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Riverfront Stadium

Former Multi Purpose Stadium in Cincinnati

For other uses, see Riverfront Stadium (disambiguation).

Riverfront Stadium in Cincinnati, Ohio.jpg

The Stadium in 1980

Former namesRiverfront Stadium (1970–96)
Cinergy Field (1996–2002)
Location201 East Pete Rose Way, Cincinnati 45202
Coordinates39°5′48″N84°30′30″W / 39.09667°N 84.50833°W / 39.09667; -84.50833Coordinates: 39°5′48″N84°30′30″W / 39.09667°N 84.50833°W / 39.09667; -84.50833
OwnerCity of Cincinnati
Capacity52,952 (baseball, 1970–2000)
59,754 (football)
39,000 (baseball, 2001–02)
Field size1970–2000
Left field – 330 ft (100 m)
Left-center field – 375 ft (114 m)
Center field – 404 ft (123 m)
Right-center field – 375 ft (114 m)
Right field – 330 ft (100 m)
Backstop – 51 ft (16 m)

Left field – 325 ft (99 m)
Left-center field – 370 ft (110 m)
Center field – 393 ft (120 m)
Right-center field – 373 ft (114 m)
Right field – 325 ft (99 m)
Backstop – 41 ft (12 m)
SurfaceAstroTurf 8 (1970–2000)
Grass (2001–2002)
Broke groundFebruary 1, 1968
OpenedJune 30, 1970
ClosedSeptember 22, 2002
DemolishedDecember 29, 2002
Construction costUS$45 million
($300 million in 2020 dollars[2])
ArchitectHeery & Heery
Structural engineerPrybylowski and Gravino, Inc.[1]
General contractorHuber, Hunt & Nichols[citation needed]
Cincinnati Bengals (NFL) (1970–1999)
Cincinnati Reds (MLB) (1970–2002)
Cincinnati Bearcats (NCAA) (1990)

Riverfront Stadium, also known as Cinergy Field from 1996 to 2002, was a multi-purpose stadium in Cincinnati, Ohio, United States that was the home of the Cincinnati Reds of Major League Baseball from 1970 through 2002 and the Cincinnati Bengals of the National Football League from 1970 to 1999. Located on the Ohio River in downtown Cincinnati, the stadium was best known as the home of "The Big Red Machine", as the Reds were often called in the 1970s.

Construction began on February 1, 1968, and was completed at a cost of less than $50 million. Riverfront's grand opening was held on June 30, 1970, an 8-2 Reds loss to the Atlanta Braves. Braves right fielder Hank Aaron hit the first home run in Riverfront's history, a two-run shot in the first inning which also served as the stadium's first runs batted in.[3] Two weeks later on July 14, 1970, Riverfront hosted the 1970 Major League Baseball All-Star Game. This game is best remembered for the often-replayed collision at home plate between Reds star Pete Rose and catcher Ray Fosse of the Cleveland Indians.

In September 1996, Riverfront Stadium was renamed "Cinergy Field" in a sponsorship deal with Greater Cincinnati energy company Cinergy. In 2001, to make room for Great American Ball Park, the seating capacity at Cinergy Field was reduced to 39,000. There was a huge in-play wall in center field visible after the renovations, to serve as the batter's eye. The stadium was demolished by implosion on December 29, 2002.


Riverfront was a multi-purpose, circular "cookie-cutter" stadium, one of many built in the United States in the late 1960s and early 1970s as communities sought to save money by having their football and baseball teams share the same facility. Riverfront, Veterans Stadium in Philadelphia, Busch Memorial Stadium in St. Louis, Atlanta–Fulton County Stadium in Atlanta, Three Rivers Stadium in Pittsburgh, Shea Stadium in New York and Robert F. Kennedy Memorial Stadium in Washington, D.C. all opened within a few years of each other and were largely indistinguishable from one another; in particular, it was often confused with fellow Ohio River cookie-cutter Three Rivers Stadium by sportscasters because of the two stadium's similar names and similar designs.

One feature of Riverfront that distinguished it from other cookie-cutters was that the field level seats for baseball were divided in half directly behind home plate, with the third-base side stands wheeled to left field and the ones on the first-base side remaining stationary for conversion to a football seating configuration.[4] The astroturf panels covering the tracks could be seen in left field during Reds games. (Shea Stadium in New York had featured a similar movable field-level seats design from its debut in 1964 until the Jets moved to Giants Stadium in New Jersey in 1984, after which the Mets retrofitted Shea for exclusive baseball use.[5] A variation of this design was also used at Three Rivers Stadium in Pittsburgh.)[6]

The site Riverfront Stadium sat on originally included the 2nd Street tenement, birthplace and boyhood home of cowboy singer and actor Roy Rogers, who joked that he was born "somewhere between second base and center field."

Riverfront Stadium's scoreboard was designed by American Sign and Indicator, but in its last years was maintained by Trans-Lux. That scoreboard would be upgraded in the 1980s with the addition of an adjacent Sony JumboTron.

Big Red Machine[edit]

The Reds moved to Riverfront Stadium midway through the 1970 season, after spending over 86 years at the intersection of Findlay Street and Western Avenue – the last 57½ of those years at Crosley Field. Riverfront quickly earned a place in Cincinnati's century-long baseball tradition as the home of one of the best teams in baseball history. The Reds had only won three pennants in their final 39 years at Crosley Field (1939, 1940, 1961) but made the World Series in Riverfront's first year (1970) and a total of four times in the stadium's first seven years, with the Reds winning back-to-back championships in 1975 and 1976. The World Series would return in 1990, with Cincinnati winning the first two of a four-game sweep of the Oakland Athletics at Riverfront.

The turf infield and dirt "slide pits" can be seen in this April 1995 photograph.

Baseball purists disliked Riverfront's artificial turf, but Reds' Manager Sparky Anderson and General Manager Bob Howsam took advantage of it by encouraging speed and line drive hitting that could produce doubles, triples and high-bouncing infield hits. Players who combined power and speed like Joe Morgan, Pete Rose and Ken Griffey, Sr. thrived there. On defense, the fast surface and virtually dirtless infield (see photo) rewarded range and quickness by both outfielders and infielders, like shortstop Dave Concepción who used the turf to bounce many of his long throws to first. CatcherJohnny Bench and first basemanTony Pérez played here. The artificial turf covered not only the normal grass area of the ballpark but also most of the normally dirt-covered portion of the infield. Only the pitcher's mound, the home plate area (in two circled areas), and cutouts around first, second and third bases had dirt surfaces (which were covered in five-sided diamond shaped areas). This was the first stadium in the majors with this "sliding pit" configuration. The new stadiums that would follow (Veterans Stadium, Royals Stadium, Louisiana Superdome, Olympic Stadium (Montreal), Exhibition Stadium, Kingdome, Hubert H. Humphrey Metrodome, B.C. Place, SkyDome) installed sliding pits as the original layout, and the existing artificial turf fields in San Francisco, Houston, Pittsburgh, and St. Louis would change to the cut-out configuration within the next few years.

Riverfront hosted the MLB All-Star Game twice: first on July 14, 1970 with President Richard Nixon in attendance (51,838 total attendance), and again on July 12, 1988 (55,837 attendance).


Despite Cincinnati's love of baseball, it was the prospect of a professional football team that finally moved the city to end 20 years of discussion and build a new stadium on the downtown riverfront. After playing for two seasons at Nippert Stadium on the University of Cincinnati campus, the Bengals built on the Reds' success in the stadium's first year when they recorded their first winning season and playoff appearance in 1970, just their third year of existence.

