Download A twist in sea urchin gastrulation and mesoderm specification

A twist in sea urchin gastrulation
and mesoderm specification
WeiWeng*.Jan Cheethamt.Jeff Hardint and Judith M.Venuti"':t:
*Department of Anatomy and Cell Biology, College of Physicians & Surgeons of
Columbia University, 630 W. I68th Street, New York, NY 10032, U.S.A. and
tDepartment of Zoology, University of Wisconsin, Madison, WI 53706, U.S.A.
Abstract
The bHLH (basic helix-loop-helix) transcription factor, sea urchin myogenic
factor-1 (SUM-1), plays an important role in myogenic determination during sea
urchin embryogenesis. SUM-1-mediated transactivation is restricted to the
mesenchyme lineages in transgenic sea urchin embryos, suggesting that other
factors, either positive or negative, influence the activity of SUM-1 in different
embryonic cell types. While post-translational regulation of vertebrate myogenic
factors has been suggested from in vitro studies, it has never been demonstrated in
vivo. The most compelling in vitro experiments have shown that the mesodermal
bHLH, twist, negatively regulates myogenic bHLHs. However, in the vertebrate
embryo, twist and myogenic bHLHs are not expressed coincidentally, and
different concentrations of twist playa role in the differentiation of different
muscle lineages (somatic versus visceral) in Drosophila embryos. The gene
expression studies in vertebrates and the genetic experiments in Drosophila
suggest disparate roles for twist in these organisms. To gain a better understanding
of the role of twist in mesodermal and myogenic specification, we cloned a sea
urchin twist homologue and characterized its role in gastrulation and myogenesis
in this simple embryo. Our data suggest that twist from Lytechinus variegatus
functions after gastrulation and initial specification of the embryonic mesoderm
of the sea urchin.
Introduction
The experiments of Horstadius in the first half of this century highlighted the
necessity for cell-cell interactions in the development of different cell types of the
sea urchin embryo [1). His experiments established a role for cell--cell signalling in
the specification of cell fates in the sea urchin . Horstadius showed that the
micromeres influenced the differentiation of other embryonic cells, setting the
stage for present day experiments which have begun to unravel the molecular
pathways involved in micromere signalling and the specification of other
embryonic lineages. It is now thought that the derivatives of the micromeres (the
skeletogenic or primary mesenchyme cells) are autonomously specified during the
tTo whom correspondence should be addressed.
153
I S4
W.Weng et al.
early cleavage stages. However, it is still not clear when and how the different
secondary mesenchymal lineages are specified during sea urchin embryonic
development [2].
Cell-lineage analysis of the vegetal plate of the mesenchyme blastula­
stage embryo suggested that lineages that arise from this region, the endoderm and
secondary mesenchyme cells, are specified prior to the initiation of gastrulation
[3]. Cell dissociation experiments have substantiated this for the endoderm [4J,
but when and how th e different secondary mesenchyme lineages are specified is
uncertam.
The only secondary mesenchyme lineage for which we have sufficient
molecular information concerning its specification is the myogenic lineage, which
is specified during early to mid-gastrulation through a programme involving a sea
urchin homologue of the vertebrate MyoD family, SUM-l [5,6]. This
transcription factor was cloned from Lytechinus va riegatus and shown to be
expressed in the presumptive muscle cells before overt myogenic differentiation
takes place towards the end of gastrulation [5,6]. When we examined the activity
of SUM-l in different embryonic lineages we found that the transactivational
activity of SUM-l was restricted to the mesenchymal lineages (results not shown) .
Further analysis of SUM -1 and its role in the specification of the myogenic lineage
has led us to examine other factors which might playa role in mesoderm specifi­
cation in the sea urchin embryo.
Studies of mesodermal development in Drosophila have identified a
gene, twist, which is essential for the earliest formation of the mesoderm in the fly
[7]. Twist, like SUM-I , is a member of the HLH family of trans crip tional
regulators which are important in a variety of determinative events during
development. Twist homologues have now been identified in the human [8],
mouse [9], frog [10], lancelet [11J, Drosophila virilis [12], Tribolium [13] leech [14]
and Caenorhabditis elegans [15] .
