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Taking Tea and Tortes With the Welsh in Distant Patagonia - The New York Times
Travel|Taking Tea and Tortes With the Welsh in Distant Patagonia
Taking Tea and Tortes With the Welsh in Distant Patagonia
By LUKE JEROD KUMMER APRIL 3, 2005
THE kettle in the center of the table anchors an elaborate Welsh tea service at the Ty Gwyn teahouse. An assortment of Celtic fruitcakes, tortes, spiced breads, fruit jams, and scones are spread on the table covered with a blue and white gingham cloth. In the corner of the room, a choir of a dozen people is softly singing the Welsh hymn "Awn Ymlaen Dan Ganu." You might almost forget that you are a whole hemisphere away from Wales until a hostess, carrying a fresh tray of pastries, smiles and asks, "Les gustaría más pasteles?" ("Would you like more pastries?")
Argentine Patagonia is home to awe-inspiring glaciers, condors, rugged wilderness -- and a community of Welsh immigrants who have preserved the culture and language of their homeland. The center of all things Welsh is a hamlet of many gardens but no stoplights called Gaimán, where both Spanish and Welsh are likely to be heard on the street. Gaimán (population 4,000 or so), lies in central Patagonia near the Atlantic in Chubut Province, where the cultures and languages have mingled for more than 100 years but remain distinct.
Charged with planning a trip to Argentina in January for my girlfriend, Nita, and me, I seized upon the possibilities of adventure travel in Patagonia's southernmost corner. Although sleeping on a glacier sounded like great fun to me, I knew Nita would prefer something a bit cozier.
When I discovered Gaimán and its picture-of-quaintness tea services, I decided taking a three-day side trip here would make an excellent bargaining chip for heading farther south and exploring the wilderness later on. After flying into the nearby town of Trelew, we spent a night in the Hotel Touring Club, an outpost of bygone grandeur and local hospitality where Butch Cassidy and the Sundance Kid once stayed while casing the bank across the street and then deciding not to rob it.
A taxi took us through fierce dust-blowing winds to a small railroad station that Gaimán has converted into a municipal museum, called Amgueddfa Hanesyddol. On exhibit are colonial artifacts, photographs and documents. When we arrived, Zoe Gauci, a native of Wales who had been jaunting around Latin America for the preceding nine months, turned with digital video camera in hand to the wizened museum curator.
"My grandmother speaks Welsh and no one else in my family does," Ms. Gauci said. "Would you send her a message?" The curator, Tegai Roberts, whose own ancestors were among the colony's earliest settlers, happily obliged. The half-dozen visitors listened to her speak in a strange musical language.
The efforts of British officials to restrict the use of Welsh in public stimulated interest in the 19th century in finding a new home in such places as the United States, New Zealand and Palestine. In the museum we learned that when Michael D. Jones, an ardent Welsh nationalist, realized that Welsh immigrants to North America had assimilated even more than the people who remained in the homeland, he looked for refuge to a nearly uninhabited place on the southern tip of the world.
In 1865, a clipper ship called the Mimosa, flying an Argentine flag with a red dragon, a Welsh national symbol, superimposed on it, sailed from Liverpool with 153 Welsh men and women. When it landed in Puerto Madryn, about 50 miles northeast of Gaimán, the passengers found a land nothing like the green hills they left. Only yellow shrub rose on the expanse of clay that an undying wind had swept dry as bone.
The first crops planted by the settlers failed because they didn't understand that the growing seasons are reversed in the Southern Hemisphere. Each night they huddled in fear of attacks by what they thought would be fierce aboriginal people. Yet it was the aboriginal Tehuelche who ultimately taught the Welsh when to plant crops and how to hunt the guanaco, or wild llama, and the ostrich-like ñandú. Despite periods of drought and famine, the community survived and dispersed throughout the region.
During our visit at the museum in Gaimán, Mrs. Roberts told us that the Welsh also brought the first irrigation system to Patagonia. Thus, the valley of the Río Chubut, which surrounds Gaimán, is an oasis of poplar trees and violet flowers in the desolate plateau. Active farming and a healthy tourism trade in Gaimán have caused it to thrive in recent years.
Before going to the museum, we took a taxi to the Moriah Chapel just outside of Trelew. Built in 1880, it is one of the oldest on a 40-mile trail of 17 Welsh chapels that wends through the Río Chubut Valley. At each stop visitors can see where colonists gave thanks for their new home. At Moriah, a cemetery has some black slate headstones, imported from Wales and engraved with the names of many founders of the colony.
Over the years since the initial settlement, Mr. Jones's dream of a Welsh-speaking realm has seen mixed results. While Welsh is widely spoken around Gaimán, it is usually a second language. Intermarriage with Spanish speakers became common, and many Welsh speakers have Spanish names. Recently, use of Welsh in local schools has led to a Welsh revival.
Each October, Trelew also is home to the Eisteddfod Welsh cultural festival featuring a two-day contest of bards, both Welsh and Spanish, that attracts more than 1,000 people from throughout Argentina and abroad.
But the most visible expression of Welshness in the area is the teahouses in Gaimán, which keep alive a tradition from the old country that starts at about 3 p.m. every day.
Mrs. Roberts's sister, Luned González, a retired headmistress of Gaimán's secondary school, said that Welsh women in Gaimán would celebrate nearly every occasion with steaming kettles.
"Every Sunday when I was a girl," she said, "the church pastors used to go from house to house having tea."
A LOCAL delicacy has helped Welsh tradition survive in Gaimán. For many years, the Welsh fruitcake, called torta negra, brought people from all over Argentina to sample its spicy sweetness. The first teahouse was a gathering place for local farmers in the 1940's, and more casas de té soon began opening. This meant that, before heading home, visitors could enjoy tea in what Argentines now call "Welsh tea service" a mix of Celtic custom and local touches.
The service includes a pot of potent black tea prepared alongside traditional Welsh desserts and Argentine favorites such as the sticky, caramel-like spread called dulce du leche. Some teahouses also offer light finger sandwiches and miniature quiches. The service costs on average $5.25 to $6.30 a person, at 2.98 Argentine pesos to the dollar. In Argentina, Welsh cuisine is mostly limited to the sugary staples of these teahouses. Other restaurants in the area serve Argentine dishes like juicy Patagonian lamb that has been charred over a fire.
During the temperate months from September to May, Gaimán's handful of teahouses provide quiet respite for adventurers on their way to glacier trekking or whale-watching sites. Punta Tombo, a colony of a half million Magellanic penguins, is only a morning's rumble away down a rock-paved road.
Among those who have dropped by Gaimán for a hot cup was Diana, Princess of Wales. Two months before her fatal accident, Diana visited a local teahouse, Ty Te Caerdydd. Now portraits of her surround the guests, who can inspect a cup that has been left unwashed since she used it. However attractive the enormous teahouse's carefully manicured gardens and souvenir-filled gift shop may be, they combine to give the grounds an unfortunate commercial flavor. For a more homestyle setting we headed to Ty Nain in the center of town. Housed in an 1890 Welsh-style brick building marked by an ivy-covered facade and door flanked by two evenly spaced sash windows, this teahouse is cluttered with old-time trinkets and remnants from frontier life, such as a puma head and skin that adorn a back wall. The confections are as rustic as the décor, with their edges miscut and powdered sugar tumbling off the side. The sign outside advertises that this is the teahouse "para una atención mas personalizada" and the owners refuse to cater to large parties.