Perhaps the most memorable football game at Riverfront was the AFC Championship on January 10, 1982. The game became known as the Freezer Bowl and was won by the Bengals over the San Diego Chargers, 27–7. The air temperature during the game was −9 °F (−23 °C) and the wind chill was −37 °F (−38 °C), the coldest in NFL history. The win earned the Bengals their first of two trips to the Super Bowl (XVI) while playing at Riverfront.

Riverfront Stadium hosted the 1988 AFC Championship, as the Bengals beat the Buffalo Bills 21–10 to advance to their second Super Bowl appearance.

During the Bengals' tenure, they defeated every visiting franchise at least once, enjoying perfect records against the Arizona Cardinals (4-0), New York Giants (4-0), and Philadelphia Eagles (3-0). They posted a 5–1 record in playoff games played in Riverfront Stadium, with victories over the Buffalo Bills (twice), San Diego Chargers, Seattle Seahawks, and Houston Oilers. Their only home playoff loss came to the New York Jets.

For most of the Bengals' tenure at the stadium, the field contained only the basic markings required for play. Until the late 1990s, there wasn't a logo at midfield or any writing in the end zone, which had long become standard in NFL stadiums.

During the 1988 season as the Bengals were making another Super Bowl run, Riverfront Stadium was nicknamed the Jungle as the Bengals went a perfect 10-0 at home during the regular season and in the playoffs. With the new stadium nickname, the fans and team adopted the Guns N' Roses song "Welcome to the Jungle" as the unofficial theme song for the Bengals. When Paul Brown Stadium opened in 2000, the Jungle theme was incorporated into the stadium design.


Between 1970 and 1990 Riverfront Stadium hosted 25 University of Cincinnati football games to accommodate higher-caliber visiting teams and local rivals which would overwhelm demand in their usual home, Nippert Stadium (which then could only hold 28,000). Among the Bearcats' opponents were the University of Maryland, University of Kentucky, University of Louisville, Boston College, West Virginia University, Penn State University, whose 1985 game took place with the Nittany Lions number one in the coaches' poll, and the University of Miami three times, twice while the Hurricanes were the defending national champions. It would be a temporary full-time home for the Bearcats during the 1990 season, when Nippert Stadium was undergoing renovations.

The Bearcats finished with a 12–13 all-time record at Riverfront.

List of college football games at the stadium
DateHome TeamOpponentScoreAttendance
September 19, 1970 CincinnatiDayton13–7 --
November 8, 1975 CincinnatiNo. 16 Maryland19–21 16,478
September 11, 1982 CincinnatiLouisville38–16 14,324
October 9, 1982 CincinnatiLong Beach State34–14 13,187
November 13, 1982 CincinnatiMorgan State52–0 --
November 18, 1982 CincinnatiMiami (OH)20–10 --
October 1, 1983 CincinnatiCornell48–20 13,840[7]
October 8, 1983 CincinnatiTemple31–16 --
October 22, 1983 CincinnatiNo. 8 Miami (FL)7–17 14,163
November 5, 1983 CincinnatiRutgers18–7 --
November 12, 1983 CincinnatiMemphis State43–10 --
October 13, 1984 CincinnatiNo. 10 Miami (FL)25–49 25,642
October 27, 1984 CincinnatiLouisville40–21 15,767
November 17, 1984 CincinnatiAlabama7–29 27,482
November 22, 1984 CincinnatiMiami (OH)26–31 --
October 5, 1985 CincinnatiTemple16–28 --
October 26, 1985 CincinnatiBoston College24–17 --
November 9, 1985 CincinnatiNo. 2 Penn State10–31 33,528
September 13, 1986 CincinnatiMiami (OH)45–38 --
September 27, 1986 CincinnatiKentucky20–37 --
October 24, 1987 CincinnatiNo. 3 Miami (FL)10–48 20,011
November 5, 1988 CincinnatiNo. 4 West Virginia13–51 21,511
September 2, 1990 CincinnatiBowling Green20–34 --
September 22, 1990 CincinnatiMiami (OH)12–16 --
November 3, 1990 CincinnatiNo. 25 Louisville16–41 23,575


Final years as a baseball-only stadium[edit]

Cinergy Field after the left and center field stands were removed.

When the Bengals moved to Paul Brown Stadium in 2000, the Reds were left as Cinergy Field's only tenant. Prior to the 2001 baseball season, the stadium was remodeled into a baseball-only configuration, and the artificial surface was replaced with grass.

Cinergy Field during a Cincinnati Reds game vs. the St. Louis Cardinals on August 23, 2001. Construction of Great American Ballpark is visible in the background.

To allow room for the construction of Great American Ball Park (which was being built largely over the grounds the stadium already sat on), a large section of the left and center field stands were removed and the distance to the fences was shortened by 5 feet (1.5 m). A 40-foot (12 m) wall was built in deep center field to prevent easy home runs. The new Great American Ball Park and old Riverfront Stadium were 26 inches apart at its closest point during this time. In the Reds' final two seasons in the stadium, ongoing construction on Great American was plainly visible just beyond the outfield walls while the team played their games. The stadium's final game was played on September 22, 2002, as the Reds lost 4-3 to the Philadelphia Phillies before a crowd of 40,964. Reds third baseman Aaron Boone hit the final home run in Riverfront's history in the loss, an eighth-inning solo home run off Phillies reliever Dan Plesac.[9]

December 29, 2002 implosion.

The stadium was demolished by implosion on December 29, 2002. Today, part of the site is now occupied by Great American Ball Park and the National Underground Railroad Freedom Center, along with several mixed-use developments and parking facilities. A small portion of the Riverfront Stadium site is now occupied by the Reds' Hall of Fame and Museum and Main Street, which was extended when the new park was built and when the old park was demolished.

Seating capacity[edit]

Years Capacity
1970 51,500
1971 51,744
1972–1974 51,726
1975–1983 51,786
1984–1991 52,392
1992–2000 52,952
2001–2002 40,007

Attendance records[edit]

Bold indicates the winner of each game.


Highest Baseball attendance at Riverfront Stadium
RankAttendanceDateGame resultNotes
1 56,393October 16, 1975Reds 6, Red Sox 21975 World Series, Game 5
2 56,079October 12, 1990Reds 2, Pirates 11990 NLCS, Game 6
3 56,040October 22, 1972Athletics 3, Reds 21972 World Series, Game 7
4 55,832October 17, 1990Reds 5, Athletics 4 (10)1990 World Series, Game 2
5 55,830October 16, 1990Reds 7, Athletics 01990 World Series, Game 1


Highest Football attendance at Riverfront Stadium
RankAttendanceDateGame result
1 60,284October 17, 1971Bengals 24, Browns 27
2 60,157December 20, 1970Bengals 45, Patriots 7
3 60,099October 10, 1970Bengals 13, Dolphins 23
4 60,084December 9, 1990Bengals 17, 49ers 20
5 60,067November 4, 1990Bengals 7, Saints 21



The logo the Reds used in 2002 for their final season at Riverfront Stadium/Cinergy Field.
  • First stadium to have its entire field covered by AstroTurf, except for the cutouts around the bases and pitcher's mound.
  • First hit: Félix Millán, June 30, 1970.
  • First home run: Hank Aaron, June 30, 1970.
  • First Presidential Visit: Richard Nixon, July 14, 1970.
  • First upper deck home run: Tony Pérez, August 11, 1970.
  • First World Series game ever played on artificial turf: October 10, 1970 (Reds vs. Baltimore Orioles).
  • First no-hitter: Ken Holtzman, June 3, 1971.
  • First pitcher ever to pitch a no-hitter and hit two home runs in the same game: Rick Wise, June 23, 1971.
  • Hank Aaron ties the all-time home run record with number 714: April 4, 1974.
  • First stadium to display metric distances on the outfield walls (100.58 meters down the lines, 114.30 to the alleys, 123.13 to center): 1976.
  • Highest season attendance, 2,629,708: 1976.
  • First rain checks issued: August 30, 1978.
  • First player to hit for the cycle: Mike Easler, June 12, 1980.
  • Pete Rose breaks the all-time hit record with number 4,192: September 11, 1985.
  • First player ever to be caught stealing four times in one game: Robby Thompson, June 27, 1986.
  • Perfect Game: Tom Browning, September 16, 1988.
  • Umpire John McSherry collapsed and died on April 1, 1996.
  • Longest home run, 473': Mark McGwire, May 5, 2000.


