While many general features of cell fate specification in the sea urchin embryo are understood, specific factors associated with these events remain unidentified. SpOtx, an orthodenticle-related protein, has been implicated as a transcriptional activator of the aboral ectoderm-specific Spec2a gene. Here, we present evidence that SpOtx has the potential to alter cell fates. SpOtx was found in the cytoplasm of early cleavage stage embryos and was translocated into nuclei between the 60- and 120-cell stage, coincident with Spec gene activation. Eggs injected with SpOtx mRNA developed into epithelial balls of aboral ectoderm suggesting that SpOtx redirected nonaboral ectoderm cells to an aboral ectoderm fate. At least three distinct domains on SpOtx, the homeobox and regions in the N-terminal and C-terminal halves of the protein, were required for the morphological alterations. These same N-terminal and C-terminal regions were shown to be transactivation domains in a yeast transactivation assay, indicating that the biological effects of overexpressing SpOtx were due to its action as a transcription factor. Our results suggest that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate.

Reference

Acampora
D.
,
Mazan
S.
,
Lallemand
Y.
,
Avantaggiato
V.
,
Maury
M.
,
Simeone
A.
,
Brulet
P.
(
1995
)
Forebrain and midbrain regions are deleted in Otx2/ mutants due to a defective anterior neuroectoderm specification during gastrulation.
Development
121
,
3279
3290
Bally-Cuif
L.
,
Gulisamo
M.
,
Broccoli
V.
,
Boncinelli
E.
(
1995
)
c-otx2 is expressed in two different phases of gastrulation and is sensitive to retinoic acid treatment in chick embryos.
Mech. Dev
49
,
49
63
Benson
S. C.
,
Sucov
H. M.
,
Stephens
L.
,
Davidson
E. H.
,
Wilt
F.
(
1987
)
A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. I. Authentication of the cloned gene and its developmental expression.
Dev. Biol
120
,
499
506
Cohen
S. M.
,
Jurgens
G.
(
1990
)
Mediation of Drosophila head development by gap-like segmentation genes.
Nature
346
,
482
485
Chomczynski
P.
,
Sacchi
N.
(
1987
)
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem
162
,
156
159
Coffman
J. A.
,
McClay
D. R.
(
1990
)
A hyaline layer protein that becomes localized to the oral ectoderm and foregut of sea urchin embryos.
Dev. Biol
140
,
93
104
Davidson
E. H.
(
1989
)
Lineage-specific gene expression and the regulativecapacities of the sea urchin embryo: A proposed mechanism.
Development
105
,
421
445
Davidson
E. H.
(
1991
)
Spatial mechanisms of gene regulation in metazoan embryos.
Development
113
,
1
26
Durfee
T.
,
Becherer
K.
,
Chen
P.-L.
,
Yang
Y.
,
Kilburn
A. E.
,
Lee
W.-H.
,
Elledge
S. J.
(
1993
)
The retinoblastoma protein associates with the protein phosphatase type I catalytic subunit.
Genes Dev
7
,
555
569
Feilotter
H. E.
,
Hannon
G. J.
,
Ruddell
C. J.
,
Beach
D.
(
1994
)
Construction of an improved host strain for two hybrid screening.
Nucleic Acids Res
22
,
1502
1503
Finkelstein
R.
,
Boncinelli
E.
(
1994
)
From fly head to mammalian forebrain: the story of otd and Otx.
Trends Genet
10
,
310
315
Finkelstein
R.
,
Perrimon
N.
(
1990
)
The orthodenticle gene is regulated by bicoid and torso and specifies head development.
Nature
346
,
485
488
Finkelstein
R.
,
Smouse
D.
,
Capaci
T. M.
,
Spradling
A. C.
,
Perrimon
N.
(
1990
)
The orthodenticle gene encodes a novel homeo domain protein involved in the development of the Drosophila nervous system and ocellar visual structures.
Genes Dev
4
,
1516
1527
Gagnon
M. L.
,
Angerer
L. M.
,
Angerer
R. C.
(
1992
)
Postranscriptional regulation of ectoderm-specific gene expression in early sea urchin embryos.
