expands and becomes the major contributor to
the adult rudiment and will develop most adult structures before
metamorphosis. The nascent coelomic pouches formed at the tip
of the archenteron by prism stage consist of 40% cells from the
SMic lineage and 60% from the macromere lineage (Cameron et
al., 1987; Cameron et al., 1991), which is a distinct lineage
derived from the second vegetal tier of the blastomeres (Veg2)
formed at the fifth cell division. Therefore, it is intriguing to
hypothesize that SMics are PGCs and Veg2 macromeres are a
somatic multipotent cell lineage.
We tested here several molecular and morphological features of
SMics that are typical of PGCs, such as autonomous expression of
germ line-related molecules, passive de-epithelialization and
translocation behavior during gastrulation, Cadherin-dependent cell
MCB Department, Brown University, 185 Meeting Street, BOX-GL173, Providence,
RI 02912, USA.
*Authors for correspondence (email@example.com; firstname.lastname@example.org)
Accepted 28 July 2012
The process of germ line determination involves many conserved genes, yet is highly variable. Echinoderms are positioned at the
base of Deuterostomia and are crucial to understanding these evolutionary transitions, yet the mechanism of germ line specification
is not known in any member of the phyla. Here we demonstrate that small micromeres (SMics), which are formed at the fifth cell
division of the sea urchin embryo, illustrate many typical features of primordial germ cell (PGC) specification. SMics autonomously
express germ line genes in isolated culture, including selective Vasa protein accumulation and transcriptional activation of nanos;
their descendants are passively displaced towards the animal pole by secondary mesenchyme cells and the elongating archenteron
during gastrulation; Cadherin (G form) has an important role in their development and clustering phenotype; and a left/right
integration into the future adult anlagen appears to be controlled by a late developmental mechanism. These results suggest that
sea urchin SMics share many more characteristics typical of PGCs than previously thought, and imply a more widely conserved system
of germ line development among metazoans.
KEY WORDS: Vasa, PGC, Germ line, Cadherin, Sea urchin, Strongylocentrotus purpuratus
Mamiko Yajima* and Gary M. Wessel*
PGC specification in sea urchin
specification and clustering, and we conclude that several
overarching mechanisms appear conserved between the SMic
lineage and the more widely studied PGCs, such as Drosophila
MATERIALS AND METHODS
Animals, embryos and larval culture
S. purpuratus were collected in Long Beach, CA, USA, and housed in
aquaria containing artificial seawater (ASW; Coral Life Scientific Grade
Marine Salt; Energy Savers Unlimited, Carson, CA, USA) at 16°C.
Gametes were acquired by 0.5 M KCl injection. Eggs were collected in
ASW and sperm were collected dry. To obtain embryos, fertilized eggs
were cultured in ASW or Millipore-filtered seawater (MFSW) at 16°C.
When early stage embryos were required for blastomere labeling,
fertilization was performed in the presence of 1 mM 3-aminotriazol
(Sigma, St Louis, MO, USA) to inhibit cross-linking of the fertilization
envelope. Before labeling, envelopes were removed by gentle pipetting.
Chemical treatment and immunolabeling
Whole-mount immunostaining was conducted as described previously
(Yajima, 2007b; Yajima and Kiyomoto, 2006). Briefly, late larvae were
fixed with methanol at –20°C for 1 hour and rinsed twice in PBS
saturated with calcite (PBSC). Washed specimens were immunostained
with 1:300 SpVasa antibody (Voronina et al., 2008) for 3 hours at room
temperature, and after rinsing with PBSC they were reacted with Cy3
goat anti-rabbit immunoglobulin G (IgG) antibody (Invitrogen) for 3
hours at room temperature. After further rinsing with PBSC under the
same conditions, larvae were mounted on a glass slide and observed by
confocal laser microscopy (Zeiss LMS510). FM1-43 (McNeil et al.,
2003) was used at a final concentration of 4 nM and embryos were
treated immediately before imaging. FM1-43 is a lipophilic, membrane-
impermeant fluorescent dye and only cells exposed to the outside of the
embryo will fluoresce in media containing FM1-43 (Covian-Nares et al.,
Injection, blastomere labeling and micromere isolation
mRNA was transcribed from Vasa-GFP (Gustafson et al., 2011) and
membrane-mCherry (Megason and Fraser, 2003) constructs using the SP6
mMessage mMachine (Ambion). SpG-cadherin morpholino (G-cad MO)
was prepared by Gene Tools (Philomath, Oregon, USA) and was designed
against the 5? UTR of SpG-cadherin: 5?-TCCACCTCGGATTTAC -
AGCCATCGT-3?. Injection into fertilized eggs was performed as described
(Yajima et al., 2007) using ~6 pl of injection mix (mRNA, MO, dye). The
blastomere injection for the Mic, SMic, mesomere or macromere was
performed iontophoretically with an Axoporator 800A (Molecular Devices,
Sunnyvale, CA, USA) either at the 16-cell or 32-cell stage as follows:
embryos were placed in a glass chamber as previously described (Yajima,
2007b) and the fluorescent dye (Fluoro-ruby, Sigma) and/or 0.1 mM G-cad
MO was injected into each blastomere while being observed by
fluorescence microscopy (Zeiss Axioplan). Mics were isolated using a
glass needle at the 16-cell stage as described (Yajima, 2007a) and were
cultured in plastic Petri dishes with MFSW at 16°C. The distance of PMC
migration and the number of cell divisions in the culture were manually
calculated from images selected randomly from five clusters of isolated
Embryos were injected with Vasa-GFP mRNA, and the resulting Vasa-GFP
protein became enriched in the SMic lineage (Gustafson et al., 2011).