  • First touchdown: Sam Wyche, September 20, 1970
  • First Field goal: Horst Muhlmann, September 20, 1970
  • Freezer Bowl: lowest wind-chill (2nd lowest temperature) in NFL history, January 10, 1982
  • Steve Largent becomes the first player in NFL History to catch 100 TD's in career, December 10, 1989.
  • Corey Dillon breaks the single-game rookie rushing record with 246 yards on December 4, 1997.


Date Artist Opening act(s) Tour / Concert name Attendance Revenue Notes
August 4, 1976Jethro TullToo Old To Rock 'N' Roll Tour
August 16, 1978The EaglesEddie Money
The Steve Miller Band
Hotel California Tour51,855
September 14, 1989The Rolling StonesLiving ColourSteel Wheels Tour53,555 / 53,555$1,522,536
July 10, 1990New Kids on the BlockPerfect GentlemenThe Magic Summer Tour48,000 / 48,000
May 5, 1993Paul McCartneyThe New World Tour38,000 / 40,000$1,156,513[12]
August 30, 1994The Rolling StonesLenny KravitzVoodoo Lounge Tour34,137 / 55,000
May 21, 2000George StraitAsleep at the Wheel
Lee Ann Womack
Mark Chesnutt
Kenny Chesney
Martina McBride
George Strait Country Music Festival42,000
July 14, 2000'N SyncSisqo
No Strings Attached Tour48,234 / 48,234$2,091,097
June 6, 2001'N SyncDreamPop Odyssey Tour36,371 / 42,285$1,947,461
  • The Kool Jazz Festival (now the Macy's Music Festival) was an annual fixture.

Religious gatherings[edit]


  • Riverfront Stadium during a Cincinnati Reds game vs. the Chicago Cubs on May 23, 1988.

  • Riverfront Stadium in 1974

  • Riverfront Stadium in 1992

  • Cinergy Field during a Cincinnati Reds game vs. the New York Mets on April 27, 2001.


  1. ^"Cincinnati Riverfront Stadium". Architectural Record. Record and Guide. 147: 54. 1970.
  2. ^1634–1699: McCusker, J. J. (1997). How Much Is That in Real Money? A Historical Price Index for Use as a Deflator of Money Values in the Economy of the United States: Addenda et Corrigenda(PDF). American Antiquarian Society. 1700–1799: McCusker, J. J. (1992). How Much Is That in Real Money? A Historical Price Index for Use as a Deflator of Money Values in the Economy of the United States(PDF). American Antiquarian Society. 1800–present: Federal Reserve Bank of Minneapolis. "Consumer Price Index (estimate) 1800–". Retrieved January 1, 2020.
  3. ^"Atlanta Braves at Cincinnati Reds Box Score, June 30, 1970". Retrieved June 7, 2021.
  4. ^
  5. ^
  6. ^
  7. ^Flynn, Terry (October 2, 1983). "UC Lets Cornell Off with 48-20 Beating". The Cincinnati Enquirer. Cincinnati, Ohio. p. B-1 – via
  8. ^"2009 University of Cincinnati Football Media Guide"(PDF). Retrieved March 27, 2020.
  9. ^"Philadelphia Phillies at Cincinnati Reds Box Score, September 22, 2002". Retrieved May 19, 2021.
  10. ^"Capsule Preview of NFL Games". Boston Globe. September 19, 1970.
  11. ^"Expect Good Game". Bryan Times. January 9, 1982.
  12. ^Paul McCartney Setlist Riverfront Stadium, Cincinnati, Ohio, USA on 5 May 1993


External links[edit]


Genomic insights into Wnt signaling in an early diverging metazoan, the ctenophore Mnemiopsis leidyi

EvoDevovolume 1, Article number: 10 (2010) Cite this article

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Intercellular signaling pathways are a fundamental component of the integrating cellular behavior required for the evolution of multicellularity. The genomes of three of the four early branching animal phyla (Cnidaria, Placozoa and Porifera) have been surveyed for key components, but not the fourth (Ctenophora). Genomic data from ctenophores could be particularly relevant, as ctenophores have been proposed to be one of the earliest branching metazoan phyla.


A preliminary assembly of the lobate ctenophore Mnemiopsis leidyi genome generated using next-generation sequencing technologies were searched for components of a developmentally important signaling pathway, the Wnt/β-catenin pathway. Molecular phylogenetic analysis shows four distinct Wnt ligands (MlWnt6, MlWnt9, MlWntA and MlWntX), and most, but not all components of the receptor and intracellular signaling pathway were detected. In situ hybridization of the four Wnt ligands showed that they are expressed in discrete regions associated with the aboral pole, tentacle apparati and apical organ.


Ctenophores show a minimal (but not obviously simple) complement of Wnt signaling components. Furthermore, it is difficult to compare the Mnemiopsis Wnt expression patterns with those of other metazoans. mRNA expression of Wnt pathway components appears later in development than expected, and zygotic gene expression does not appear to play a role in early axis specification. Notably absent in the Mnemiopsis genome are most major secreted antagonists, which suggests that complex regulation of this secreted signaling pathway probably evolved later in animal evolution.


The overwhelming majority of phylogenetic studies identify four clades of metazoan animals that branched off before the origin of the Bilateria. These include cnidarians (corals, sea anemones and 'jellyfish'), poriferans (sponges), placozoans (Trichoplax) and ctenophores (comb jellies) (Figure 1A). Often referred to as 'basal metazoans', 'diploblasts' or non-bilaterians, these four clades display radically different adult body plans and developmental programs from one another. The exact relationship of these early-branching taxa to one another remains contentious. Although morphological data suggest that poriferans and placozoans were the earliest metazoan lineages, followed by cnidarians and then ctenophores (Figure 1B) [1, 2], molecular studies have led to a number of different hypotheses regarding early animal evolution. Studies using 18S ribosomal RNA have suggested that sponges were the earliest branch, followed by ctenophores, thereby making them more basal compared with the classification based on morphological studies (Figure 1C) [3–6]. With the dawn of phylogenomics, the position of the ctenophores has continued to be contentious. The ctenophores have been positioned as sister to all other metazoans (Figure 1D) [7, 8], grouped with the cnidarians in a clade known as the Coelenterata (Figure 1E) [9], and considered sister to the clade comprising Bilateria, Placozoa and Cnidaria (Figure 1F) [10]. Additionally, a combined morphological and phylogenomic analysis has even suggested a monophyly of the basal metazoans in the clade 'Diploblastica', which is sister to the Bilateria (Figure 1G) [11]. As yet, there is very little consensus as to the placement of Ctenophora in the animal tree of life.

Non-bilaterian animal relationships. (A) Representative images of non-bilaterian animals, Ctenophora (Mnemiopsis leidyi), Cnidaria (Nematostella vectensis), Placozoa (Trichoplax adhaerens) and Porifera (Dysidea spp.). Photos courtesy of William E. Browne and Eric Roettinger. (B-G) Alternate hypotheses on early animal evolution and the placement of the ctenophores, based on (B) morphological data, (C) 18S ribosomal RNA results, (D-F) different phylogenomic analyses and (G) a combined morphological and phylogenomic approach.

