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Gold rocks are not the only ones that you may mine in RuneScape. There's Iron Ore, Gems, Granite, Mithril, Coal, Copper, Adamant, Runite and another mineral that can be used to craft various items of Silver, Black, Steel or Dragon rarity A golden rock is an item required to build the Statue of Rhiannon in the Shattered Heart Distraction and Diversion. In order to start receiving the golden rocks, one must first toggle the ability by talking to Auron Ithell who is standing by a golden plinth in the Tower of Voices in Prifddinas
Item list:- pickaxe, 1k gp, 4 gold bars, hammer- ammo mould (can be bought during quest for 5 gp)- a weapon to kill approx 5 lvl 14 scorpions- weight reducin.. A golden rock is an item required to build the Statue of Rhiannon in the Shattered Heart Distraction and Diversion. To start receiving golden rocks, one must first toggle the ability by having the required levels and talking to Auron Ithell who is standing by a golden plinth in the Tower of Voices in Prifddinas Take 3 Gold bars and Hammer to an anvil. Use one of the bars on it to make a helmet. Now, withdraw a pickaxe and prepare yourself for the battle with a level 75-125 enemy. Once ready, put on the Gold helmet and head back Dondakan the Dwarf. Give him the assmebled Schematic and you will be shot into the rock OSRS Gold Mining. The delicate nature of gold makes it unfitting for mining training. However, if you're interested in making jewelry or selling it, it is good to know where to mine it. First things first, though - you need to have level 40 Mining in order to mine gold ore and the gold veins respawn 60 seconds after depletion The rock golem is a skilling pet that can be obtained while training Mining. The chances of getting it are dependent on the player's Mining level, and the time it takes to gather a resource. When a player receives the pet, it will automatically try to appear as their follower
Rock crabs vs Experiments., For one, Ive never been to experiments before. So I have no idea if its a good place although I have heard it is. I hate quests, but if experiments is a g, Archives, Archives, RuneScape Pictures & Videos, RuneScape Server Development, Characters: Level 100-149, Characters: Levels 100-149, Characters: Levels 150 - 200, RS2 Guide Sales, Runescape Private Servers. OldSchool RuneScape (OSRS) Guide To Sarachnis - A Great Supplies Source About the boss: Sarachnis is a brand new, mid-difficulty boss which was released on 4 of July 2019 and is notable for wide range of supplies drops like seeds, herbs, uncuts high tier ores etc 1-99 OSRS Mining Training Guide for 2020. Mining is a skill that lets you obtain gems & ores that later can aid in training a variety of skills. It is a gathering skill and for that reason the items you gather while training it can bring you a lot of money. In this Ezrsgold OSRS 1-99 mining guide we will talk about these topics . Old School RuneScape has one primary currency, and that is gold! Whether you're a new player or a veteran at the game, one thing is undeniable: gold is extremely valuable in the game. Certain skills such as Construction and Prayer can take hundreds of millions of OSRS GP to fully train to level 99
With 40+ Attack and Strength, you can expect to obtain 150-250K+ OSRS Gold per hour by killing these monsters. The OSRS Gold that you will make comes from the consistent Grimy Herb drops, ranging from values of 13gp - 15K OSRS Gold per drop. The most popular locations to kill these are in Edgeville Dungeon and Taverly Dungeon OSRS.mobile strives to provide cheapest Runescape 2007 gold for players, and we firmly believe that you can get the best service and the lowest price on our site. 3.Fastest Delivery. OSRS.mobile promise that we will deliver the gold as soon as possible. 4.100% Satisfaction Guarantee. OSRS.mobile offer 24/7 customer service
RuneScape Item and Name Sales (25 viewing) Buy and sell RuneScape Items and Display Names here. Discussions: 44,791. Posts: 575,642. RuneScape Name Sales RuneScape 3 Item Exchange RuneScape 2007 Item Exchange. [$2,007+ Donor] [1,400+ Vouches]⭐Bert Gold⭐⚡Buy/Sell OSRS Items⚡Instant Payment/Delivery⚡ by Bert OSRS GOLD Buy&Sell. 186 likes · 2 talking about this. Osrs Gold Buy&Sel Welcome to Old School RuneScape! Relive the challenging levelling system and risk-it-all PvP of the biggest retro styled MMO. Play with millions of other players in this piece of online gaming heritage where the community controls the development so the game is truly what you want it to be Gold Bars are used in the process of making gold items such as Jewellery. Gold mining as a money-maker in Mining Skill. If you wish to start making money on mining gold in OSRS, then this section will guide you on how to do that. The recommended level to start with is 85 7484:Rocks //Copper 7485:Rocks //Tin 7486:Rocks //Unknown, Blurite? 7487:Rocks //Clay? 7488:Rocks //Iron 7489:Rocks //Coal 7490:Rocks //Silver 7491:Rocks //Gold 7492.