Ty Gwyn provides a comforting blend of warmth and elegance. The oldest active teahouse in the area, it has expanded to include a second dining room to accommodate the crews of cruise ships that sometimes visit. Yet it remains a charming place, with cheerful service and touches of Welsh culture like the occasional serenade by a Welsh choir. Perfect torta negra and other specialty sweets that the owner, Maria Elena Naso de Sanchez, and her daughter, Sonia Irene Sanchez, prepare are some of the tastiest in town.
For our final night, Nita and I stayed at Ty Gwyn, one of the few teahouses that offer bed-and-breakfast accommodations upstairs, in one of four clean and spacious blond wood-trimmed rooms with balconies. Drifting into sleep that night with freshly kneaded pastries baking in the oven one floor below, we inhaled the sweetly toasted air and pondered whether we should start the next day with apricot squares or bite-sized custard pies. I seemed to recall that one is a traditional Welsh favorite and the other Argentine. Luckily, they go together quite nicely on the same plate.
In mid-March, fares for round-trip flights from New York to Ministro Pistarini International Airport near Buenos Aires in April ranged from $795 to $868. Domestic flights leave from Jorge Newberry Airport, a 45-minute cab ride from Ministro Pistarini that costs about $10.50 at 2.98 Argentine pesos to the United States dollar. Bus fare between the airports is $7.
Aerolíneas Argentinas is the only airline that flies from Buenos Aires to Trelew. Travelers flying into the country on Aerolíneas Argentina may be able to buy domestic tickets for the local price, which would reduce the round-trip fare to Trelew from $540 to $383. City buses (about 90 cents) and cabs (about $8) are available from the Trelew Airport to Gaimán.
There is a tourism information counter, (54-2965) 420-139, and tour and car rental agencies at the airport.
These teahouses are open from midafternoon to evening.
At Ty Nain, Hipólito Yrigoyen 283, (54-2965) 491-126, on the Web at www.cpatagonia.com/gaiman/ty-nain, descendants of the original Welsh settlers serve guests sweets such as torta negra and custard pie, in an 1890 ivy-covered building. Tea service with pastry is about $6.65.
Ty Te Caerdydd, Finca 202, (54-2965) 491-510, , is a large teahouse surrounded by gardens and fields that was once visited by Diana, Princess of Wales. Tea service with pastry is $6.30.
Ty Gwyn, 9 de Julio 111, (54-2965) 491-009, on the Web at www.cpatagonia.com/gaiman/ty-gwyn, offers a refined tea service that features savory snacks alongside traditional pastries. It is also a bed-and-breakfast inn with four rooms that cost $31.50 a person with breakfast. Tea service is $6.30.
In Trelew, the Hotel Touring Club, Avenida Fontana 240, (54-2965) 433-998, or e-mail htouring@internet.siscote.com, is an interesting place to kick off dust from the local roads. It looks as if it has not seen a remodeling or a repainting since it was Patagonia's most luxurious hotel 100 years ago. A room with breakfast is $14 for a single, $22.75 for a double.
Hotel Galicia, 9 de Julio 214, (54-2965) 433-802, centrally located in Trelew, is notable for its stately foyer. The clean and basic rooms are $17.50 for a single and $21 for a double, including breakfast. LUKE JEROD KUMMER

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a community for 6 yearsmessage the moderatorsMODERATORSqgyh2noname99tubersmjChunmastakylevmattlDimeShakeAutoModerator242526Linux laptops with >4 hours of battery life. (self.linux)submitted 1 year ago * by CorinnaBrodez61 commentssharecancelsorry, this has been archived and can no longer be voted onloading...sorted by: bestbesttopnewhotcontroversialoldrandomyou are viewing a single comment's thread.view the rest of the comments →[–][deleted] 14 points15 points16 points 1 year ago (5 children)sorry, this has been archived and can no longer be voted onX230, bigger battery, i5, ssd gets me 7+ with cinnamon on mint. The monitor sometimes tells me I have 9, but I don't believe it really. Longer on xfce. I just used it to watch two full length movies on a plane permalinkparent[–]ParadigmComplex 0 points1 point2 points 1 year ago (0 children)sorry, this has been archived and can no longer be voted onIf I'm not pushing it to hard - just taking notes in class or browsing the web - my X230 (with a spinny harddrive and 94Wh battery) regularly gets 11-12 hours in real-world conditions on my Linux setup. It'd probably get even more with an SSD. Your monitor may not be lying - it may just expect that you'll continue using your laptop at the current power rate, which may be low because its relatively idle.
permalinkparent[–][deleted] -1 points0 points1 point 1 year ago (3 children)sorry, this has been archived and can no longer be voted onYea I would say an i5. Ya know so it doesn't go obsolete next year permalinkparent[–]cowlicks 1 point2 points3 points 1 year ago* (2 children)sorry, this has been archived and can no longer be voted onEdit: An i3 does necessarily save power.
I have an i3 and I'm perfectly happy, in fact I think it gives me better battery life than an i5 or i7. However, I don't "game". When I do require serious computing power, I don't do it on a laptop. permalinkparent[–]songyiyuan 0 points1 point2 points 1 year ago (1 child)sorry, this has been archived and can no longer be voted onWith Turboboost, i5 and i7 CPUs will over/underclock as needed (or shift the power intake across the cores). This actually saves power if you aren't using it heavily.
permalinkparent[–]cowlicks 0 points1 point2 points 1 year ago (0 children)sorry, this has been archived and can no longer be voted onThanks, I did not know this.
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Conditional Controls at Runtime in Silverlight DataGrid - CodeProject 11,637,176 members (60,417 online)
C#.NETDevIntermediateSilverlight Conditional Controls at Runtime in Silverlight DataGrid Manas_Patnaik, 9 Aug 2011
CPOL 16.5K 611 17
How to add events to the controls added at runtime? Vipul H Mistry20-Sep-12 21:01
Vipul H Mistry20-Sep-12 21:01 How to add events to controls added at runtime? this event has to be handled in actual class and not in the Converter class file.
Your datagrid owenthebrit13-Apr-12 7:36
owenthebrit13-Apr-12 7:36 Absolutely the most fantastic thing i have seen so far in silverlight. This has enabled me to do so many things.
Run Time changes alshameer rawther18-Aug-11 1:02
alshameer rawther18-Aug-11 1:02 hi i liked your control but can we change the mood @ runtime so that it gets reflected @ the control...if so pls help me
Re: Run Time changes Manas_Patnaik20-Aug-11 11:12
Manas_Patnaik20-Aug-11 11:12 Hello alshameer If you want to change the Mood at run time then why dont you implement a combo box using it as CellEditTemplete.
modified on Monday, August 29, 2011 12:53 PM
My vote of 5 pgmckillop17-Aug-11 2:50
pgmckillop17-Aug-11 2:50 clear and concise. Very useful for a particular problem i was addressing. Thanks for your efforts.