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Fortunately, genomic data (gene content and complexity) and information on overall genomic structure can prove useful in resolving the relationship of these clades to one another. The genomes of the anthozoan cnidarian Nematostella vectenis [12], the hydrozoan cnidarian Hydra magnipapillata [13], the placozoan Trichoplax adhaerens [14] and the sponge Amphimedon queenslandica [15] have already proven to be invaluable resources in the effort to understand the genomic makeup of the earliest metazoans. Along with data from other sponges [16], the genomic data from choanoflagellates [17, 18] (the sister group of metazoans) have provided significant insight into the molecular complexity present in the closest extant unicellular ancestor of animals. Nonetheless, the available data from ctenophores (that is, the modest expressed sequence tag (EST) sets from two species, Mnemiopsis leidyi and Pleurobrachia pileus) is far from sufficient to resolve the placement of this enigmatic lineage.

Unlike the other non-bilaterians, ctenophores display a stereotypical developmental program (Figure 2A), with a well-studied cell lineage [19, 20]. The first two cleavages are equal and meridional, whereas the third cleavage is unequal and oblique. At this stage, the eight macromeres divide unequally to give off micromeres at the future aboral pole. Many of the early blastomeres in ctenophore embryos display a precocious determination of cell fate that is consistent with segregation of cytoplasmic determinants, although some inductive interactions are known to occur [21]. Unfortunately, no good molecular candidates for cell fate specification determinants have been identified in ctenophores. The primary adult body axis, the oral--aboral axis, is established at the time of the first cleavage [22] and early cleavages are important for localizing developmental potential [23]. The oral-aboral axis of larval (or cydippid) and adult ctenophores is demarcated by the mouth at the oral pole and the apical sensory organ at the aboral pole (Figure 2B). Additionally, there are two planes of rotational symmetry: the tentacular plane, which passes through the two tentacles, and the oesophageal or sagittal plane, which is perpendicular to the tentacular plane. Ctenophores also possess complex features, such as a well-developed muscular system composed of non-epithelial muscle cells and a nervous system that comprises sensory cells and a subepidermal nerve net [reviewed in [24]].

Ctenophore development and body plan. (A) Early cleavage from egg to 60-cell stage, based on Martindale and Henry [20] and others. The top row shows the view from the aboral (or vegetal) pole and the bottom row shows the lateral view, with the oral pole at the bottom. The first two divisions are equal and meridional, and the third cleavage is unequal and oblique, giving rise to middle (M) and end (E) macromeres. Subsequent divisions are unequal, with micromeres given off at the aboral pole. (B) Basic ctenophore body plan, as shown during the cydippid stage. The oral pole is the location of the mouth, which opens to the pharynx. The pharynx leads internally to the gut and endodermal canals (yellow). Also shown are paired tentacle bulbs (from which the tentacles grow), the eight comb rows, and the apical sensory organ located at the aboral pole.

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Although ctenophores have proven to be exceptional experimental embryological material, very little is known about the identity of the exact genes and proteins involved in specifying the body axes. To date, work on ctenophores has focused mainly on different families of transcription factors, including Sox [25], Fox [26], T-box [27] and Homeobox [28, 29], yet nothing is known about the cell signaling pathways. Bilaterian model systems have identified a limited number of cell signaling pathways, including the Wnt/β-catenin, TGF-β, Hedgehog, Notch, receptor tyrosine kinase, and Jak/STAT pathways. These pathways generally involve an extracellular (and often diffusible) ligand, transmembrane receptor, intracellular signal transduction/amplification system and, interestingly, a system of antagonists that can be used to further regulate informational content. These systems are used repeatedly in different tissues throughout the life history of organisms [30], with the basic elements of these systems arising early in animal evolution [31].

In this study we examined the Wnt/β-catenin signaling pathway in the ctenophore Mnemiopsis leidyi (Figure 3). In this pathway, the absence of a Wnt ligand results in the shunting of cytoplasmic β-catenin into a 'destruction complex' of axin, adenomatous polyposis coli (APC) and glycogen synthase kinase 3 (GSK-3) [32]. GSK-3 phosphorylates specific residues in the amino terminus of β-catenin, thereby targeting β-catenin for degradation via ubiquitination. T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) interacts with the repressor Groucho to suppress specific target genes. When the Wnt ligand is present, it activates the signaling cascade by first binding to the seven-transmembrane receptor Frizzled (Fzd). Along with a co-receptor, lipoprotein receptor-related protein 5/6 (LRP5/6), Wnt binding results in the phosphorylation of Dishevelled (Dsh), thereby activating it. Dsh inhibits GSK-3 activity, which allows active, non-phosphorylated β-catenin to accumulate in the cytoplasm. Increasing levels of cytoplasmic β-catenin promotes translocation to the nucleus, where it interacts with TCF/LEF (and other cofactors) to enhance transcription of target genes. Recent work in a number of cnidarian species has shown that the Wnt pathway is evolutionarily highly conserved and plays important roles in axis and cell fate specification [33–40]. Work in the sponge Amphimedon has shown polar localization of a Wnt ligand, suggesting a role in axial specification [41]. In another species of sponge, Oscarella lobularis, Wnt/β-catenin signaling has been implicated in adult epithelial patterning and ostia formation [42]. Some components of this pathway are known to be present in Trichoplax [14, 31], but their expression patterns and function are not yet known.

Overview of Wnt/β-catenin signaling Ctenophore pathway. (A) When Wnt signaling is inactive, cytoplasmic β-catenin protein is bound by the 'destruction complex' of axin, glycogen synthase kinase 3 (GSK-3) and adenomatous polyposis coli (APC). While sequestered, GSK-3 phosphorylates β-catenin, which targets β-catenin for ubiquitination and degradation. (B) In the presence of a Wnt ligand, the pathway is activated. Wnt binds to the seven-transmembrane receptor Frizzled and its co-receptor lipoprotein receptor-related protein 5/6 (LRP5/6), which causes Dishevelled (Dsh) to be activated. Dsh inhibits GSK-3, thereby allowing β-catenin to accumulate in the cytoplasm. Eventually, β-catenin gets translocated to the nucleus, where it interacts with the transcription factor T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) to activate target genes. The diffusible antagonists (Secreted Frizzled-related (Sfrp), Dickkopf (DKK), Wnt Inhibitory Factor (WIF) and Cerberus (CER)) can modulate Wnt activity by preventing the binding of Wnt to its receptors.

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We recently used next-generation technologies to sequence the genome of the lobate ctenophore Mnemiopsis leidyi, in an effort to better understand early animal evolution. In this paper, we look at one particular aspect, the evolution of the canonical Wnt signaling pathway. We found a near-complete Wnt signaling pathway present, including four Wnt ligands. However, part of the 'destruction complex' appears to be incomplete, and many Wnt antagonists are not recognizable in the genome. In situ hybridization studies showed that transcripts for all four Wnt genes are detected relatively late in development in discrete domains of the developing tentacles and apical organ.


Wnt/β-catenin pathway

A preliminary assembly of the genome of the lobate ctenophore Mnemiopsis leidyi, totalling 156 megabases in 5,100 scaffolds was generated by the National Institutes of Health (NIH) Intramural Sequencing Center using 454 and Illumina sequencing techniques. Using reciprocal BLAST searches of the Mnemiopsis genome, reverse transcriptase PCR cloning, and subsequent phylogenetic analyses, we identified and isolated nearly all of the essential members of the canonical Wnt/β-catenin signaling pathway (Table 1).