Development
114
,
457
467
Gan
L.
,
Klein
W. H.
(
1993
)
A positive cis-regulatory element with a bicoid target site lies within the sea urchin Spec2a enhancer.
Dev. Biol
157
,
119
132
Gan
L.
,
Mao
C.-A.
,
Wikramanayake
A.
,
Angerer
L. M.
,
Angerer
R. C.
,
Klein
W. H.
(
1995
)
An orthodenticle-related protein from Strongylocentrotus purpuratus.
Dev. Biol
167
,
517
528
Gan
L.
,
Wessel
G. M.
,
Klein
W. H.
(
1990
)
Regulatory elements from the related Spec genes of Strongylocentrotus purpuratus yield different spatial patterns with a lacZ reporter gene.
Dev. Biol
142
,
346
359
Gan
L.
,
Zhang
W.
,
Klein
W. H.
(
1990
)
Repetitive DNA sequences linked to the sea urchin Spec genes contain transcriptional enhancer-like elements.
Dev. Biol
139
,
186
196
Hanes
S. D.
,
Brent
R.
(
1991
)
A genetic model for interaction of the homeodomain recognition helix with DNA.
Science
251
,
426
430
Hardin
P. E.
,
Angerer
L. M.
,
Hardin
S. H.
,
Angerer
R. C.
,
Klein
W. H.
(
1988
)
The Spec2 genes of Strongylocentrotus purpuratus: structure and differential expression in embryonic aboral ectoderm cells.
J. Molec. Biol
202
,
417
431
Hickey
R. J.
,
Boshar
M. F.
,
Crain
W. R.
(
1987
)
Transcription of three actin genes and a repeated sequence in isolated nuclei of sea urchin embryos.
Dev. Biol
124
,
215
227
Kingsley
P. D.
,
Angerer
L. M.
,
Angerer
R. C.
(
1993
)
Major temporal and spatial patterns of gene expression during differentiation of the sea urchin embryo.
Dev. Biol
155
,
216
234
Li
X. X.
,
Shou
W.
,
Kloc
M.
,
Reddy
B. A.
,
Etkin
L. D.
(
1994
)
Cytoplasmic retention of Xenopus nuclear factor 7 (xnf7) prior to the MBT utilizes an anchoring mechanism involving a cis-acting retention domain and several phosphorylation sites.
J. Cell Biol
124
,
7
17
Makabe
K. W.
,
Kirchhamer
C. V.
,
Britten
R. J.
,
Davidson
E. H.
(
1995
)
Cis-regulatory control of the SM50 gene, an early marker of skeletogenic lineage specification in the sea urchin embryo.
Development
121
,
1957
1970
Mao
C.-A.
,
Gan
L.
,
Klein
W. H.
(
1994
)
Multiple Otx binding sites required for expression of the Strongylocentrotus purpuratus aboral ectoderm-specific Spec2a gene.
Dev. Biol
165
,
229
242
Matsuo
I.
,
Kuratani
S.
,
Kimura
C.
,
Takeda
N.
,
Aizawa
S.
(
1995
)
Mouse Otx2 functions in the formation and patterning of rostral head.
Genes Dev
9
,
2646
2658
Niemeyer
C. C.
,
Flytzanis
C. N.
(
1993
)
Upstream elements involved in the embryonic regulation of the sea urchin CyIIIb actin gene: Temporal and spatial specific interactions at a single cis-acting element.
Dev. Biol
156
,
293
302
Nemer
M.
,
Wilkinson
D. G.
,
Traviglini
E. C.
(
1985
)
Primary differentiation and ectoderm-specific gene expression in the animalized sea urchin embryo.
Dev. Biol
109
,
418
427
Nocente-McGrath
C.
,
Brenner
C. A.
,
Ernst
S. G.
(
1989
)
Endo16, a lineage-specific protein of the sea urchin embryo, is first expressed just prior to gastrulation.
Dev. Biol
136
,
264
272
Pannese
M.
,
Polo
C.
,
Andreazzoli
M.
,
Vignali
R.
,
Kablar
B.