Re: My vote of 5 Manas_Patnaik20-Aug-11 11:09
Manas_Patnaik20-Aug-11 11:09 Thanks Manas Patnaik
Last Visit: 31-Dec-99 18:00 Last Update: 30-Jul-15 9:22Refresh1

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Off the grid permaculture farm kickstarter : homesteadjump to contentmy subredditsannouncementsArtAskRedditaskscienceawwblogbookscreepydataisbeautifulDIYDocumentariesEarthPornexplainlikeimfivefoodfunnyFuturologygadgetsgamingGetMotivatedgifshistoryIAmAInternetIsBeautifulJokesLifeProTipslistentothismildlyinterestingmoviesMusicnewsnosleepnottheonionOldSchoolCoolpersonalfinancephilosophyphotoshopbattlespicsscienceShowerthoughtsspacesportstelevisiontifutodayilearnedTwoXChromosomesUpliftingNewsvideosworldnewsWritingPromptsedit subscriptionsfront-all-random | AskReddit-pics-news-funny-worldnews-gifs-todayilearned-videos-nottheonion-aww-gaming-television-Showerthoughts-movies-TwoXChromosomes-Jokes-mildlyinteresting-LifeProTips-photoshopbattles-OldSchoolCool-space-food-Futurology-Art-tifu-sports-Music-Documentaries-IAmA-UpliftingNews-personalfinance-WritingPrompts-books-EarthPorn-science-explainlikeimfive-dataisbeautiful-creepy-history-nosleep-gadgets-DIY-askscience-InternetIsBeautiful-GetMotivated-philosophy-listentothis-announcements-blogmore » homesteadcommentsother discussions (9)Want to join? Log in or sign up in seconds.|Englishlimit my search to /r/homesteaduse the following search parameters to narrow your results:subreddit:subredditfind submissions in "subreddit"author:usernamefind submissions by "username"site:example.comfind submissions from "example.com"url:textsearch for "text" in urlselftext:textsearch for "text" in self post contentsself:yes (or self:no)include (or exclude) self postsnsfw:yes (or nsfw:no)include (or exclude) results marked as NSFWe.g. subreddit:aww site:imgur.com dogsee the search faq for details.advanced search: by author, subreddit...this post was submitted on 07 Dec 20130 points (38% upvoted)shortlink: remember mereset passwordloginSubmit a new linkSubmit a new text posthomesteadsubscribeunsubscribe63,999 readers~30 users here now
created by paulwheatona community for 6 yearsmessage the moderatorsMODERATORSpaulwheatontrevbillionpaulwheaton12Wild_Ass_MommyJpasholkabout moderation team »discussions in /r/homestead<>X33 · 6 comments Preparing for Pigs - Some of the things we have learned22 · 1 comment Canning Pantry Finished and Organized3 · 4 comments Question - Raising Broiler Chickens8 Sustainable farming conference in the UK podcastWhat's the best building software?37 · 32 comments What are the negatives to having a waterfall on your property..?59 · 21 comments What sites do y'all use to look for land?9 · 1 comment SitRep Sunday4 · 16 comments Need help with picking gear for pulling trees w/ truck3 · 4 comments I'm finishing high school. what are some good courses to look out for post secondary*about to look at stuff with some counsellor*This is an archived post. You won't be able to vote or comment.
000Off the grid permaculture farm kickstarter (kickstarter.com)submitted 3 years ago by BreezyMeadowsOrchardcommentshareloading...no comments (yet)sorted by: besttopnewcontroversialoldrandomq&alive (beta)there doesn't seem to be anything herePosts are automatically archived after 6 months.Got Itaboutblogaboutsource codeadvertisejobshelpsite rulesFAQwikireddiquettetransparencycontact usapps & toolsReddit for iPhoneReddit for Androidmobile websitebuttons<3reddit goldredditgiftsUse of this site constitutes acceptance of our User Agreement and Privacy Policy (updated). © 2017 reddit inc. All rights reserved.REDDIT and the ALIEN Logo are registered trademarks of reddit inc.create a new accountusername:password:verify password:email: (optional)remember mesign uplog inusername:password:remember mereset passwordlog inBy signing up, you agree to our Terms and that you have read our Privacy Policy and Content Policy.English [en]Afrikaans [af] (*)العربية [ar] (*)Беларуская мова [be] (*)български език [bg]বাংলা [bn-IN] (*)বাংলা [bn-bd] (*)Bosanski [bs]català [ca]česky [cs]Cymraeg [cy] (*)dansk [da]Deutsch [de]Ελληνικά [el]English (Australia) [en-au]English (Canadian) [en-ca]English (Great Britain) [en-gb]English [en-us]Esperanto [eo] (*)español [es]español [es-ar]Español [es-mx]eesti keel [et] (*)Euskara [eu]فارسی [fa]suomi [fi]Filipino [fil] (*)français [fr]Français [fr-ca]Frysk [fy-NL] (*)Gaeilge [ga-ie] (*)Gàidhlig [gd]Galego [gl] (*)עברית [he] (*)मानक हिन्दी [hi] (*)hrvatski [hr]Magyar [hu]Հայերեն լեզու [hy]Bahasa Indonesia [id] (*)íslenska [is]italiano (Italy) [it]日本語 [ja]ಕನ್ನಡ [kn_IN]한국어 [ko]Latin [la] (*)1337 [leet]LOL [lol]lietuvių kalba [lt] (*)latviešu valoda [lv]Bahasa Melayu [ms] (*)Malti [mt-MT]Nederlands [nl]Nynorsk [nn]Norsk [no]Arrrrrrrr! [pir]polski [pl]português [pt] (*)português [pt-pt]português brasileiro [pt_BR]română [ro]русский [ru]slovenčina [sk]slovenščina [sl] (*)српски језик [sr]Srpski [sr-la]Svenska [sv]தமிழ் [ta]ภาษาไทย [th]Türkçe [tr]українська мова [uk]Tiếng Việt [vi]中文 [zh]简化字 [zh-cn] (*) incomplete volunteer to translateπ Rendered by PID 4941 on app-414 at 2017-01-23 20:23:05.926323+00:00 running cf19881 country code: US.


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Search Nantucket Cars
https://www.expedia.com/Car-Rentals-In-Nantucket.d6063111.Car-Rental-Guide
Last year, excursions to Nantucket were a popular activity. In fact, Nantucket is so great that scores of folks have put down roots here. From the moment you get to Nantucket, you'll have your eye on new experiences and be prepared to check out the city. You could rely on public transportation to explore after you are settled into your accommodations. And yes, you can always get into the taxi queue after you deplane and grab your luggage. Plus, you might find traveling through the city center is easiest done on foot. We say you should bid farewell to staying beholden to the public transit schedule and howdy to the benefits of a private ride. Renting a car gives you more independence during your vacation. With a rental car you can visit all the attractions in Nantucket on your desired schedule.
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Patent US7984849 - Portable magnetic stripe reader for criminality security applications - Google Patents
A security device comprises a portable magnetic stripe reader and computing functionality associated with the magnetic stripe reader, such as a credit card reader, drivers license reader, debit card reader, club card reader, or any other element that can be mechanically read. A single device may be needed...http://www.google.com/patents/US7984849?utm_source=gb-gplus-sharePatent US7984849 - Portable magnetic stripe reader for criminality security applications
Publication number US7984849 B2
Application number US 11/504,483
Also published as US20070040019, WO2008021428A2, WO2008021428A3
Publication number 11504483, 504483, US 7984849 B2, US 7984849B2, US-B2-7984849, US7984849 B2, US7984849B2
Inventors Hal Berghel, Dennis Cobb, Michael Sthultz
Original Assignee University of Nevada, Las Vegas
Patent Citations (37), Referenced by (28), Classifications (7), Legal Events (2)
US 7984849 B2
US6832003 * Feb 14, 2003 Dec 14, 2004 Mcgrew Stephen P. Method and apparatus for reading and verifying holograms
US20020031266 * Jun 13, 2001 Mar 14, 2002 Mcgrew Stephen P. Method and apparatus for reading and verifying holograms
US20040034604 Jan 10, 2003 Feb 19, 2004 Klebanoff Victor Franklin Method and system for assisting in the identification of merchants at which payment accounts have been compromised
U.S. Classification 235/380, 235/375, 235/449, 235/493

value: This website is a search engine provided by Google. Handling over 3.5 billion searches per day, it has a 92% share of the global search engine market.