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The four non-paralogous Wnt genes identified in the Mnemiopsis genome were cloned by rapid amplification of cDNA ends (RACE) PCR, using mixed-stage embryonic cDNA. Phylogenetic analyses including the sequences of Wnt genes from representative taxa having fully annotated genomes showed moderately strong support for the ctenophore Wnt genes grouping in the Wnt6, Wnt9 and WntA families (Figure 4A). The fourth Wnt gene MlWntX does not group with any of the recognized Wnt families. It should be noted that the ctenophore Wnt genes tend to be somewhat more divergent than other animal genomes, as evidenced by their longer branch lengths in our phylogenies, and this is consistent with previous analyses of homeobox genes ([29] and Ryan et al, submitted). In addition, except for MlWnt6, the support for the other Wnt genes is relatively low. When phylogenetic analyses included additional non-bilaterian taxa (such as Amphimedon, Oscarella and Trichoplax), family-level classification of some Mnemiopsis sequences showed variation, perhaps due to artifacts caused by long branch attraction (see Additional file 1). The genomic complement of Wnt genes varies greatly across the Metazoa; however, there are 13 well-supported and described families, with a few orphans [40, 43]. The four Mnemiopsis Wnt genes are comparable in number with Amphimedon and Trichoplax, which both possess three, whereas cnidarians and bilaterians possess nearly the full complement of Wnt genes [35, 43].

Wnt gene orthology. Bayesian analysis to determine orthology of Mnemiopsis Wnt genes. Shown is a consensus tree of four independent runs of 5 million generations each. Posterior probability support is shown at each node, as well as maximum likelihood (PhyML with WAG model) bootstrap support (posterior probability/ML bootstrap). Asterisks at each node indicate ≥ 99%. support. A dash (-) indicates that the node was not supported by maximum likelihood analyses. Mnemiopsis Wnt genes are shown shaded and marked with arrows. Ate = Archaearanea tepidariorum; Bfl = Branchiostoma floridae; Cte = Capitella teleta; Hsa = Homo sapiens; Mle = Mnemiopsis leidyi; Nve = Nematostella vectensis; Pdu = Platynereis dumerilii; Sko = Saccoglossus kowalevskii; Spu = Strongylocentrotus purpuratus; Tca = Tribolium castaneum.

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Each Mnemiopsis Wnt gene has a predicted signal peptide at the 5' end. MlWntA, MlWnt9 and MlWntX have the 24 conserved cysteine residues, whereas MlWnt6 has only 22. Comparison of the coding regions with that of genomic data reveals two important intron positions (Figure 5A) that are well conserved across all metazoan Wnt genes analyzed to date [43]. These correspond to introns 3 and 5 in MlWntA, MlWnt6 and MlWnt9, and to introns 2 and 4 in MlWntX. In the current assembly, none of the Wnt genes are on the same scaffold, so there is no evidence of genomic linkage. MlWntA is currently on a 182 kb scaffold, spanning positions 64,343 to 70,583, with predicted genes 4 kb upstream (highest BLAST hit was XP_001624925.1) and 10 kb downstream (XP_002018102). MlWnt9 is on a 283 kb scaffold, spanning 131,863 to 139,362, with adjacent genes located 4 kb upstream (XP_001620894.1) and 12 kb downstream (XP_002755989.1). MlWntX is on a 285 kb scaffold, region 26,134 to 12,946, with no genes detected downstream and the closest hit 12 kb upstream (ZP_03967055). MlWnt6 is currently on a 48 kb contig with no other genes predicted, spanning the region 35,863 to 8,886. Extrapolating, the closest possible distance between any two genes is 20 kb.

Wnt gene structure and domains of Wnt pathway members. (A) Intron-exon structure of the four Mnemiopsis Wnt transcripts that were cloned. Turquoise shading indicates the coding region and the diagonal lines show the 5' and 3' untranslated regions. The start (ATG) is indicated, and the vertical lines represent intron positions. The conserved intron positions are marked with arrows. (B) Predicted protein domains present in the other Wnt components that were cloned out. Specific domains and other regions of interest are colored and named.

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Receptors and downstream components of the Wnt signaling pathway

In addition to the four Wnt ligands, the Mnemiopsis genome contains the receptors Fzd and LRP5/6 (Table 1). We were able to clone and identify two Fzd genes (MlFzdA and MlFzdB), both containing the extracellular Frizzled cysteine-rich domain (CRD), which binds Wnt ligands, and the transmembrane domain (Figure 5B). MlFzdA also has a signal peptide and the intracellular KTXXXW motif (KTASNW), which is thought to bind the PDZ domain of Dsh and is therefore required for canonical Wnt signaling [44]. MlFzdB does not appear to have the signal peptide or the KTXXXW motif based on cloned RACE PCR fragments. There was only a single LRP5/6 identified in the genome.

For functional canonical Wnt signaling, key intracellular components of the pathway are required. In Mnemiopsis, there are single genes encoding Dishevelled (MlDsh) and β-catenin (MlBcat). MlDsh contains all three key domains found in Dishevelled proteins of other animals (DIX, PDZ and DEP) (Figure 5B). The full-length MlBcat sequence was cloned from a mixed stage cDNA template. Similar to β-catenin from other metazoans, there is a highly conserved GSK-3 phosphorylation site and a conserved N-terminal motif (Figure 5B). Centrally, there are 12 armadillo repeats that are clearly detectable but widely divergent compared with other metazoan sequences. Surprisingly, based on the homology of predicted protein sequences, MlBcat appears to lack both C-terminal motifs (motifs A and B), which are thought to serve as transactivational domains [45]. When Wnt signaling is inactive, the 'destruction complex', composed of axin, APC and GSK-3, binds cytoplasmic β-catenin and targets it for degradation [32]. Although we found a clear GSK-3 ortholog, in silico searches found only a partial match to APC (low similarity to armadillo repeat domain and lacking all other domains) and did not find any evidence of axin. It is known that GSK-3 can phosphorylate β-catenin without requiring the other members of the complex [46]. We did find that the transcription factor TCF/LEF (MlTcf), the binding partner of stabilized nuclear β-catenin, is required for the activation of downstream target genes. MlTcf contains the β-catenin binding domain at its amino terminus and also contains the the Sox-Tcf high mobility group domain, which binds DNA (Figure 5B).

Although we found Wnt pathway genes from all parts of the pathway, including ligand modification/secretion, receptors and other membrane-associated proteins, and cytoplasmic and nuclear factors (Table 1; see Additional file 2), we failed to identify the important antagonists Dickkopf (DKK), Wnt Inhibitory Factor (WIF) and Cerberus (CER), which are characteristic of bilaterian Wnt signaling. We were able to identify a possible Secreted Frizzled-related gene (MlSfrp) that may be involved in regulating Wnt signaling; it contains the extracellular Frizzled CRD but not the transmembrane domain (Figure 5B). Unlike bilaterian Sfrp, MlSfrp lacks a Netrin-like (NTR) domain.

Wnt/β-catenin expression patterns

We examined the mRNA expression patterns of key components of the Wnt signaling pathway by whole-mount in situ hybridization. All four Wnt genes are detected at relatively late stages of development after gastrulation. MlWnt9 is detected the earliest, at about 3 to 4 hours post-fertilization (HPF) in four rows of cells in the aboral ectoderm derived from micromeres born at the vegetal pole (Figure 6A). As development proceeds, these cells form four clusters within the forming tentacle bulb, which appear to approach the aboral midline and fuse into two groups. Similarly, MlWntA is expressed in four groups of cells in the anlage of the tentacle apparati, beginning at about 9 HPF (Figure 6B). These cells are located below the surface ectoderm and are adjacent to the forming tentacle bulb, in the most lateral regions. In comparison with the MlWnt9+ cells, these are located slightly deeper below the surface and are positioned more towards the distal extremes of the tentacular axis.