P2P OSRS Money-Making: The Best Methods. Published by RuneDeal on January 31, 2021 . There are several ways to make money in the OSRS P2P worlds, unlike the F2P worlds, where the money-making methods are minimal and not very profitable The Leading Runescape Gold Store Since 2006, You Can Shop Cheap Runescape Gold,Buy Osrs Gold, Manual Runescape Powerleveling with 100% No Ban Guaranteed, All Runescape Accounts Are Secure To Buy Which Never Get Recovered! With 24*7 Live Service, You Will Enjoy The Instant Delivery Experience At Gamerluck.com RuneScape® Bot for Old-School (OSRS®) Desktop and Mobile to train your account, harvest gold, do quests, and more! TRiBot will do anything you want in OSRS® Browse thousands in-game items offers. Buy PoE Orbs, OSRS Gold and other items. 24/7 Customer support! Fast and safe delivery
One of the great things about the OSRS gold is that it is cheap to buy, yet expensive to sell. If you ever decide to say goodbye to your gold, you can put it up for sale and Food4Rs.com will offer you best price. Let your doubts never stop you from buying OSRS gold again! Go and get what you want. Less hesitation, more gold Ability to teleport to Dondakan's rock using a Ring of Wealth You get to keep your gold helmet and can continue using it to fire yourself into the rock to access the Arzinian Mine where there a lots of gold rocks and gold veins, and a nearby dwarf banks the player's gold ore for a charge (he keeps 20% of the ores, rounded up, or 10% of the ores if the player has the ring of charos (a)) Ranged is one of the three combat classes in Old School RuneScape. This 1-99 Ranged guide is going to teach you everything you need to know about training your Ranged level in Old School RuneScape. MmoGah also provides cheap OSRS gold for people who want to skip grind and reach 99 fast and easily. Content for this article was inspired by Theoatrix OSRS's video OSRS gold can make your gaming experience smooth and painless! The OSRS experience. OSRS game play is mainly you watching your character chop a tree, mine a rock or kill 100+ mobs for your slayer task. As you get to the later stages of gaming in OSRS, these moments are fewer as it takes longer to get a new level,.