text: Patent US5726975 - Switching system capable of performing alternative routing in accordance ... - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsIn a switching system including an alternative routing function, a maintenance terminal compiles an alternative routing scenario into a compiled alternative routing scenario. Connected to the maintenance terminal and an exchange switch for carrying out an exchange operation, an exchanging CPU carries...http://www.google.com/patents/US5726975?utm_source=gb-gplus-sharePatent US5726975 - Switching system capable of performing alternative routing in accordance with an alternative routing scenario assembled in a maintenance terminalAdvanced Patent SearchTry the new Google Patents, with machine-classified Google Scholar results, and Japanese and South Korean patents.Publication numberUS5726975 APublication typeGrantApplication numberUS 08/442,156Publication dateMar 10, 1998Filing dateMay 16, 1995Priority dateMay 17, 1994Fee statusLapsedPublication number08442156, 442156, US 5726975 A, US 5726975A, US-A-5726975, US5726975 A, US5726975AInventorsKiyotsugu ItoOriginal AssigneeNec CorporationExport CitationBiBTeX, EndNote, RefManPatent Citations (12), Referenced by (2), Classifications (21), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetSwitching system capable of performing alternative routing in accordance with an alternative routing scenario assembled in a maintenance terminal


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Characterization of the Dictyostelium Homolog of Chromatin Binding Protein DET1 Suggests a Conserved Pathway Regulating Cell Type Specification and Developmental Plasticity
Journal ListEukaryot Cellv.10(3); Mar 2011PMC3067478
Eukaryot Cell. Mar 2011; 10(3): 352–362. doi: 10.1128/EC.00196-10PMCID: PMC3067478Characterization of the Dictyostelium Homolog of Chromatin Binding Protein DET1 Suggests a Conserved Pathway Regulating Cell Type Specification and Developmental Plasticity†Manu J. Dubin,‡ Sonja Kasten, and Wolfgang Nellen*Department of Genetics, Kassel University, Heinrich-Plett-Str. 40, 34132 Kassel, Germany*Corresponding author. Mailing address: Abt. Genetik, Universität Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany., Phone: 49 (0) 561 804 4805. Fax: 49 (0) 561 804 4800. E-mail: nellen/at/uni-kassel.de.‡Present address: Gregor Mendel Institute, Dr.-Bohr-Gasse 3, 1030 Vienna, Austria.Author information ► Article notes ► Copyright and License information ►Received August 11, 2010; Accepted December 18, 2010.Copyright © 2011, American Society for Microbiology.This article has been cited by other articles in PMC.AbstractDET1 (De-etiolated 1) is a chromatin binding protein involved in developmental regulation in both plants and animals. DET1 is largely restricted to multicellular eukaryotes, and here we report the characterization of a DET1 homolog from the social amoeba Dictyostelium discoideum. As in other species, Dictyostelium DET1 is nuclear localized. In contrast to other species, where it is an essential protein, loss of DET1 is nonlethal in Dictyostelium, although viability is significantly reduced. The phenotype of the det1− mutant is highly pleiotropic and results in a large degree of heterogeneity in developmental parameters. Loss of DET1 results in delayed and abnormal development with enlarged aggregation territories. Mutant slugs displayed cell type patterning with a bias toward the prestalk pathway. A number of DET1-interacting proteins are conserved in Dictyostelium, and the apparently conserved role of DET1 in regulatory pathways involving the bZIP transcription factors DimB, c-Jun, and HY5 suggests a highly conserved mechanism regulating development in multicellular eukaryotes. While the mechanism by which DET1 functions is unclear, it appears that it has a key role in regulation of developmental plasticity and integration of information on environmental conditions into the developmental program of an organism.INTRODUCTIONThe social amoeba Dictyostelium discoideum provides a simple model for studying developmental processes. When their food supply is exhausted, Dictyostelium amoebae exit their unicellular proliferative phase and display facultative multicellularity. A few amoebae in the population start emitting cyclic AMP (cAMP) pulses, which act as a chemoattractant for others (7). These will relay the signal, chemotax toward the cAMP source, and aggregate into mounds of approximately 100,000 cells. The amoebae in the mound enter a developmental pathway, differentiating into a number of different cell types to form a multicellular slug. The slug consists of prestalk cells at the anterior fifth and mainly prespore cells, anterior-like cells (ALC), and circulating sentinel cells (10) in the posterior four-fifths, with a small zone of prestalk-like rear guard cells at the posterior extremity. The slugs migrate toward light and away from heat, and the prestalk cells differentiate further into different subtypes (pstO, pstA, pstB, pstAB, and the tip organizer region) (47). Eventually, after a period of migration, the slugs culminate and form a stalk on which a spore head is perched, thus favoring spore dispersal (6, 40).Aggregation is regulated by cAMP, which induces differentiation of prespore cells. Prespore cells in turn produce a chlorinated polyketide, DIF-1, that induces prestalk cell differentiation (9). While the entire cascade downstream of DIF-1 is not known, DIF-1 ultimately induces the nuclear accumulation of the bZIP transcription factors DimA and DimB, which bind the promoter of the prestalk ecmA gene (51) and regulate its expression. In addition, Myb, MADS-box, Cbl, STAT, and the amoebozoan-specific CudA transcription factors are involved in the regulation of Dictyostelium development (18, 21, 30, 33, 42, 48). Microarray analysis has shown that roughly a third of the genome displays a more than 2-fold change in expression during early development (26).The Dictyostelium genome encodes several homologs of proteins involved in developmental regulation in plants and animals, including components of a Cul4 (cullin 4)-based ubiquitin ligase complex. Cullins are a small family of evolutionarily conserved proteins that together with substrate adaptors catalyze the conjugation of ubiquitin chains to their targets, resulting in their degradation by the 26S proteasome (25). Cullins have been shown to have critical roles in transducing hormone signals, developmental regulation, environmental signaling, and cell cycle regulation (25, 27). In agreement with this, the loss of either culA or culB in Dictyostelium results in developmental defects (34, 44). Cul4 (CulD in Dictyostelium) has been implicated in the ubiquitination of various chromatin components, including the transcription factor c-Jun, the cell cycle regulator E2F, and the DNA replication licensing factor CTD1 (46), and is involved in developmental regulation in plants, including light response (5, 49, 50). It is also part of the DNA damage repair complex containing DDB1 (RepE in Dictyostelium), which can catalyze the monoubiquitination of histone H2A (12, 29) and/or histones H3 and H4 (45). In Schizosaccharomyces pombe, Cul4 also forms part of a complex with the DDB1 homolog Rik1, which acts to target the H3K9 methyltransferase Clr4 to pericentromeric heterochromatin (24, 28). In plants and animals, DDB1 and Cul4 occur together in a complex with the DET1 protein (39). DET1 was identified in mutant screens in Drosophila (41) and light-signaling mutants in Arabidopsis (11) and tomato (35). DET1 binds to chromatin via the N-terminal tail of histone H2B and acts as a transcriptional repressor at its target genes (3, 4). While DDB1 and Cul4 are highly conserved and appear to be ubiquitous in eukaryotes, DET1 has a much more limited distribution and occurs almost exclusively in multicellular plants and