Wnt gene expression during development. Whole- mount in situ hybridization analyses of all Wnt genes during development. The timeline at the top depicts hours post-fertilization (HPF). All images are oriented laterally, unless otherwise specified (aboral). For lateral views, the oral pole is at the bottom and aboral pole at the top, with an asterisk (*) marking the blastopore/mouth. For the aboral views, the tentacular plane is horizontal and the sagittal plane vertical. Gene expression is detected colorimetrically and shown by the blue/purple staining. (A)MlWnt9 is first detected after gastrulation in four aboral regions of the future tentacle bulb (arrows). After approximately 9 HPF, these four groups of cells converge along the tentacular plane and form two groups of cells within the tentacle bulb. (B)MlWntA is also expressed in four groups of cells of the forming tentacle bulb, slightly more internal than MlWnt9. It remains expressed in these four groups of cells at the periphery of the tentacle bulb. (C)MlWnt6 is expressed in both the tentacle bulb and the floor of the apical organ (ao). The tentacular staining is fainter in the cydippid stage; however, the apical organ staining remains prominent. (D)MlWntX is expressed both in the apical organ floor and in the ciliated groove (cg), which is the structure that connects the apical organ to the individual comb rows.

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Whereas MlWntA and MlWnt9 are primarily expressed in small regions of the tentacle bulb, the other two Wnt genes are associated with the apical sensory organ and its surrounding regions. The apical organ is primarily a gravity-sensing organ [47], although it possibly also acts as a photoreceptor [48], and it is highly innervated, as evidenced by ultrastructural analysis [24]. MlWnt6 is expressed in the apical organ floor, primarily in a central region along the tentacular plane (Figure 6C). There is also faint diffuse expression of MlWnt6 within the tentacle bulb. MlWntX is expressed in a region surrounding the floor of the apical organ, except for two areas in the pharyngeal plane, where it is excluded (Figure 6D). There is also expression in cells of the ciliated groove, the ciliated connection pathway between the gravity-sensing cells of the apical organ, and the locomotory comb rows.

To understand to which cells these Wnt ligands are signaling, we also looked at the expression patterns of the Frizzled-related genes and other intracellular components (Figure 7). MlFzdA is expressed maternally, in cleavage stages, and through gastrulation in a uniform manner (Figure 7A). After gastrulation and through cydippid formation, expression becomes concentrated primarily in the pharynx, tentacle bulb, and two ectodermal domains between the comb rows in the sagittal plane. MlFzdB, which lacks the intracellular motif, is initially expressed after gastrulation in the ectoderm (Figure 7B). However, later in development, most of the ectodermal expression is downregulated (except in the pharynx), and there is an additional expression domain in the muscle cells that connects the two tentacle apparati. The Secreted Frizzled-related gene, MlSfrp, is expressed after gastrulation in the pharynx and also in the mesoderm, which becomes two diffuse regions of the tentacle bulb (Figure 7C). By the cydippid stage, only faint tentacle bulb staining can be detected.

Expression of Wnt pathway components. Whole-mount in situ hybridization of other members of the Wnt pathway, including (A)MlFzdA, (B)MlFzdB, (C)MlSfrp, (D)MlDsh, (E)MlBcat and (F)MlTcf. The timeline above the images denotes the different stages of embryos below, from 0-3 hours post-fertilization (if applicable) to 24 HPF or the cydippid stage. Unless noted, all images are lateral views, with the asterisk marking the blastopore or mouth. Blue/purple staining shows where the genes are expressed. (A)MlFzdA is detected uniformly from egg, through early cleavage stages and gastrulation. From 9 HPF onward, it is expressed mainly in the tentacle bulb (arrows) and pharynx (ph). (B)MlFzdB is not detected until 3-4 HPF in cells of the ectoderm (ec). At 5-6 HPF, it is expressed in the tentacle bulb and around the blastopore, in cells that will invaginate to form the pharynx. Later, it is additionally expressed in the trans-tentacular muscle (white arrow), which connects the two tentacles. (C)MlSfrp is expressed in the invaginating pharynx and in the presumptive mesoderm (mes). This mesodermal expression becomes confined to two regions of the tentacle bulb, which becomes barely detectable in the cydippids. The pharyngeal expression is also not detected in cydippid stages. (D)MlDsh is expressed uniformly from egg to cydippid stages. (E)MlBcat is first detected after gastrulation (about 4 HPF) in ectodermal cells around the blastopore. This blastoporal expression continues however, at 6 HPF there is MlBcat expression everywhere, except in the cells that form the comb plates (arrowheads). (F)MlTcf is expressed primarily in the ectoderm after gastrulation but excluded from cells that form the comb plates. At cydippid stages, it is expressed in discrete regions of the apical organ floor (ao) and in the tentacle bulbs.

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The key Wnt modulator MlDsh is expressed maternally in a uniform pattern and throughout development in almost all cells (Figure 7D). By contrast, MlBcat is first detected during mid-gastrulation, and is localized to the region surrounding the blastopore (Figure 7E). However, the blastopore, which corresponds to the animal pole and the future location of the mouth, is already fully formed, and the endodermal macromeres have already been internalized by the time zygotic transcripts are detectable. The cells that express MlBcat remain on the surface, and expression is not seen in endodermal precursors. We are not able to detect any transcripts before this stage by in situ hybridization; however, we cannot rule out that there are low levels of expression or maternally deposited protein present. As development proceeds, MlBcat is expressed almost ubiquitously in both the ectoderm and the endoderm. The only region in which it is not expressed (or is expressed at low levels) is in the cells that form the comb plates. It is possible that this widespread expression is due to the role of β-catenin in cell adhesion. The onset of MlBcat expression occurs earlier than that of all four Wnt genes and, in contrast to the expression of MlBcat, which is initially localized to the oral (animal) pole, all four Wnt genes are localized primarily to the aboral (vegetal) pole of the embryo. Finally, the transcription factor MlTcf is expressed after gastrulation diffusely in the ectoderm and more intensely around the blastopore (Figure 7F). Similar to MlBcat, it also is not expressed in cells that form the comb plates. Late expression of MlTcf is confined to individual cells of the apical organ and parts of the tentacle bulb.


To date, existing studies have offered only a partial view of a limited number of gene families in ctenophores [25–29, 49–51]. Using next-generation sequencing, we were able to investigate complete gene families and signaling pathways in the ctenophore Mnemiopsis leidyi. Although results from full genome analyses of gene families are not yet available, we examine in this paper the comprehensiveness of an important developmental signaling pathway: the Wnt/β-catenin pathway. To ensure that the genomic searches were complete and that false negatives were minimized, we examined the published 15,752 Mnemiopsis ESTs and found that there was a match of approximately 97 to 99% to genomic contigs, depending on stringency conditions (data not shown), suggesting that our genomic sequencing is fairly complete.

Ancestral metazoan gene complement

When compared with other animals (Table 2), Mnemiopsis appears to have a Wnt complement that is more similar to poriferans (3 genes) and Trichoplax (3) than to cnidarians and bilaterians (7-20), which have up to 13 distinct Wnt family members. This suggests that the expansion of Wnt genes occurred after the divergence of ctenophores, poriferans and Trichoplax, and that this expansion was nearly complete in the cnidarian-bilaterian ancestor. However, if Trichoplax is the sister group of bilaterians [10], this would suggest a significant loss of Wnt genes in the placozoan lineage. The uncertainty of assigning orthology of sponge Wnt genes [42] and to some of the ctenophore genes makes it difficult to unequivocally determine the evolutionary history of the Wnt gene family. Wnt genes have yet to be found in any non-metazoan (including choanoflagellates), indicating that these ligands are likely to be specific to metazoans. There is not enough phylogenetic information to ascertain the branching order within the Wnt family; however, there is moderate support for the groupings of Wnt1 and 6, Wnt2 and 7, Wnt4 and 11, and Wnt9 and 10, based on our own analyses (Figure 4) and on those performed by others [40, 43], suggesting these families resulted from early duplications.