. Home. OSRS Tools . Home MyRSGP Buy OSRS Gold Contact Us Skill Calculators Other Calculators Hiscores Look-Up Equipment Compare Best In Slot XP Table Signature Generator OSRS Map Monsters Database Items List. OSRS Questing. OSRS Gold The Rock is going to be depleted after mining a single Ore out of it unless you're using the Expert mining gloves. A quick way to get to 99 is to smelt Gold Bars in the Blast Furnance. anglers and other skilling outfits found in osrs
OSRS How do gold farmers use NMZ without a Rock cake? Discussion in 'OSRS' started by blood1000, Mar 3, 2021. Sponsored Ad ? blood1000. Joined: clearly used NMZ to get the stats however I'm a bit confused how they would have done this given the lack of a Dwarfen rock cake or the thing you get for during DS2 to reduce your health In our OSRS Motherlode Mine guide, we'll explain what this new feature in Old School RuneScape is, what requirements are needed, and how you can take advantage of it to increase your mining level.. Hopefully, by the time you have finished this guide, you will have learned everything there is to know about the Motherlode Mine. What's included in this OSRS Motherlode Mine guide
OSRS money making Method #2: Mining Gem Rocks(500k+/hourly) Mining gem rocks sounds like an odd one, however these rocks coupled with the new OSRS gem bag allowing easy access to more gem space and easier clicking and smoothness allow you to tick some amazing profit per hour. According to the wiki the average gem rock is worth 600 gp per rock. (#74) Between a Rock... is a quest about helping the old dwarf Dondakan, who is trying to access a legendary gold mine south of Keldagrim, but is unable to penetrate inside.Hidden away from the fearsome trolls and the roaming Fremennik, the dwarves delve deep beneath the mountains in search for the ore they need to support their home of Keldagrim Rs2hot —— The dwarven rock cake is a quest item used in Freeing the Mountain Dwarf subquest, from Recipe for Disaster, to free Rohak's son from the Culinaromancer. Players must pry the recipe from Rohak with 4 glasses of Asgoldian ale before he of..
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Time: 1.5 hours. Required Skills:None. Required Quests:None. Reward: Two experience lamps which provide 500 XP each. An instant 20% favour increase in any house within Great Kourend. Permanently doubled rate of gaining favour in all houses within Great Kourend. Quest points gained upon completion: 1 OS-Unity is a rebuilt OSRS server with amazing features. We are a newly formed OSRS Emulation. Start on December 1, 2016 We have loads of content to offer and a very enjoyable community in which to enjoy it with. The running of the server is flawless and you will find we support all platforms and . 33
r/2007scape: The community for Old School RuneScape discussion on Reddit. Join us for game discussions, weekly events, and skilling competitions! Join 501.4k+ other OSRS players who are already capitalising on the Grand Exchange. Check out our OSRS Flipping Guide (2021), covering GE mechanics, flip finder tools and price graphs. Login Register. Ring of endurance (uncharged) ID: 24844. Contact.
Mining concentrated gold rocks RuneScape Wiki Skilfully manoeuvring around the monsters and hiding behind the rock spawns can also keep you from getting attacked Using Sign of the porter IV can mine more than 900 gold ores per hour at level 80 but it would require the use of a Beast of Burden to carry more materials to create porters (cannot hold porters itself) It is not recommended however. Everyone would like to see their OSRS Gold delivered to them. All excellent sites should mention that your OSRS Gold needs to take to reach you. A fantastic OSRS Gold transaction takes between 3 to 15 minutes to be complete. If from is getting normal time you need to be warned of the If you're here to Buy OSRS Gold, Buy RS3 Gold, Sell RS gold, or swap Runescape gold, we hope you enjoy your time using RSGoldMine for your RS Gold needs. It's our aim as a company and group to reach out to as many people as possible and supply them with the best services on the market, and our trained 24/7 English speaking live chat team reflect that love and gratitude every day Buy Osrs Gold.We provide 24/7 service to our customers. Over 500 Feedbacks at trustpilot. Get your cheap OSRS gold delivered within 3 minutes of purchasing. Check out the best gold provider on the market Gold in RuneScape may refer to Gold rock, Gold ore, Gold bar, Focused gold rocks, Coins, Toban's gold, Gold leaf that is used in building and construction, Golden Barrows devices, Golden mining suit, Golden warpriest armour, Golden rock (located while skilling), Golden chinchompa, Golden Hammer, Golden cracker, Golden katana, Jeweler onslaughts and also Nuggets (gold pieces discovered at Digsite)