animals. Interestingly a homolog of DET1 appears to be present in the social amoebae Dictyostelium discoideum and Dictyostelium purpureum but not their unicellular relative Entamoeba histolytica. Given its role in developmental regulation in higher eukaryotes and its presence in Dictyostelium, DET1 is a promising candidate for a regulator of development in this species.MATERIALS AND METHODSStrains and growth. For cloning and plasmid isolation, the Escherichia coli strain DH5α was cultured at 37°C in Luria-Bertani (LB) broth or on LB agar plates supplemented with 50 μg ml−1 ampicillin.The Dictyostelium discoideum strain Ax2-214 (axeA2 axeB2 axeC2) was cultured in petri dishes or shaking culture at 20°C in HL5 medium (ForMedium, Hunstaton, United Kingdom) supplemented with 100 μg ml−1 ampicillin, 100 μg ml−1 amphotericin B, and the appropriate selective agent (10 μg ml−1 Geneticin and/or 10 μg ml−1 blasticidin). Alternatively, Dictyostelium cells were grown on bacterial lawns of Klebsiella aerogenes on SM agar plates.Cloning. The N- and C-terminal halves of the DetA (DDB_G0277075) gene were cloned separately with the primers MJD29 (5′-GTCGACAATGATGGATCCATCCACTATATC-3′) and MJD32 (5′-CATGTAAAAGCTTTTTCTCGTGAG-3′) for the N-terminal half and MJD31 (5′-CTCACGAGAAAAAGCTTTTACATG-3′) and MJD30 (5′-CTCGAGTTATGATACTGTATTATTATTATTGTTGTTGTTATTA-3′) for the C-terminal half. After sequencing to confirm the absence of mutations, the N-terminal half was excised with the SalI and HindIII restriction sites, and the C-terminal half was excised with the HindIII and XhoI restriction sites. These fragments were cloned into the green fluorescent protein (GFP)-expressing vector pDneo2a-GFP digested with SalI/XhoI by three-fragment ligation. The C-terminal conserved region was PCR amplified with the primers MJD184 (5′-AGTCGACAACGATGAATTATCAACAAATGTTTGGACAAAC-3′) and MJD30 (5′-CTCGAGTTATGATACTGTATTATTATTATTGTTGTTGTTATTA-3′) and cloned into the vector pDneo2a-3×HA, containing a 3-hemagglutinin (3×HA) tag, by using the SalI and XhoI restriction sites. To create an expression construct for the N-terminal half of DetA, the N-terminal domain described above (amplified with the primers MJD29 and MJD32) was digested with HindIII, blunted with T4 DNA polymerase, and then excised from the cloning vector by SalI digestion. The resulting fragment was cloned into the vector pDneo2a-GFP, which had been XhoI digested, blunted with T4 DNA polymerase, and then digested with SalI.RepE (DDB_G0286013) and CulD (DDB_G0292794) were amplified from genomic DNA with a mixture of Pfu/Taq (1:30) polymerases by using the primers: MJD169 (5′-AGTCGACAATGTATAATTTTGTATCAACTGTTCAAAAACC-3′)and MJD170 (5′-TCTCGAGTTAACGAATATATTGCATTAAAGATTCAATTCTTC-3′) for RepE and MJD171 (5′-AGTCGACAATGAATTTTAATAATAACAATAACAATAATAATAATAATAATAA-3′) and MJD172 (5′-TCTCGAGTTAAGCCATGTAATTATATATCATTGCATTTTC-3′) for CulD. After sequencing to confirm the absence of mutations, they were cloned into the vector pDneo2a-3×HA or pDneo2a-GFP (13) by using SalI and XhoI restriction sites introduced during PCR amplification. All accession numbers refer to DictyBase (20).Disruption of detA. The N-terminal domain of Dictyostelium detA described above (amplified with the primers MJD29 and MJD32) was cloned into vector pGEM-T Easy (Fermentas). pGEM-T-DetAn was double digested with PstI/SmaI, blunted with T4 polymerase, and then religated with T4 ligase in order to remove the NheI site within the vector backbone. The resulting vector, pGEM-T-DetAn (-NheI), was then digested with NheI (which is present within the detA gene), blunted with T4 polymerase, and treated with alkaline phosphatase. A SmaI-digested fragment from the vector pLPBLP (19) containing a blasticidin cassette flanked by LoxP sites was ligated in, resulting in the vector pGEM-T-detA-LPBLP. The vector was linearized by digestion with EcoRI and used to transform Ax2 cells. Blasticidin-resistant cells were subcloned. Putative disrupted clones were screened with the primers Act15R (5′-CCAACCCAAGTTTTTTTAAACC-3′) and MJD36 (5′-GCAAATAAAGGATTCATTGGGGTG-3′). The disruption of the detA gene was confirmed by Southern blotting, as described in reference 38.Western blotting. A total of 107 cells were pelleted and resuspended in 100 μl 1× Laemmli buffer and boiled for 5 min, and 10 μl of the soluble supernatant was run on an 12% SDS–PAGE gel. Proteins were transferred to polyvinylidene difluoride (PVDF) membrane by semidry transfer. Homogeneous loading and transfer of proteins were verified by staining with Ponceau-S dye (0.1% in 5% acetic acid). Excess dye was removed by washing in 1% acetic acid. The membrane was then blocked with 5% bovine serum albumin (BSA) and incubated with the primary antibody overnight at 4°C. After being washed, the membrane was incubated with the appropriate secondary antibody conjugated to alkaline phosphatase (Dianova, Hamburg, Germany) at a dilution of 1:10,000. Staining was done with 0.2 mg ml−1 BCIP (5-bromo-4-chloro-indolylphosphate).Identification of homologs and alignments. Dictyostelium homologs of characterized genes were identified in the Dictyostelium genome (16) by using BLAST search (2); a reciprocal BLAST search against the NCBI databank (nonredundant protein sequences) was performed to confirm identity (1).Immunofluorescence. Cells were grown overnight on coverslips to approximately 80% confluence and fixed in 4% (wt/vol) paraformaldehyde (PFA) dissolved in 20 mM phosphate buffer (pH 6.7) for 10 min at 22°C. The PFA solution was removed, and cells were permeabilized for 5 min in 0.2% (vol/vol) Triton-X 100 in 20 mM phosphate buffer (pH 6.7). Cells were blocked with 2% (wt/vol) BSA in phosphate-buffered saline (PBS; pH 7.6) for 30 min at room temperature. The coverslips were incubated overnight at 4°C with the primary antibody diluted in 1% (wt/vol) BSA in PBS (pH 7.6). The coverslips were washed 3 times for 5 min each in PBS and then incubated with the secondary antibody diluted in 1% (wt/vol) BSA in PBS (pH 7.6) for 1 h at room temperature. The coverslips were washed 3 times for 5 min each in PBS and then mounted on a slide with a drop of mounting medium: 250 ng ml−1 4′,6-diamidino-2-phenylindole (DAPI) in a mixture of 90% (vol/vol) glycerol, 20 mM Tris-HCl, and 1 μg ml−1 1,4-diazabicyclo[2.2.2]octane (pH 8.3). Cells were examined by fluorescence microscopy.Coimmunoprecipitation. A total of 5.5 × 108 cells were harvested, washed with phosphate buffer, and cross-linked in 10 ml phosphate buffer with 5 mM dimethyl 3,3′-dithio-propionimidate dihidrochloride for 30 min. Cells were washed and resuspended in 1 ml lysis buffer (10 mM Tris-Cl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, 25 mM MgCl2, 1 tablet of Roche proteinase inhibitor cocktail mini). Sonication was done three times for 15 s each with a Dr. Hielscher UP200S sonifier using the S1 tip and 50% amplitude/50% cycle. After centrifugation (10,000 × g, 15 min), the supernatant was incubated with 20 μl of GFP-Trap (Chromotek, Martinsried, Germany) Sepharose beads for 90 min on a rotating wheel at 4°C. The supernatant was removed, and the beads were washed twice with a mixture of 10 mM Tris-Cl (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 26 mM MgCl2, and Roche proteinase inhibitor cocktail. Bound protein was boiled off the beads in 25 μl Laemmli buffer. Aliquots were taken from the intermediate steps and compared to bound protein by SDS-PAGE and subsequent Western blotting.Microscopy. Images were acquired on a Leica DMIRB inverted microscope equipped with a DC350 camera and IM50 acquisition software (Leica Microsystems; Wetzlar, Germany). Images were prepared for presentation with ImageJ v1.42n-v1.43b (http://rsbweb.nih.gov/ij/).Developmental assay. Ax2 or strains of interest were grown in shaking culture to a density of 1 × 106 cells ml−1. A total of 1 × 107 cells were centrifuged for 2 min at 200 × g