Drosophila twist homozygous-null embryos fail to make mesoderm [16],
suggesting a role in the earliest specification of this tissue. However, initial analysis
of mouse embryo twist mutants suggests that murine twist functions after gastru­
lation [17]. Whereas twist from Drosophiu. was shown in vivo to act as a transcrip­
tional activator for early mesoderm-specific genes, in vitro analysis of murine twist
demonstrated that it ca n act as a repressor of myogenic genes [18]. Gene expression
studies in vertebrates [19] and genetic experiments in Drosophila [20] suggest
disparate roles for twist in these different organisms . We therefore wish to use our
transac tivation assay as a functional test to better understand the role of twist in
mesodermal and myogenic specification. To begin, we have identified a sea urchin
twist homologue and characterized its role in gastrulation and myogenesi s.
Surprisingly, higher levels of Lv -twist (twist from L. variegatus) transcripts are
expressed following the initiation of gastrulation, suggesting that the primary
function of sea urchin twist occurs after the early specification of the mesoderm.
Materials an
Sea urchin er
Lytechinus vari
U.S.A.) or Beau
variegatus embl
[6,7].
Molecular cI(J
Two degenerat(
(G/A/T/C)ATG
(T/C)TT(G/ AfT
used to PCR-am
stage endomeso(
University, Pro"
of each primer v
KCI, 100 jJ.g/ml
(Fisher Scientific
(30 s at 94°C and
extension at 72°1
itated with ethar
with SalI, gel pur
U.S .A.). Clones 1
and were analyse
Reverse tram
RNA
L. variegatus
e~
Millipore-filterel
using Tri-reagem
30 min of DNa:
(25 :24:1, by vol.
RNA was anneal
TTGATCTTCT
carried out using
(Gibco, BRL). ,
amplification wi!
CCACAATATI
with 30 cycles at
Immunocytol
Embryos were s
minimum of 20 r
temperature WI I
phosphate buffe
Embryos fixed i
(PBST); subseql
'I!lI.................
Sea urchin gastrulation and mesoderm specification
the different
. n embryonic
,w-
y me blastula­
endoderm and
of gastrulation
eendoderm [4J,
are specified is
' have sufficient
lineage, which
einvolving a sea
-1 [5,6]. This
td shown to be
I differentiation
r ed the activity
ra nsactivational
ults not shown).
I
. 1meage
Iyogemc
soderm specifi­
0
lve identified a
[)derm in the fl y
transcriptional
events during
the human [8J ,
'J [13] leech [14]
mesoderm [16J,
" initial analysis
,ns after gas tru ­
:t as a transcrip­
of murine twist
;ene expression
14 [20J suggest
wish to use our
role of twist in
ied a sea urchin
d myogenesiso
transcrtpts are
at the primary
~ mesoderm.
Materials and methods
Sea urchin embryo culture
Lytechinus variegatus adults were obtained from Susan Decker (Davie, FL,
U.S.A.) or Beaufort Biologicals (Duke Marine Lab, Beaufort, NC, U.S.A.). L.
variegatus embryos were cultured by stirring at 17°C as described previously
[6,7].