. It can be created by using a vessel carrying water such as a jug of water or a bucket of water on clay, by using the Humidify spell, or by mining the clay rock whilst wearing a bracelet of clay. It is also used to create magic tablets at a player-owned house. If you. The gold is a reward for completing different challenges and doing well in the game. The coins can also be used in different ways within the game. Importance of Osrs Gold in RuneScape. The gold can be used in the following manner within the game; Skills; You can use Osrs gold to amp up your skills within the game. The game features a total of. The Fourth test is crafting. You will end up on a plane full of crafting substances, non-agressive monsters, and two chests. The testing agent will provide you a crossword puzzle, to fillout that OSRS gold will tell you the number of items you require, and what you want to make. As soon as you fix it, get the crafting tools you want from a chest you'll need to cast air strike on to open A pet rock is a members only item, and can be obtained from askeladden during and after the fremennik trials quest. This used to be a pet for a skritt. 741,600 iron if you are in need of osrs gold for a skilling pet, you can always buy rs 07 gold cheap on rsorder. Easiest pets to get in osrs (fastest drop rate)
I really could, but you would need an extremely strong furnace to smelt it. I've planned for an extreme heat-producing machine. Should you bring me Armanite Ore, and 5 bits of molten glass, I can smelt you a bar. Ok. Talk to him again. There is a master g DreamBot's # Rock Crabs script Look at the product reviews below to find out why DreamBot users love this script! Script brought to you by @Hashtag Features Smart aggressivity reset system Waterbirth Island and Rellekka Eats food automatically Drinks potions automatically Banking Special attacks Roam-mode AFK-mode Looting Very informative paint Active customer support Gallery Save 25%
M any OSRS beginners are wondering how to start making tons of gold, so that they can buy high-tier gear, and enjoy the game like the rest of the end-game players. Actually there are many methods to start making money , but in my opinion most of them are not worth it , based on the time needed , the complexity , how boring they are, and the low profits they offer, while being hard to master RuneScape Support How can we help you? [06-May] Lockout account returns. Browse help topic 31 Top Photos Pet Rock Osrs / Menagerie | RuneScape Wiki | Fandom powered by Wikia. A pet rock is a members only item, and can be obtained from askeladden during and after the fremennik trials quest. Check out our pet rock selection for the very best in unique or custom, handmade pieces from our figurines & knick knacks shops
In OSRS, gold is used in just about every task and training your skills is no exception. For example, smithing requires a large amount of ore to level up. You will either have to spend a long period of time mining all the ore yourself, or you buy it from an ore seller in the Grand Exchange using your OSRS gold This Old School Quest Guide was written by Piro_Omar.Thanks to Cabola, Pipson, Im4eversmart, Alfawarlord, and McSwindler for corrections. This Old School Quest Guide was entered into the database on Tue, Nov 28, 2006, at 03:46:13 PM by Im4eversmart, and it was last updated on Wed, Jun 28, 2017, at 12:07:38 AM by L1ttleR3d.. If you see this guide on any other site, please report it to us so we. Sep 26, 2019 - shoutouts to whoever wrote the Pet Rock article on the OSRS wiki. Sep 26, 2019 - shoutouts to whoever wrote the Pet Rock article on the OSRS wiki. Pinterest. Today. Explore. When autocomplete results are available use up and down arrows to review and enter to select
Among the challenges that OSRS gold players should conquer early on is currently making a good amount of money. Money is just one of the most powerful items in Old School Runescape and often determines what RuneScape players will have the ability to perform and how well equipped they are to do it Cheap OSRS Gold, RS 2007 Gold and RS3 Gold with Fast Delivery. When RS3 players to vote No on Promotions. Old School RuneScape is inhabited by a lot of different enemies, with monsters and other foes of all different shapes and sizes. In this short guide, we will be discussing the best way for you to overcome trolls. Many players will opt to play this rendition of Runescape to gather up as much OSRS Gold as they can, where they focus on OSRS gold buying and selling rather than slaying beasts