at 4°C. Cells were washed twice in phosphate buffer and then adjusted to a cell density of 4 × 106 cells ml−1. Fifty microliters of each strain was spotted in triplicate on phosphate agar plates, so that each spot had an area of approximately 1 cm2. Plates were left open in a sterile hood for 30 min to allow excess buffer to evaporate. Plates were examined at regular intervals to examine development.GFP reporter constructs. Ax2 or the mutant of interest was transformed with [ecmAO]:GFP or [cotB]:GFP reporter constructs (44) and subcloned. Cells were grown in shaking culture to a density of 1 × 106 cells ml−1. A total of 1 × 107 cells were centrifuged for 2 min at 200 × g at 4°C. Cells were washed 3 times in ice-cold water and then resuspended in a minimal amount (approximately 100 μl) of water and deposited in 3 or 4 small drops on freshly prepared 1% phosphate agar plates. The plates were placed in a dark humid chamber to develop for 18 to 20 h at 20°C. Slugs were examined with a fluorescence microscope, taking care to minimize the exposure of the plates to light beforehand.LacZ reporter constructs. Ax2 or the mutant of interest was transformed with a [ecmB]:LacZ, [ecmA]:LacZ, [ecmO]:LacZ, or [ecmAO]:LacZ reporter construct (15), as described above, except that selection was carried out with 50 μg ml−1 Geneticin. β-Galactosidase staining was performed as described in reference 17.Anti-EB4 staining of developing Dictyostelium. Staining of developing Dictyostelium with the anti-EB4 antibody was carried out as described for LacZ staining, up to the washing steps with Z buffer. After being washed twice with Z buffer, the cells were incubated with 5% (wt/vol) nonfat milk powder in PBS for 30 min at room temperature. The blocking solution was removed, and the filters were incubated with anti-EB4 antiserum (1:1,000) in 3% (wt/vol) nonfat milk powder in PBS overnight at 4°C. Filters were gently washed with 3× 800 μl PBS and then incubated with alkaline phosphatase-conjugated goat anti-rabbit (1:10,000) in 3% (wt/vol) nonfat milk powder in PBS for 60 min at room temperature. Filters were gently washed with 3× 800 μl PBS and then incubated with development buffer (0.2 mg ml−1 BICP, 100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2, 0.05% Tween 20 [pH 9.5]) until blue staining was apparent, at which point the filters were washed twice with PBS.RESULTSDET1 is a nuclear localized protein of approximately 60 kDa in plants, vertebrates, and Drosophila and has no homology with any other known protein (37). Based on sequence conservation between different species, a DET1 domain of approximately 400 amino acid residues has been defined in the PFAM database (PF09737). A BLAST search against the predicted Dictyostelium proteome using human DET1 as a query sequence revealed one protein (DDB_G0277075) with homology to human DET1 (Fig. 1A). This protein, which we have named DetA, is significantly longer than the other DET1 homologs (1,462 amino acids compared to approximately 550 amino acids for other characterized DET1 proteins). A February 2007 BLAST search of the Swiss-Prot protein database (43) with DetA as the query sequence gave hits against human, mouse (1e−49), and Arabidopsis (1e−45) DET1 (Fig. 1A). A BLAST search of the closely related Dictyostelium purpureum genome with DetA as the query sequence revealed a homolog (1e−130), DPU0059753, while S. pombe and Saccharomyces cerevisiae, as well as the unicellular amoeba Entamoeba histolytica, lack a DET1 homolog. Dictyostelium discoideum detA is located toward the distal end of chromosome 2, has a short intron of 90 bp, and encodes a protein of 169 kDa. Expressed sequence tags (ESTs) (36) span part of the coding region and support the presence of the intron. DetA consists of an asparagine-rich 500-amino-acid region containing homopolymer stretches followed by a region of approximately 200 amino acids, homologous to the N-terminal domain of characterized DET1 proteins (Fig. 1B) and a further asparagine- and glutamine-rich 400-amino-acid stretch of homopolymer sequences followed by another 250-amino-acid region homologous to the C-terminal domain of DET1 (Fig. 1C). The C-terminal domain (CTD) is conserved to a slightly higher degree than the N-terminal domain (NTD) among the different species examined. Dictyostelium appears to be one of the few species outside the metazoan and plant lineages in which a DET1 homolog is present (data not shown; Fig. SA1a in the supplemental material). With the exception of Dictyostelium, DET1 homologs in all other species are small, with a continuous DET1 domain and no additional domains on the protein (data not shown). Though asparagine- and glutamine-rich insertions are frequent in Dictyostelium genes, the presence of a separated NTD and CTD in DetA may suggest that DET1 consists of two separate domains with distinct functions.Fig. 1.Putative Dictyostelium DET1 homolog. (A) Schematic presentation of domain organization of DDB_G0277075 (DetA) aligned with DET1 from Homo sapiens and Arabidopsis. (B) Alignment of the N-terminal domain of Dictyostelium DetA (starting at amino acid [aa] ...DetA localization. The detA gene was cloned from genomic DNA, inserted into the C-terminal cloning site of pDneo2a-6×MYC or pDneo2a-GFP (13), and used to transform Dictyostelium. Western blotting confirmed that the six-MYC-tagged DetA construct (6×MYC-DetA) was expressed and gave a band of the expected size (180 kDa; see Fig. SA1b in the supplemental material). GFP-tagged DetA localized to the nucleus in both live and fixed cells (see Fig. SA1c and d in the supplemental material), as has been previously described for DET1 in plants and Drosophila (37). Double immunostaining of 6×MYC-DetA-expressing cells with antibodies against H3K9me2 or H3K9me3 showed that the overexpressed fusion protein was largely, but not exclusively, nuclear localized and excluded from the pericentromeric heterochromatin region when examined by wide-field or confocal microscopy (Fig. 2; see Fig. SA1e in the supplemental material).Fig. 2.Localization of DetA. Immunofluorescence on fixed cells expressing 6×MYC-DetA with an anti-MYC antibody (green) and an antibody against the heterochromatin marker H3K9me2 (red) counterstained with DAPI. Scale bar, 2 μm.Knockout of detA. A knockout construct was made by introducing a blasticidin resistance cassette into the 5′ half of the coding sequence of detA (see Fig. SA2a in the supplemental material). This construct was then used to transform Ax2 (wild-type) cells. Potential knockout lines were identified by PCR screening. Southern blotting confirmed that the detA locus was disrupted in three of the putative knockout lines. However, it was also evident that in one of the lines (8D), in addition to the disrupted locus, a second wild-type copy of the gene was present, suggesting that a duplication event had occurred (see Fig. SA2b in the supplemental material). Line 8D was used in further experiments as an internal control since the disruption was compensated for by the gene duplication. The disrupted lines 8A and 9B, but not the duplicated line 8D, displayed an increased doubling time (12 h compared to 10 h for the wild type) when grown in liquid medium, suggesting a mild growth or cell cycle defect (Fig. 3A). When developed on phosphate agar plates, clones 8A and 9B displayed a slight delay in the onset of development (Fig. 3B). In addition to the developmental delay, clones 8A and 9B displayed enlarged aggregation territories, and streaming could still be observed after 10 h. In these experiments, the duplicated line 8D displayed a wild-type phenotype (data not shown). Compared to the wild type, clones 8A and 9B formed fewer and larger slugs. The lower number of slugs appeared to be partly due to the larger aggregation territories and partly because up to 50% of the mounds are arrested at this stage for