Molecular cloning of the bHLH domain of the Lv-twist
Two degenerate primers [primer 1: 5'-CCCTCGAG(C/A)G(G/A/T/ C)GT
(G / A/T/ C)ATGGC (GI AlT/C)AA(T/C)GT-3' and primer 2: 5'-CCGTCGAC
(TIC)TI(GI Aff IC)A(GIA)(GI Aff IC)GT(T/C)TG(GIAff)AT(T/C)TI-3'J were
used to PCR-amplify a twist-specific bHLH domain from an L. variegatus prism
stage endomesoderm enriched eDNA library (provided by Gary Wessel, Brown
University, Providence, RI, U.S.A.). A 1 f.LI portion of eDNA library and 0.5 f.Lg
of each primer were mixed in 20 mM Tris-HCI (pH 8.3), 2 mM MgCI 2, 25 mM
KCI, 100 f.Lg/ml gelatin, 50 mM dNTP and 5 units of Taq DNA Poly merase
(Fisher Scientific, Pittsburgh, PA, U.s.A.) in a 50 f.L1 reaction volume. Forty cycles
(30 s at 94°C and 3 min at 50°C) of PCR reaction was followed by a single 8 min
extension at 72°e. PCR reaction products were extracted with phenol, precip­
itated with ethanol and resuspended in deionized H 20 . The DNA was digested
with Sail, gel purified and ligated into pBluescriptIIKS+ (Strata gene, La Jolla, CA,
U.S.A.). Clones were sequenced using the dideoxy chain-termination method [21J
and were analysed with the BLAST search engine (NCB I).
Reverse transcriptase-PCR (RT-PCR) analyses of embryonic
RNA
L. variegatus embryos were cultured to appropriate developmental stages in
Millipore-filtered artificial sea water (MFASW) at 17°e. Total RNA was prepared
using Tri-reagent (Molecular Research Center, Inc., Cincinnati, OH), followed by
30 min of DNase I treatment at 37°C, phenollchloroform/iso-amyl alcohol
(25:24:1, by vol.) extraction and ethanol precipitation. A 10 f.Lg portion of total
RNA was annealed with 5 pg of a sequence-specific oligonucleotide (5' -ATCTI­
TTGATCTICTICGTCACTC-3 ' ) at 65°C for 2 min. Reverse transcription was
carried out using SuperScript II RTase, following the manufacturer's instructions
(Gibco, BRL). About 2 f.LI of the 20 f.LI total RT reaction was used for PCR
amplification with the reverse primer mentioned above and a forward primer (5'­
CCACAATATITCGAAGAACTICAG-3'). PCR amplification was performed
with 30 cycles at 94°C for 2 min, 55°C at 1 min and 72°C at 1.5 min.
Immunocytochemistry
Embryos were stained as whole mounts after fixation in - 20°C methanol for a
minimum of 20 min, or in 3.7% paraformaldehyde in MFASW for 30 min at room
temperature with similar results. Methanol-fixed embryos were w;lshed in
phosphate buffered saline (PBS) and incubated with antibodies diluted in PBS .
Embryos fixed in paraformaldehyde were washed in PBS with 0.1 % Tween 20
(PBST); subsequent incubations and washes were performed in PBST. Primary
155
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W.Weng et al.
antibodies were incubated for 1 h to overnight, followed by 3 X 5 min washes.
Secondary antibodies were incubated for a minimum of 30 min, followed by 3 X 5
min washes . Embryos were mounted in PBS/glycerol (50:50, v/v) and viewed with
a Zeiss Axiovert 100 Epifluorescence microscope. Embryos were photographed
with an Olympus OM-1 35 mm camera using Kodak 400 ASA Gold print film , or
a Dage video camera attached to a Power Computing computer equipped with
Scion imaging software.
Antibodies
Anti-(Drosophila twist) polyclonal antibodies were obtained from Bruce
Patterson (NCI, NIH, Bethesda, MD, U .S.A.) and Han Nyugen (Albert Einstein
College of Medicine, Bronx, NY, U .S.A .) and used at 1:200 dilution . Goat anti­
mouse FITC secondary antibody was diluted 1:1 00 in PBS (Cappel, ICN
Pharmaceuticals, Inc., Costa Mesa, CA, U .S.A.).
Results and discussion
Isolation of a twist homologue in L variegatus
To isolate a twist homologue from L. variegatus, degenerate primers were designed
to two conserved regions of the Drosophila twist bHLH domain (Figure 1A)
Figure 1
A.