Membrane-mCherry mRNA was co-injected to better visualize the
membrane movements of the SMics and other cells in the embryo. For
prolonged visualization, embryos were embedded in a 0.5% soft agar plate
to immobilize them during the recording. For time-lapse recordings,
images were taken every 25 minutes for 10-15 hours, and five to seven z-
stacks were collected with a depth of 35 m for each time point using a
Zeiss 710-2 photon confocal microscope at MBL, Woods Hole, MA, USA.
The resultant z-stack images were projected using Zen 200 software (Zeiss)
and movies were made using ImageJ software (NIH).
Small micromeres autonomously express and
maintain germ line determinants
Small micromeres (SMics) are formed at the fifth division in the S.
purpuratus embryo and contribute to germ cell formation in the
adult (Yajima and Wessel, 2011a). To delineate the mechanism of
SMic specification, Vasa-GFP mRNA was injected into fertilized
eggs, and the micromeres (Mics), which are parent blastomeres of
SMics, were isolated at the fourth cell division using a glass needle
and were cultured (n25; Fig. 1A) in a plastic Petri dish with
Millipore-filtered natural seawater (MFSW), which is the same
medium in which the embryos were grown and the SMics were
exposed. This culture approach was previously used to document
that the large micromeres (LMics) develop exclusively and
autonomously into the skeletogenic lineage (Endo, 1966; Okazaki,
1975a; Okazaki, 1975b). The LMics divide several times in culture
and migrate to form spicules in the presence of serum, just as
programmed in vivo. The fate of the sibling cells of LMics, the
SMics, in these culture conditions is unknown. Depending on the
condition of the dissection, all four Mics were isolated together
(Fig. 1B) or one or two of the four were isolated and cultured (Fig.
1C). In both cases, 45-60 minutes after the beginning of the culture
procedure, these cells asymmetrically divided and formed the
sibling LMics and SMics. Vasa protein selectively accumulated
asymmetrically into the SMics, just as it does in vivo (Fig. 1B,C,
arrows) (Yajima and Wessel, 2011b), such that the skeletogenic
LMics only have background levels of Vasa whereas the SMics
possess strong Vasa signal. Vasa accumulation increased in the
SMics following this unequal division and Vasa accumulated in its
typical, perinuclear and granular-like structure (Fig. 1B,C, right two
panels). This post-translational mechanism of Vasa regulation as
seen in vivo (Gustafson et al., 2011) was faithfully replicated in
these isolated culture conditions.
We cultured isolated Mics up to 5 days postfertilization in
MFSW. At 48 hours postfertilization (hPF) (day 2), the LMic
lineage demonstrated weak (background level) Vasa-GFP
expression and migrated extensively with extended filopodia (in
five explants the minimum migration distance was 12±1 m and
maximum was 66±6 m), whereas the SMic lineage cells remained
adherent to each other in their original aggregate, did not divide,
and maintained the typical (perinuclear and granular) Vasa
expression (Fig. 1D). From day 3 to day 5, the LMic descendants
underwent further cell divisions, reaching 14-36 cells per cluster
(up to five cell divisions, n5) with a corresponding decrease in
size, whereas the SMic rarely underwent a cell division (once,
maximally), remained within their original site, and retained
perinuclear Vasa expression (Fig. 1E). This in vitro phenotype of
no migration and slow cell division of the SMics further replicated
the in vivo phenotype. Additionally, we tested the ability of these
cells to undergo new transcriptional activation. nanos is a
translational repressor and is usually found in PGCs. In the sea
urchin, nanos is expressed and maintained specifically within the
SMic lineage (Juliano et al., 2006; Juliano et al., 2010; Fujii et al.,
2009). We tested the ability of SMics to activate nanos
transcription in culture by qPCR. nanos mRNA accumulation was
quantified in isolated Mic descendants after 1 day in culture and
immediately after isolation at the 16-cell stage. These values were
then compared with embryos both before nanos expression (2-cell
stage) and at the late blastula stage when nanos accumulation is
maximal. We found that nanos transcriptional activation in vitro
closely replicates its activation in embryos (Fig. 1F) (Juliano et al.,
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