,
Barsacchi
G.
,
Boncinelli
E.
(
1995
)
The Xenopus homologue of Otx2 is a maternal homeobox gene that demarcates and specifies anterior body regions.
Development
121
,
707
720
Ransick
A.
,
Davidson
E. H.
(
1993
)
A complete second gut induced by transplanted micromeres in the sea urchin embryo.
Science
259
,
1134
1138
Royet
J.
,
Finkelstein
R.
(
1995
)
Pattern formation in Drosophila head development: the role of the orthodenticle homeobox gene.
Development
121
,
3561
3572
Simeone
A.
,
Acampora
D.
,
Guilisano
M.
,
Stornaluolo
A.
,
Boncinelli
E.
(
1992
)
Nested expression domains of four homeobox genes in the developing rostral brain.
Nature
358
,
687
690
Simeone
A.
,
Acampora
D.
,
Mallamaci
A.
,
Stornaluolo
A.
,
D'Apice
M. R.
,
Nigro
V.
,
Boncinelli
E.
(
1993
)
A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm in the gastrulating mouse embryo.
EMBO J
12
,
2735
2747
Summers
R. G.
,
Musial
C. E.
,
Cheng
P. C.
,
Leith
A.
,
Marko
M.
(
1991
)
The use of confocal microscopy and STERECON reconstructions in the analysis of sea urchin embryonic cell division.
J. Electron Microsc. Tech
18
,
24
30
Theze
N.
,
Calzone
F. J.
,
Thiebaud
P.
,
Hill
R. L.
,
Britten
R. J.
,
Davidson
E. H.
(
1990
)
Sequences of the CyIIIa actin gene regulatory domain bound specifically by sea urchin embryo nuclear proteins.
Mol. Reprod. Dev
25
,
110
122
Tomlinson
C. R.
,
Klein
W. H.
(
1990
)
Temporal and spatial transcriptional regulation of the aboral ectoderm-specific Spec genes during sea urchin embryogenesis.
Mol. Reprod. Dev
25
,
328
338
Wang
D. G.-W.
,
Britten
R. J.
,
Davidson
E. H.
(
1995
)
Maternal and embryonic provenance of a sea urchin embryo transcription factor, SpZF12-1.
Molec. Mar. Biol. Biotech
4
,
148
153
Wang
D. G.-W.
,
Kirchhamer
C. V.
,
Britten
R. J.
,
Davidson
E. H.
(
1995
)
SpZF12-1, a negative regulator required for spatial control of the territory-specific CyIIIa gene in the sea urchin embryo.
Development
121
,
1111
1122
Wei
Z.
,
Angerer
L. M.
,
Gagnon
M. L.
,
Angerer
R. C.
(
1995
)
Characterization of the SpHe promoter that is spatially regulated along the animal-vegetal axis of the sea urchin embryo.
Dev. Biol
171
,
195
211
Wessel
G. M.
,
McClay
D. R.
(
1985
)
Sequential expression of germ layer specific molecules in the sea urchin embryo.
Dev. Biol
111
,
451
463
Wieschaus
E.
,
Perrimon
N.
,
Finkelstein
R.
(
1993
)
Orthodenticle activity is required for the development of medial structures in the larva and adult epidermis of Drosophila.
Development
115
,
801
811
Wikramanayake
A. H.
,
Brandhorst
B. P.
,
Klein
W. H.
(
1995
)
Autonomous and non-autonomous differentiation of ectoderm in different sea urchin species.
Development
121
,
1497
1505
Yuh
C.-H.
,
Ransick
A.
,
Martinez
P.
,
Britten
R. J.
,
Davidson
E. H.
(
1994
)
Complexity and organization of DNA-protein interactions in the 5-regulatory region of an endoderm-specific marker gene in the sea urchin embryo.
Mech. Dev
47
,
165
186
Zeller
R.W.
,
Britten
R. J.
,
Davidson
E. H.
(
1995
)
Developmental utilization of SpP3A1 and SpP3A2: Two proteins which recognize the same DNA target site in several sea urchin gene regulatory regions.
Dev. Biol
170
,
75
82
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