RT-PC
L. variegah
embryon
stage
RT-PCR analysis
RNA isolated from dij
primers (rom those I
gastrulation has cea
mesoderm. Abbreviati(
M-Gast, early and mi
control.
and used to am
mesoderm-enric
123 bp fragment
species (Figure 1
observed for the'
to be most similal
addition, high sin
bHLH proteins,
eHAND [25].
5' -<X:C'l.'CGIIGC~-3 ' --.
A
C
Temporal exp
Prbnet ... J
Dm-Twist
QRVMANVIIBRQRTQSLIIDAFKS LQQ lIP - -- -'rLP--- --s DXLS:.t QTLKLII!l'R YID FL
3'
5'
-=
T
AAG'l"l"l'G IIGAIII'rl".l'CAGC'I.'OC C­
G
C
A
C
-..
B.
"'liz I
LOOP
B.JJJr u
QRVLARVRBRQRTQSLRDAFARLRKIIP----'rLP-----sDKLSKIQTLK
Primcr-l ... .1c
Lv-Twist
M-'l'IfiBt
R-'l'Ifist
X-'l'IfiBt
Rro-Twist
Bb-Twist
Dv-Twist
Dm-Twist
Ce-twi8t
QRV M AHVRERQR~SLREAFAaLRKIIP----TLP-----SDXL8KIQTLK
QRVMAll\lRERQ~SLll£All'AaLRKI IP- --- TLP --- --SDXLSItI QTLK
QRVM AHVRERQ~SLREAP SS LRKIIP----TLP-----SDKLSKIQTLK
QRVLARVRBRQRTQSLRDAFPQLRKI VP--- -'rLP-- --- SDKLSKIQTLK
QRVLARVRBR -RTQSIMEAFSSLRKI IP- ---TU' --- --SDKLSKI QTLK
QRVMAll\lRERQ~SLRDAF KAI.QQ IIP- --- TLP --- --SDXLSKIQTLK
QRVM All\lRERQ~SLIIDAF KS
IIP----TLP-----SDKLSKIQTLK
QRACARRRBRQRTKELRDAFTLLRKL IP----SMP-----SDKMSKIHTLR
.-­
92\
92\
92\
92\
90\
88\
88\
65\
Primer-2
RNA from diffe
when Lv-twist t
Lv-twist are pre:
reappear during:
The zygotic expr
the completion
subsequent to the
role in later devel,
Spatial expres
Immunocytoche
antibodies gener;
localization of tw
is spatially restrict
potential role in t!
PCR cloning ofthe Lv-twist bHLH domain
(A) Degenerate oligonucleatide primers to conserved regions o( the Drosophila twist bHlH were used to
Summary
ampli(y lv-twist (rom a prism-stage sea urchin mesendoderm-enriched eDNA library. These
oligonucleotide primers recognize residues in the bHlH that are specific to twist (B) Comparison o( the
lv-twist bHLH sequence with those o(the murine (M-Twist), human (H-twist), Xenopus laevis (X-twist),
leech (Hro-Twist),lancelet (Bb- Twist), D. virilis (Dv-twist), D. melanogaster (Om-twist), and C. elegans (Ce­
The sea urchin tv.
of other species,
dimerization mo
twist) twist sequences shows the twist bHlH is highly conserved amongst these different species.
i l r l l. .. .~~. . . . . .. .~
--- Sea urchin gastrulation and mesoderm specification
5 min washes.
owed by 3 X 5
od viewed with
•
photographed
d print film, or
~quipped with
Figure 2
RT-PCR
L. variegatus
embryonic
HB
E l
MB
E
M
l
Gast
l'
Pr
+­
Coni
stages
RT-PCR analysis of Lv-twist expression
RNA isolated from different-stage L variegatus embryos was analysed by RT-PCR using a different set
of
of twist transcripts are found after
gastrulation has ceased. This suggests that Lv-twist functions after the initial specification of the
primers from those used to clone the bHLH. The highest levels
d from Bruce
~lbert Einstein
ion. Goat anti­
,( Cappel, ICN
5 were
designed
in (Figure 1A)
~_r-2
~c-
!LK
!Lit
!LIt
!LIt
92'
92\
92\
~
92'
!LIt
!Lit
!LK
90'
88\
88'
65'
lUI.