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Core components of the Wnt pathway are present in all non-bilaterians (Table 2) [31], suggesting the existence of functionally active signaling at the base of the animal tree of life. Many genes in the Wnt pathway appear to be animal-specific novelties. However, proteins containing Armadillo repeats (such as those in β-catenin) are found in all eukaryotes; these proteins have cytoskeletal functions in fungi and protists, and are involved in intracellular signaling in plants [52]. Certain pathway components are also present in organisms such as the slime mold Dictyostelium, which contains a β-catenin-like gene called aardvark, a GSK-3 homolog and Frizzled-like receptors [53, 54]. Aardvark has 10 armadillo repeats (compared with 12 in β-catenin), potential N-terminal GSK-3 phosphorylation sites, and no C-terminus motifs. Functional work has shown aardvark to have roles in both adherens junctions and cell signaling (in the form of stalk formation); however this is independent of GSK-3 activity [55]. All other metazoan β-catenin proteins examined to date have the two C-terminus motifs (A and B) that are thought to be transactivational domains, except for Caenorhabtidis elegans, which seems to have lost or modified them [45]. Thus, depending on the phylogenetic positions, ctenophores either lost these two motifs or they evolved after the ctenophore divergence. Additionally, bilaterian β-catenins also have other C-terminal motifs, which appear to be lineage-specific innovations.

The lack of certain Wnt pathway components in Mnemiopsis that are present in other non-bilaterians is an intriguing result. For instance, axin is found in Amphimedon, Trichoplax, Nematostella and bilaterians, but appears to be missing from Mnemiopsis (Table 2). Whether this gene appeared after ctenophores diverged from later metazoan lineages or was lost in the Mnemiopsis lineage is not yet clear. Likewise, there seems to be a paucity of diffusible antagonists in Mnemiopsis, Amphimedon and Trichoplax. Whereas Amphimedon has several Sfrp-like genes [15, 31], Mnemiopsis has only a single Sfrp; however, in both species the netrin domain is lacking. A DKK ortholog has been reported only for the sponge Oscarella carmela [18], as well as cnidarians and deuterostomes [56]. Trichoplax does not appear to have any of the known antagonists. Whereas DKK appears to be relatively ancient and lost in the protostome lineage, WIF is probably a bilaterian novelty and CER is only found in vertebrates. It is likely that antagonists were relatively recent additions to the pathway, providing an extra mechanism to control the activity. Alternatively, there could be additional novel antagonists in Mnemiopsis or in the other early lineages, whose identities can only be discovered through functional experiments.

Based on the gene content observed in the early-branching phyla, we can begin to deduce the key steps that led to the complexity observed in the bilaterian Wnt signaling pathway. It appears that the core components were present in the metazoan ancestor, including a Wnt ligand, Frizzled receptor, Dsh and β-catenin. Before the cnidarian-bilaterian ancestor developed, a series of duplication and divergent events, especially among the Wnt genes themselves, led to significant expansion of the components in the pathway. This expansion, coupled with the origin of the Wnt antagonists DKK, WIF and CER was probably the catalyst for the acquisition of additional roles of the pathway.

Based on gene content and diversity, our results are incongruent with a sister relationship between cnidarians and ctenophores (that is, the Coelenterata hypothesis). Firstly, in our phylogenies the genes of ctenophores do not group closely with those of cnidarians. Moreover, if cnidarians and ctenophores were sister phyla, a tremendous amount of gene loss (including the loss of multiple Wnt ligands, axin and DKK) would have been required in the Mnemiopsis lineage. These results are consistent with previous analyses of homeobox and nuclear receptor gene families (Ryan et al., submitted; Reitzel et al., submitted) in rejecting the Coelenterata hypothesis. Unfortunately, the comparison of Wnt signaling components between Mnemiopsis, Amphimedon and Trichoplax is not sufficient to identify the relationships between Ctenophora, Porifera and Placozoa. Additionally, it is not known how well Mnemiopsis represents the ancestral ctenophore gene complement, therefore data from other ctenophores would be of great benefit.

Expression patterns

In Mnemiopsis, all of the Wnt genes are expressed at the aboral (vegetal) pole in a striking pattern that suggests they are playing some role in patterning the body. However, they are expressed at such a late stage in development that many cell fates have already been specified. The expression patterns of the Wnt genes in the apical organ and tentacle bulb would suggest they might be involved in neural specification. In cnidarians, it has been suggested that Wnt genes expressed in staggered ectodermal and endodermal domains are patterning the oral-aboral axis in a 'Wnt code' [35, 56]. By contrast, most of the Wnt genes in Nematostella are primarily expressed at the oral pole, whereas in Mnemiopsis, they are expressed at the aboral pole. In the sponge Amphimedon, a Wnt gene is expressed at the posterior pole of the swimming larvae [41]. A major similarity is that Wnt genes appear to be expressed at the posterior pole of most animals [57, 58]. Mnemiopsis locomotes primarily with the oral end to the front (as do most ctenophores), as this aids in feeding; however, they are capable of moving in both directions. Cnidarians, such as Nematostella, swim in the direction of the aboral end, as this is the location of their apical tuft. The observed Wnt expression patterns could suggest that the aboral pole of ctenophores corresponds to the posterior pole of bilaterians.

It is difficult to determine whether the Wnt/β-catenin pathway is functioning early in development based on in situ expression patterns alone. Whereas MlFzdA and MlDsh are both expressed maternally and persist through early cleavage, MlBcat and the Wnt genes are not expressed until after gastrulation. Furthermore, whereas the Wnts are detected primarily at the aboral pole, MlBcat is initially expressed at the oral (or animal) pole. Either this pathway is not involved in early axis specification or there may be a maternal β-catenin protein that is functioning before onset of zygotic expression. Protein localization (particularly that of β-catenin) would help to determine whether the pathway is involved in axial patterning; however, we have not generated an antibody to β-catenin and have yet to find one that crossreacts. Because MlBcat appears to lack transactivational domains (at least as determined by sequence comparison), further experiments are necessary to determine whether it can actually function as a transcriptional activator. Attempts to activate Wnt signaling via GSK-3 inhibition (for example, with lithium chloride or alsterpaullone treatments) have not produced any obvious phenotype [Pang K, personal observation]. Functional experiments to knock down gene expression (morpholino antisense oligonucleotides or dominant negative constructs) would provide much-needed insight into whether canonical Wnt signaling is actually active in the developing embryo.


The canonical Wnt signaling pathway evolved at the base of the animal tree of life. We searched through the genome of the ctenophore Mnemiopsis leidyi, and identified most of the components of this well-known developmental signaling pathway. Conspicuously absent from ctenophores is axin, a member of the 'destruction complex', which is present in all other animals. Wnt antagonists also appear to be lacking or scarce in early diverging metazoans, with Sfrp present only in ctenophores, sponges and cnidarians, and DKK present only in sponges and cnidarians, with vertebrates possessing the entire array of Wnt antagonists (Sfrp, DKK, WIF and CER). Wnt genes evolved early in animal evolution, but did not radiate and diversify until the Cnidarian-Bilaterian ancestor. However, it is also not clear if Wnt signaling has direct effects on the regulation of gene expression in ctenophores, as key transactivational domains in a downstream target of the Wnt pathway, β-catenin, appear to be absent, and pharmacological treatments that lead to the stimulation of β-catenin activity in other metazoans produce no visible phenotype with these.