Top Runescape Gold Sites. RSGilded, founded in 2017, is the #1 place to find the most trusted Runescape Gold Sellers on the market. We rank our sellers by the best price so you can purchase your gold at the best prices possible A gold bar is a bar of refined gold created through the Smithing skill at level 40 by using a gold ore with a furnace, or by casting the Superheat item spell on gold ore, granting 22.5 Smithing experience per bar. If goldsmith gauntlets are equipped, obtained after completing the Family Crest quest, the experience is boosted to 56.2 per bar. After an update at some point, smelting gold will. OSRS How do gold farmers use NMZ without a Rock cake? blood1000, Mar 3, 2021. Replies Locked. OSRS Illegalargumentation. XiXu, Feb 24, 2021. Replies: 1 Views: 119. CuppaJava Feb 24, 2021. OSRS How do i use the runemate autoclicker for teleport mage spells. range ko u1 OSRS Transferring gold/items onto Ironman account. Buy Runescape Gold Cheap RS Gold For Sale Here at RSGOLDB2C.com you can buy cheap Runescape gold for OSRS and RS3. We have billions of Runescape gold Kudos Guide for OSRS Kudos can be obtained in the oldschool Runescape by helping the varrock museum staff. Depending on the player, the staff gives out certain kudos as a reward for the help. The fos
However, many sellers of RS gold are oriented only in RS3 players - they don't sell OSRS gold. We are not like them. Our team supports your decision to play OSRS. That's why we can offer you to get Old School RuneScape gold from us. You can buy Old School RuneScape gold from us at any time - we have a lot of it One of the easiest, and most iconic methods of training Magic in OSRS is using the High Alchemy spell. At level 55, you can cast this spell on noted items to turn them into gold using a Nature rune and fire staff. You can do this anywhere in the game, and it gives you a decent amount of experience per hour When you ask her about dwarves and ale, she will offer to tell you a secret recipe for Asgoldian ale in exchange for 200 coins. If you have a bar of chocolate, she will take that plus 50 coins. She will tell you to add gold to an Asgarnian ale to get an Asgoldian ale The pleasure comes as OSRS gold there are strict laws against bringing it out of getting the rum back into the mainland. It's a fun mind teaser with a solution. It is as easy as following digging and the map, after obtaining the rum. A standard Doric's Quest that has for completing, a nice payout


text:
"To us, this is not a political issue."
Despite its measured success, social and political detractors of nearly everything “progressive” still have the audacity to lament the bill’s fictitious and/or largely over-embellished shortcomings while consistently ignoring the efforts of the Republican base to effectively sabotage recovery.
“War on Men” rape comments are perfect example of hate and loathing in right wing media | One White Duck
Wall Street Journal columnist James Taranto – who has also argued previously that sexual assault in the military is all part of a vast, leftist conspiracy to “criminalize male sexuality” – suggests in his latest column that the onus of NOT being raped lies with the woman; a sentiment highly reminiscent of the anti-women propaganda all too prevalent in Right wing circles. His correlation between such defensive behavior – drink less, dress different, know your place – and the avoidance of rape is one of the main reasons why rape victims are all too often either reluctant to report assaults or shamed into self-blame afterwards.

domain: sociology


A stunning performance from Livingston saw them pick up a first ever victory against Celtic as goals from Scott Robinson and Lyndon Dykes ensured they defeated the champions 2-0 at the Tony Macaroni Arena.
Livi boss Gary Holt admitted he was 'over the moon' with the three points and hailed his side's defensive efforts as they put an end to a run of four successive defeats in all competitions.
He said: "It's another tick in the box and I'm over the moon to be honest.
"The first goal was great. It was a great bit of skill, great movement, great pass and a great finish and I think the game deserved that.
"I'm delighted for the back-line because I questioned them, and the whole team, about who wants to get hurt at set-pieces. Who is going to put their body on the line? I thought they were tremendous today because Celtic got a lot of balls into the box. As a team we defended great."
It was the type of spirited, robust display that has been Livi's hallmark over the past few seasons while Neil Lennon's side are unlikely to produce such an abject performance again any time soon with a string of players looking as if they'd rather be anywhere but The Tony Macaroni Arena, in particular Christopher Julien who was second best to Dykes all afternoon.