mesoderm. Abbreviations:HB, hatching blastula; £- and L-MB, early and late mesenchyme blastula; £- and
M-Gast, earfy and mid-stage gastrula; L-Gast and L' -Gast, two separate late gastrula; Pr, prism; Cont,
control.
and used to amplify a -123 bp DNA fragment from a late gastrula-stage
mesoderm-enriched cDNA library. The deduced amino acid sequence of the
123 bp fragment was compared with the bHLH regions of twists from other
species (Figure 1B). An average of 87 % identity and over 90% similarity was
observed for the bHLH regions of twists from other species. Lv-tw ist was found
to be most similar to the bHLH regions of Xenopus, mouse and ascidian twists. In
addition, high similarities were shared with other vertebrate mesodermal-specific
bHLH proteins, including Dermo-1 [22], Paraxis/Meso1 [23], Scleraxis [24] and
eHAND [25].
Temporal expression pattern of Lv-twist
~YIDFL
liLH were used to
RNA from different embryonic stages was analysed by RT-PCR to determine
when Lv-twist transcripts are expressed during development. Transcripts of
Lv-twist are present at low levels at the blastula stage but then disappear and
reappear during gastrulation, with highest levels at the pluteus stage (Figure 2).
The zygotic expression pattern of Lv-twist transcripts reveals highest levels after
the completion of gastrulation . These data suggest that Lv-twist functions
subsequent to the establishment of the different germ-layers and has an important
role in later developmental events.
SpatiaJ expression of Lv-twist
Immunocytochemical analysis of Lv-tw ist expression employed polyclonal
antibodies generated to the Drosophila twist protein. The immunocytochemical
localization of twist in developing sea urchin embryos suggests that most Lv-twist
is spatially restricted to the mesendoderm of the gastrula stage embryo, indicating a
potential role in the specification of lineages that arise from this region (Figure 3C).
Summary
IIA library. These
Comporison of the
us laevis (X-twist),
WId C. elegans (Ce­
• species.
157
The sea urchin tw ist homologue, Lv-tw ist, has high sequence similarity to twists
of other species, suggesting conserved function through its DNA binding and
dimerization motifs. Expression of Lv-twist suggests that its primary function
IS.8
W.Wengetal.
Figure 3
Neural
cranial
in the t
mexico.
Indirect immunofluorescent localization
Polyclonal antibodies to !he full length Drosophila twist protein was used to examine !he spatial pattern
Lennart Olssol1
of Lv-twist expression at different embryonic stages. No expreSSion is observed in !he blastula stage (A)
Evolutionary Biol4
and only weak expreSSion is observed in the vegetal plate at the early gastrula (B). Highest levels of
Sweden
protein were deteaed in !he mesendoderm at !he gastrula stage (q.
occurs after the initial specification of the embryonic mesoderm of the sea urchin.
While our data do not preclude the possibility that low levels of maternal or
zygotic Lv-twist protein may function to specify the embryonic mesoderm, high
levels of expression after the initial events of gastrulation suggest that Lv-twist
also serves a later function. It remains to be determined if Lv-twist influences the
activity of the sea urchin myogenic bHLH SUM-I.
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Introduction
The neural crest
an important rol,
features, such a~
neural crest is ah
[3-5].
Surpris
Ambystoma me;
Sellman and HOI
for a long time s.
the development
to gain deeper in ~
not been used. V(
the role of the n~
during axolotl he
Theim
amphibian crani;
perturbing neur
development, as
this should be tt
been thought no
effects on muscle
the connective ti!
[8]. Maybe this is
In this
formation of the
extirpation and /
indirect method
tissues are derive
neu ral crest cell
':'To whom correspOl
! 1~ 1I""""""""·