a considerable period of time without forming tips. The slugs also tended to be thinner than the wild type, a phenotype reminiscent of the dimB− mutant (51). The enlarged aggregation territories in the detA− mutants were only observed when the cells were plated at low densities (2 × 105 cells cm−2) but were not apparent when cells were plated at higher densities (above approximately 5 × 105 cells cm−2) (data not shown). When plated at high density, mutant slugs were often smaller than the wild type (see, for example, Fig. 5). Line 8A and its derivatives were used in all subsequent experiments. To confirm that the observed phenotypes were linked to the disruption of the detA locus, we attempted to complement the detA− knockout. When grown on bacterial lawns, the detA− mutant displayed a growth defect which could be rescued by supertransforming the knockout with a construct expressing 6×MYC-DetA, GFP-DetA, or TAP-6×MYC-DetA (Fig. 4A). The developmental assay was repeated using the wild-type strain (Ax2), the detA− mutant, and the detA− mutant complemented with either GFP alone (empty vector control) or GFP-DetA. In Ax2 and the detA−/GFP-DetA strain, the first mounds were visible at 5 h of development, but not in the detA− strain nor in the detA−/GFP control strain, where the onset of aggregation was delayed approximately 30 min with respect to the wild type (Fig. 4B). In wild-type cells, aggregation was largely complete by 8 h, but it continued until 9 h in the detA− and detA−/GFP strains. In the detA−/GFP-DetA strain, aggregation was even faster than in the wild type and was complete after 7 h. As previously observed, the detA− mutant formed fewer and larger aggregates, many of which arrested at this stage, resulting in fewer fruiting bodies. Except for a slightly accelerated development, the detA−/GFP-DetA strain displayed a wild-type phenotype, confirming that the loss of the DetA protein was responsible for the phenotype observed in the detA− mutant and that epitope-tagged DetA was functional in rescuing the mutant.Fig. 3.Phenotype of the detA− mutant. (A) Doubling time of wild-type (Ax2) and putative disruption strains in HL5 shaking culture. Clone 8D contained a gene duplication. (B) Development of the wild type and a disruption strain on phosphate-buffered agar ...Fig. 4.Complementation of the detA− mutant. (A) Growth (plaque size) of wild-type strain Ax2, the detA− mutant strain, and the detA− mutant strain complemented with different DetA expression constructs on bacterial lawns. The pictures ...Fig. 5.Developmentally regulated gene expression. (A) Fluorescence microscopy of wild-type (Ax2) or detA− slugs transformed with the prestalk reporter [ecmAO]:GFP 17 h after the onset of starvation; (B) fluorescence microscopy of wild-type or detA− ...Role of DetA in cell-type specification. Given the role of DET1 in developmental regulation in other organisms and the developmental defects observed in the detA− mutant, the role of DetA in cell-type specification was investigated. Wild-type (Ax2) and detA− (clone 8A) cells were transformed with the prespore [cotB]:GFP or prestalk [ecmAO]:GFP reporter construct. Lines expressing the reporter constructs were developed at high density on phosphate agar for 17 h in the dark and observed by fluorescence microscopy. In Ax2, the [cotB]:GFP reporter labeled the prespore cells comprising the posterior 80% of the slug, while the [ecmAO]:GFP reporter labeled the anterior 20% of the slug comprising the prestalk cells. Compared to the wild type, detA− slugs expressed [ecmAO]:GFP more strongly, and the prestalk region was larger (approximately 25 to 30% of the slug) than in Ax2 (Fig. 5A). In agreement with this, the expression level of the endogenous ecmA gene also appeared to be significantly upregulated in the detA− background compared to that in the wild type (see Fig. SA3a in the supplemental material). [cotB]:GFP staining in the detA− mutant was much reduced, and the anterior third of the slug, comprising the enlarged prestalk zone, was free of the label. Surprisingly, staining was also absent in the very posterior of most slugs (Fig. 5B). As [ecmAO]:GFP did not appear to label these posterior cells, their identity remains unknown. We also performed immunostaining on slugs with an antibody against the PsvA prespore markers (EB4 and SP35) (22, 23). In wild-type slugs, the antibody labeled the prespore cells in a rather uniform manner. In contrast, in the detA− mutant, the pattern was quite variable from slug to slug (see Fig. SA3b in the supplemental material), with some cells displaying expression over the entire length of the slug. In many slugs, however, it was largely absent from the anterior third, which corresponded to the enlarged prestalk region, and it was also absent from the posterior of the slug, as was observed for the [cotB]:GFP line.Analysis of prestalk subtypes. The prestalk region of Dictyostelium is comprised of several zones of different prestalk subtypes (47). To determine the architecture of the enlarged prestalk zone in the detA− mutant, the different subtypes were more closely examined. Ax2 and detA− cells were transformed with a reporter for PstB (prestalk B) cells ([ecmB]:LacZ), PstA cells ([ecmA]:LacZ), or PstO cells ([ecmO]:LacZ) and the reporter [ecmAO]:LacZ (15). Slugs were allowed to develop for 12 to 18 h and then were fixed and stained. The ecmA gene is expressed in almost all prestalk cells, but different cell types express it using either the distal part of the promoter [ecmO] or the proximal part [ecmA]; these cell types are referred to as PstO and PstA, respectively. ecmB is expressed in the PstB cells, which are present at the base of the prestalk zone in migrating slugs and will form the basal disk and lower cup in mature culminants, and also in the rearguard cells. A small region within the PstA zone also expresses ecmB, and this region is called the PstAB zone. The [ecmAO]:LacZ marker labels essentially the entire prestalk region: i.e., the PstA, PstO, and PstAB cells (Fig. 6A). As previously observed for the [ecmAO]:GFP construct, the zone expressing [ecmAO]:LacZ was expanded in the mutant compared to that in the wild type. Although variable, it typically stained the front 25 to 30% of the slug instead of the anterior 20% in the wild type. Staining of the slug posterior and also within the prespore region was also stronger in the detA− mutant. Compared to the wild type, [ecmB]:LacZ staining in the detA− mutant appeared to be slightly weaker after 12 and 14 h of development, but was otherwise similar to that in the wild type (Fig. 6B). It was also apparent that after 16 to 18 h, the mutant has a developmental delay of approximately 4 h compared to the wild type. The [ecmA]:LacZ reporter, which contains the proximal portion of the ecmAO promoter, was much more active in the detA− mutant (Fig. 7A). Some scattered staining was visible well into the prespore zone and at the slug posterior region. Staining of the stalk was strong and homogeneous in the detA− mutant but weak and punctate along the stalk in wild type. Compared to Ax2, [ecmO]:LacZ expression in the detA− mutant appeared to be diffuse, more extended, and possibly weaker (Fig. 7B). Also, the staining did not end abruptly at the prespore region, but continued throughout the slug, suggesting an increased number of anterior-like cells (ALCs), which became more abundant toward the posterior of the slug. Many of the detA− mutant aggregates that arrested at the mound stage showed a rather homogeneous staining of prestalk and prespore markers (Fig. 6 and 7; see Fig. SA3b in the supplemental material), indicating that they were able to differentiate into prestalk and prespore cells. Some of these mounds eventually formed slugs, while others remained arrested 48 h after the onset of development (data not shown).Fig. 6.Effect of DET1 on the formation