Holt made a bold move with his team selection in dropping goalkeeper Ross Stewart in favour of Aston Villa loanee Matija Sarkic, who made his first league appearance of the campaign.
The hosts created the first half chance of the game on seven minutes when the impressive Dykes teed up Robinson on the edge of the box, but the former Hearts man saw his strike blocked while Celtic will be thinking they should have taken the lead moments later.
Odsonne Edouard bulldozed his way through the Lions' defence before finding James Forrest but the winger's poked effort was wide of the mark.
Nicky Devlin's cross was steered wide by John Guthrie as the hosts were giving as good as they got. Steven Lawless was next to go close but his neat one-two with Robinson ended in the former firing straight at Fraser Forster.
Edouard was well marshaled all game but did have a glorious opportunity to send his side into lead but Sarkic was more than a match after Julien had strolled up the park before finding his fellow Frenchman.
The turning point arrived just before the half-hour mark when Celtic's Ryan Christie was shown a straight red card for a late, and high, lunge on Robinson. The midfielder could have no complaints about the decision.
The hosts needed barely two minutes of the second half to find the opener when Robbie Crawford's delightful through ball caught a static Celtic defence with Robinson showing composure that had been lacking from Livi this term to stroke the ball home beyond Forster.
That sparked the champions into action with Vakoun Bayo being introduced while the previously ineffective Mohamed Elyounoussi went close to grabbing a leveller on the hour mark only for Sarkic to pull off a great save and the Livi defence to react first to the rebound and clear the danger.
Having kept his team in front, the Montengrin keeper played a crucial role in the second 17 minutes from time when his free-kick was launched up the park for Dykes to sneak in behind the once again dithering Celts defence and loop a sumptuous effort over Forster for his fifth goal of the campaign.
From then on it was a case of seeing the game out, which simply played into Livi's hands as they camped all 11 men behind the ball while Celtic completely ran out of ideas as slumped to their first domestic defeat of the season.
Holt added: "I actually thought we were better when it was 11 versus 11. We were at it and it was a really good game and I think we got a wee bit disjointed when we got the extra man and we spoke to them at half-time about being patient.
"I asked them before the game, as I do every game, to go toe-to-toe with their opposite man. Look them in the eye and say 'you're going to be in a battle today. Do you want it? And I think we got that all over the park.
"The performances have been excellent and it's been the fine lines that have cost us so to take our chances was pleasing."


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Understanding how complex organ systems are assembled from simple embryonic tissues is a major challenge. Across the animal kingdom a great diversity of visual organs are initiated by a ‘master control gene’ called Pax6, which is both necessary and sufficient for eye development. Yet precisely how Pax6 achieves this deeply homologous function is poorly understood. Using the chick as a model organism, we show that vertebrate Pax6 interacts with a pair of morphogen-coding genes, Tgfb2 and Fst, to form a putative Turing network, which we have computationally modelled. Computer simulations suggest that this gene network is sufficient to spontaneously polarise the developing retina, establishing the first organisational axis of the eye and prefiguring its further development. Our findings reveal how retinal self-organisation may be initiated independently of the highly ordered tissue interactions that help to assemble the eye in vivo. These results help to explain how stem cell aggregates spontaneously self-organise into functional eye-cups in vitro. We anticipate these findings will help to underpin retinal organoid technology, which holds much promise as a platform for disease modelling, drug development and regenerative therapies.
Positional cues that govern cell fate decisions in the embryo may arise at multiple organisational levels: cell intrinsically (e.g. asymmetric cell divisions), tissue intrinsically (e.g. reaction-diffusion mechanisms), tissue extrinsically (e.g. inductive tissue interactions) or some combination of these. Historically, the early patterning of cell fates within the vertebrate eye has emphasised inductive interactions, stemming from Spemann's seminal work on lens induction (Spemann, 1901). These inductive interactions furnish positional information to coordinate self-assembly of the various tissues that make up the vertebrate camera eye, including the optic vesicle of the forebrain, which generates the retina, and the overlying presumptive lens ectoderm (Gunhaga, 2011). In the embryo, interactions with neighbouring tissues help to remodel the hemi-spherical optic vesicle into a bi-layered optic cup (Fig. 1A). Yet this vesicle-to-cup transformation is spontaneously recapitulated by stem cell-derived retinal organoids in vitro (Eiraku et al., 2011), revealing that a hitherto unsuspected tissue-intrinsic mechanism suffices to self-organise the primary retinal axis. Here, we provide evidence for a self-organising mechanism centred on the transcription factor-coding gene paired box 6 (Pax6).