Although most of the canonical Wnt pathway components are present, their mRNA expression patterns would suggest that this pathway is not involved in early axis specification in Mnemiopsis. Both the late expression patterns (after the axes have been specified) and the expression of Wnt and β-catenin at opposite poles of the embryo suggest that this pathway may not required for fate specification. The rapid development of ctenophores could imply that asymmetric segregation of maternally loaded protein, rather than zygotic gene expression, is responsible for precocious cell fate specification in these embryos. Further genomic, expression and functional analyses are necessary to determine what genes and/or determinants are involved in axis specification in this unique early diverging animal lineage. Moreover, once the Mnemiopsis axial patterning system has been deciphered, it will become increasingly important to reach a consensus regarding the branching position of Ctenophora relative to other early-branching metazoans to place this unique developmental program within a phylogenetic context.

Materials and methods

Animal collection and gene expression

Mnemiopsis leidyi adults were collected (from Eel Pond or the NOAA Rock Jetty, Woods Hole, MA, USA) during the months of June and July and spawned as previously described [59]. RNA was extracted from embryos at regular intervals from fertilization to 36 hours (TRI Reagent; Molecular Research Center, Cincinnati, OH, USA) [59]. RNA was reverse transcribed to generate cDNA (SMART RACE cDNA Amplification Kit; BD Biosciences, San Jose, CA, USA). This cDNA was used as template to isolate the genes of interest. The following genes were isolated and fully sequenced, and are described in this paper: MlWntA (HM448813), MlWnt6 (HM448814), MlWnt9 (HM448815), MlWntX (HM448816), MlBcat (HM448817), MlDsh (HM448818), MlFzdA (HM448819), MlFzdB (HM448820), MlSfrp (HM448821) and MlTcf (HM448822). Additionally sequences were isolated for MlPygopus (HM448823), MlChibby (HM448824), MlPorc (HM448825) and MlDIXD (HM448826).

For whole-mount in situ hybridization, embryos were fixed at various stages from freshly collected uncleaved embryos (0 HPF) to cydippids (24 to 36 HPF). They were stored in methanol at -20°C until used. Digoxygenin-labeled riboprobes (0.1 ng/ul) (Ambion/Applied Biosystems, Austin, TX, USA) were hybridized for 48 hours at 60°C, and detected using an alkaline phosphatase-conjugated antibody (Roche Applied Science, Indianapolis, IN, USA) and the colorimetric substrate nitro-blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) [59]. After detection, specimens were washed with phosphate-buffered saline and transferred through a glycerol series up to 70% glycerol. They were then mounted, viewed under a compound microscope (Zeiss Axioskop 2, Jena, Germany), and imaged using a digital imaging system (AxioCam HRc with Axiovision software; Zeiss). Color balance and brightness were adjusted using Photoshop software (Adobe Systems Incorporated, San Jose, CA, USA). The only modification to the in situ protocol was a change in acetic anhydride treatment (treated in 0.1 mol/L triethanolamine rather than 1% w/v) (for most recently updated protocols, contact the authors). All in situ images presented here and additional developmental stages and/or views, are available online via the comparative gene expression database, Kahikai

Genome sequencing and searches

Mnemiopsis genomic DNA was collected from the self-fertilized spawning of two separate adult animals. One pool of genomic DNA was used to construct a library for 454 sequencing and the other used for Illumina paired-end sequencing. The 454 sequencing resulted in 8.1 million reads (2.7 Gb), which were assembled into contigs using the Phusion assembler [60]. The Illumina run resulted in 2.8 million paired end reads, which combined with the 454 data, was used to generate 5,100 scaffolds (scaffold N50 of 187 kb), resulting in a total coverage of ~50×.

The Mnemiopsis genome was scanned in silico for genes of interest using a reciprocal BLAST approach. Human, frog, Drosophila and Nematostella orthologs were used as queries for TBLASTN searches. Candidate matches were then used in BLASTP searches of the human genome to find the closest hit. If the closest match was not the original ortholog or if the E-value was greater than 0.001, then it was coded as being absent from the genome. A gene model was created by scanning the genomic region using Genscan [61]. This predicted protein sequence was then searched for conserved Pfam domains using SMART [62]. For certain genes of interest, gene-specific primers were designed for RACE PCR (MacVector, Cary, NC, USA). RACE PCR fragments were then conceptually spliced and aligned back to genomic contigs for comparison of exon-intron boundaries, using Sequencher (Gene Codes, Ann Arbor, MI, USA).

Phylogenetic analyses

The Mnemiopsis predicted amino acid sequences were aligned with the sequences of other organisms. The predicted domains or regions of interest were trimmed and aligned using Muscle, then corrected by hand for alignment errors (see Additional file 3, Additional file 4). Bayesian phylogenetic analyses were performed using MrBayes 3.1.2 [63] using the 'mixed' amino acid model with four independent runs of 5 million generations each, sampled every 100 generations with four chains. A summary consensus tree was produced in MrBayes from the last 49,000 trees of each run (196,000 trees in total), representing 4,900,000 stationary generations. Posterior probabilities were calculated from this consensus. Maximum likelihood analyses were performed using PhyML [64], using the WAG model with 1000 bootstraps. Alignments and nexus files are available upon request.


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TCF Bank
Nearest Banks and ATM Locations  <div><h2>                                                   COVID-19 Community Vaccination Program                                        </h2><div><p><img src=

Minnesota’s COVID-19 Community Vaccination Program brings easy, free, and safe vaccine opportunities to familiar community settings. 

Walk-ins and appointments for Minnesotans age 12-and-older are available at the Bloomington (Mall of America) community vaccination site, which is administering the Pfizer vaccine. Parent or guardian consent is required for COVID-19 vaccination for people younger than 18 years old.

To find a vaccination location near you, use the Find Vaccine Locations map.

For State Community Vaccination sites:

  • 12-15 year olds must be accompanied by a parent or guardian.
  • 16 and 17 year olds must receive permission from a parent or guardian, either in person through being accompanied by a parent or guardian, or online beforehand through the appointment scheduling and registration process.
  • Families are highly encouraged to join 12-17 year olds and get vaccinated at the same time.

To make an appointment, follow the links below.

Note: Masks are required at all Community Vaccination Program locations.

image Safe Travels, Minnesota

Safe Travels, Minnesota!

Get your vaccine at MSP-Terminal 2 between October 8 and December 29 and you could win a $200 Sun Country flight voucher!

Get your shot at Terminal 2 at the Minneapolis-Saint Paul International Airport between October 8 and December 29 and enter for a chance to win a $200 travel voucher through Sun Country Airlines! A new winner will be announced each week for twelve consecutive weeks. Undrawn entries do not rollover into subsequent weeks.

The one-dose Johnson & Johnson vaccine is being administered at Terminal 2. Minnesotans must be 18 or over to receive the shot and enter for a chance to win.

This opportunity is open to the general public. No boarding pass or appointment are required.

Terms and conditions are available on the Safe Travels page.


Tcf National Bank - Coon Rapids Cub Branch

The following are this Tcf National Bank branch's opening and closing hours:

10:00 AM - 8:00 PM

10:00 AM - 8:00 PM

10:00 AM - 8:00 PM

10:00 AM - 8:00 PM

10:00 AM - 8:00 PM

10:00 AM - 5:00 PM

10:00 AM - 5:00 PM

The Coon Rapids Cub Branch location of Tcf National Bank was established Oct 15, 1990 (31 years and 1 months ago). They are one of 331 branch locations operated by Tcf National Bank. For ATM locations, drive-thru hours, deposit info, and more information consider visiting their online banking site at:
Bank's Headquarters:

2508 South Louise Avenue
Sioux Falls, South Dakota 57106

Became FDIC Insured:

Mar 25, 1936


watch the thematic video

Friday Nights With TCF Ep. 4