of prestalk cell subtypes. (A) Wild-type or detA− slugs transformed with the prestalk subtype reporter [ecmB]:LacZ were fixed and stained for β-galactosidase at the indicated times after the onset of starvation. ...Fig. 7.Effect of DET1 on the formation of prestalk cell subtypes. (A) Wild-type or detA− slugs transformed with the prestalk subtype reporter [ecmA]:LacZ were fixed and stained for β-galactosidase at the indicated times after the onset of starvation. ...Possible DetA-interacting partners. The Dictyostelium genome encodes homologs of the DET1-associated proteins Cul4 (CulD; DDB_G0292794), RepE (DDB1; DDB_G0286013), and COP1 (for constitutive photomorphogenesis 1; DDB_G0288453). These were cloned and expressed as GFP or 3×HA epitope-tagged fusions. All three were nuclear localized and displayed a localization pattern similar to DetA (Fig. 8A and B) (data not shown). When they were expressed in the detA− mutant background, no noticeable changes in their distribution were observed, suggesting that DetA is not primarily responsible for their nuclear localization (data not shown). DET1 is known to bind directly to DDB1 (14, 39). While DET1 and Cul4 can also be detected in the same complex (5, 46), the interaction appears to be weaker and occur via DDB1. The 3×HA-tagged CulD and RepE were both expressed at the expected size (Fig. 8C), although for CulD, a number of bands probably representing degradation products were observed, while for RepE, higher-molecular-weight species were apparent, which may correspond to ubiquitinated RepE. Loss of DetA appeared to result in a relative increase in the higher-molecular-weight species of RepE, while epitope-tagged CulD was unaffected (Fig. 8C). Overexpression of GFP-DetA in the detA− mutant background resulted in a significant increase in the levels of both CulD and RepE (Fig. 8C).Fig. 8.Putative DetA interaction partners. (A) Immunofluorescence on fixed detA−/GFP-DetA cells supertransformed with a 3×HA-RepE construct; (B) Immunofluorescence on fixed detA−/GFP-DetA cells supertransformed with a 3×HA-CulD ...In order to more directly test the interaction between DetA, RepE, and CulD, coimmunoprecipitations were performed with detA−/GFP-DetA cells supertransformed with 3×HA-CulD or 3×HA-RepE. As shown in Fig. 9, HA-tagged RepE was found on the GFP-Trap Sepharose beads. The predicted indirect interaction of DetA via RepE with CulD was too weak to be seen in the reproduction of the Western blot.Fig. 9.RepE interacts with DetA. DetA−/GFP-DetA cells expressing 3×HA-RepE (left) or 3×HA-CulD (right) were processed for coimmunoprecipitation. The anti-HA-stained protein bands are indicated by arrows. C, cells before processing; L, ...DISCUSSIONWe report here the identification and characterization of DetA, the Dictyostelium homolog of DET1, a chromatin binding protein involved in developmental regulation in both plants and animals. DET1 homologs are almost exclusively restricted to multicellular organisms. Consistent with this, among the amoebozoa, a DET1 homolog is present in the multicellular species Dictyostelium discoideum and Dictyostelium purpureum but not in the obligatory unicellular species Entamoeba histolytica.In contrast to all other known DET1 homologs, Dictyostelium DetA is split by a large homopolymer stretch. It is thus significantly larger than DET1 proteins from other organisms. This organization, together with the observation that both parts were independently able to localize to the nucleus (data not shown), suggests that DET1 may consist of two separate domains.Loss of DetA resulted in a slight increase in doubling time in vegetative cells, delayed aggregation and enlarged aggregation territories suggesting a defect in quorum sensing. Up to 50% of the mounds had arrested development, but some resumed development later, indicating a role of DetA in the transition from mounds to slugs. The prespore cotB promoter showed a reduced zone of expression in detA− slugs, while the prestalk zone labeled by [ecmAO]:GFP was extended. The PstB zone was rather unaffected, but the PstA zone was enlarged, and expression of [ecmA]:LacZ was stronger in the mutant. DetA− cells thus have an altered prespore/prestalk ratio and a misproportioning within the prestalk population. As the posterior of the detA− slugs failed to express [cotB]:GFP or stain with an antibody against the SP35 protein (PsvA/EB4), these cells are not well defined: they are probably prestalk like, with at least some PstO characteristics. Since DET1 appears to act as a transcriptional repressor, it seems probable that the increase in the percentage of prestalk cells in the detA− mutant is due to the inability to repress the prestalk genes.The DET1-associated proteins RepE, CulD, and Cop1 are conserved in Dictyostelium and localized to the nucleus in a manner similar to DetA. No significant change in their localization was observed in the detA− mutant, but substantial overexpression of RepE and CulD in the GFP-DetA overexpression mutant pointed to at least regulatory interactions. The binding of DetA to RepE could be shown by coimmunopreciptitation, while the indirect interaction with CulD was only barely detectable.Plant development is highly plastic in order to adapt to changing environmental conditions (1). While DET1 was originally described as a negative regulator of photomorphogenesis, however, further analysis revealed extensive and rather variable organ patterning defects, somewhat reminiscent of polycomb mutants (32). Likewise, loss of the Drosophila DET1 homolog ABO1 also results in extensive but highly variable patterning defects during embryogenesis (41). Developmental patterning of most animals displays a certain degree of plasticity patterning. For example, cell fate been specified in different segments of Drosophila embryos by morphogen gradients, while mammalian embryos go through a number of cell divisions before cell fate is specified (31). An interesting exception is Caenorhabditis elegans, which relies on asymmetry that exists prior to the first cell division to establish polarity. In contrast to most other organisms, it exhibits almost no developmental plasticity, always going on to form an organism with exactly the same number of cells (1,031 for males) and the same proportion of cell types (8). C. elegans is one of the few multicellular animals that lacks a DET1 homolog. It is tempting to speculate that the loss of DET1 in this organism is linked to the lack of plasticity in its developmental program. In contrast, Dictyostelium development is highly plastic, with cell fate only been specified late in development after the individual amoebae have aggregated into a mound and in response to morphogen gradients such as DIF-1 (40). Being among the most simple organisms that harbor a DET1 homolog, Dictyostelium provides an ideal system to study developmental plasticity and the functions of this evolutionarily conserved chromatin regulator.Supplementary Material
ACKNOWLEDGMENTSWe thank Rick Firtel for providing the GFP reporter constructs and the Dictyostelium Stock Center for the LacZ reporter constructs and bioinformatics resources. We thank Markus Maniak for helpful comments and critical reading of the manuscript.This work was supported by grants (Ne285/10 and Ne285/12) from the Deutsche Forschungsgemeinschaft to W.N.Footnotes†Supplemental material for this article may be found at http://ec.asm.org/.Published ahead of print on 30 December 2010.REFERENCES1. Alabadí D., Blázquez M. A. 2009. Molecular interactions between light and hormone signaling to control plant growth. Plant Mol. Biol. 69:409–417. [PubMed]2. Altschul S. F., et al. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389–3402. [PMC free article] [PubMed]3. Benvenuto G., Formiggini F., Laflamme P., Malakhov M., Bowler C. 2002. 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