Pax6 has been called an eye master control gene (Gehring, 1996) and is necessary for eye development across much of the animal kingdom, from flies to humans (Hill et al., 1991; Hodgson and Saunders, 1980; Hoge, 1915; Nakayama et al., 2015). Mis-expression of mammalian or cephalopod Pax6 genes triggers the spontaneous development of ectopic compound eyes in arthropods (Halder et al., 1995; Tomarev et al., 1997), as well as supernumerary camera eyes in vertebrates (Chow et al., 1999). This deeply homologous function, whereby a shared Pax6 genetic apparatus builds eye structures that are morphologically and phylogenetically distinct (Shubin et al., 1997), is poorly understood.
The transforming growth factor β (Tgfβ) signalling pathway (Massagué, 1998) is transduced by ligand dimers that assemble hetero-tetrameric receptor complexes. The activated receptor complex then phosphorylates Smad2 and Smad3 proteins, which assemble with Smad4 before translocating to the nucleus where they interact with transcription factors to regulate gene expression. Whereas Smad2, Smad3 and Smad4 transduce Tgfβ, activin and Nodal signals, an inhibitory Smad7 antagonises this pathway cell-autonomously. Additionally, secreted antagonists, such as follistatin (Fst) act non-cell-autonomously by blocking ligand-receptor interactions (Iemura et al., 1998; Nakamura et al., 1990; Nogai et al., 2008). Smad4 is shared with the parallel bone morphogenetic protein (Bmp) signalling pathway, the signals of which are transduced by Smad1, Smad5 and Smad8, and inhibited by Smad6. We have previously reported that Pax6 protein function, and thus autoregulation, is inhibited via a direct Tgfβ-dependent interaction with Smad3, which inhibits Pax6-DNA binding (Grocott et al., 2007). Subsequently, we showed that Tgfβ signals emanating from the peri-ocular neural crest mesenchyme suppress Pax6 to align the lens with the optic vesicle (Grocott et al., 2011).
The molecular mechanisms by which tissues spontaneously generate patterns was first considered by Turing, who coined the term ‘morphogen’ to describe such molecules and devised reaction-diffusion models to simulate them (Turing, 1952). Gierer and Meinhardt later independently conceived of their activator-inhibitor model – a Turing network in which a slow-diffusing activator morphogen drives both its own production and that of a faster diffusing inhibitor morphogen, which suppresses the activator (Fig. 1B) (Gierer and Meinhardt, 1972). Thus, there arises a molar excess of activator over inhibitor at their source, where positive-feedback dominates, but a molar excess of inhibitor away from their source, where negative-feedback dominates (Fig. 1C).
Here, we describe a putative self-organising Turing network (Turing, 1952) comprising Pax6 and a pair of morphogen-coding genes: transforming growth factor β2 (Tgfb2) and follistatin (Fst). Using reaction-diffusion modelling we show how this gene network may spontaneously polarise the optic vesicle to trigger self-organisation of the vertebrate retina.
Extrinsic Bmp signals drive Pax6 expression in the distal optic vesicle
Optic vesicle polarisation is apparent from Hamburger and Hamilton (1992) stage HH10 in the chick, evidenced by differential gene expression along a proximal-distal axis (Fig. 1D): Pax6 and visual system homeobox 2 (Vsx2; formerly Chx10) are expressed distally (Fig. 1E,F), whereas microphthalmia-associated transcription factor (Mitf) and Wnt family member 2b (Wnt2b; formerly Wnt13) are expressed proximally (Fig. 1G,H). We additionally report that two further genes, transforming growth factor β2 (Tgfb2) and follistatin (Fst) are co-expressed with Pax6 in the distal optic vesicle (Fig. 1I,J). Neither Tgfb2 nor Fst expression is detected in the overlying presumptive lens ectoderm.
As the optic vesicles evaginate between stages HH8 and HH10, they encounter bone morphogenetic protein (Bmp) family growth factors from the overlying surface ectoderm (e.g. Bmp4; Fig. 1K). Bmps are implicated in establishing both distal and proximal cell identities within the optic vesicle; Bmp alone promotes distal character (Pandit et al., 2015), whereas combined with canonical Wnt signalling it was proposed to induce proximal character (Steinfeld et al., 2013). Consistently, we found that exposing HH10 optic vesicle explants to Bmp4 ligand for 16 h in vitro led to an upregulation of distal Pax6 (2.35±0.19 fold, mean±standard deviation; P<0.01; n=4) as measured by RT-QPCR (Fig. 1L). The remaining distal (Vsx2) and proximal (Wnt2b and Mitf) markers were not significantly affected (Fig. 1L). Following combined exposure to both Bmp4 and the Wnt agonist BIO (6-bromoindirubin-3′-oxime; GSK3 inhibitor) (Meijer et al., 2003), Pax6 (1.88±0.38 fold; P<0.05; n=5) was similarly affected (Fig. 1M), while the proximal marker Wnt2b was additionally upregulated (9.28±7.89 fold; P<0.05; n=5), suggesting that Wnt2b may auto-regulate. Wnt activation alone induced proximal Wnt2b (3.69±1.43 fold; P<0.01; n=4) without significantly affecting distal markers (Fig. 1N), while exposure to DMSO (carrier for BIO) had no impact (Fig. 1O). These data do not support a direct synergism between Bmp and Wnt signalling in establishing proximal-distal polarity, as their combined action is merely additive.
To validate the interaction between Bmp signalling and Pax6 expression in vivo, we performed electroporation-mediated gene transfer to mis-express the cell-autonomous Bmp inhibitor Smad6 in single optic vesicles, while unelectroporated contralateral vesicles served as internal negative controls (Fig. 2A). In comparison with mis-expression of a benign enhanced green fluorescent protein (GFP; 1.13±0.37 fold; n=7; Fig. 2C,D), Smad6 caused an asymmetric reduction in the area of Pax6 expression between transfected and contralateral control vesicles (0.56±0.31 fold; P<0.05; n=13; Fig. 2C,E). This confirms that distal Pax6 expression in vivo requires upstream Bmp.
Auto-regulation of Pax6 has been reported in a number of tissues, including the lens (Ashery-Padan et al., 2000). To test for Pax6 auto-regulation in the optic vesicle, a C-terminally truncated dominant-negative Pax6 gene (dnPax6) (Grocott et al., 2007) was mis-expressed unilaterally, while a C-terminal riboprobe was used to selectively detect endogenous Pax6 expression (Fig. 2B). dnPax6 did not disrupt endogenous Pax6 expression (0.75±0.36 fold; P>0.05; n=9; Fig. 2C,F) compared with the GFP control, nor could we distinguish a difference between dnPax6 and Smad6 mis-expression (Fig. 2C; P>0.05). To confirm that dnPax6 was overexpressed relative to endogenous Pax6, an N-terminal riboprobe was used to collectively detect both endogenous Pax6 and exogenous dnPax6 expression (Fig. 2G). Thus, although distal Pax6 expression in the optic vesicle requires Bmp signalling in vivo, we cannot exclude the possibility that upstream Bmp action may mask subsequent Pax6 auto-regulation.
Pax6 drives expression of Tgfb2 and its antagonist Fst in the distal optic vesicle
Migratory neural crest cells reach the optic vesicle at stage HH10, where they contribute to the periocular mesenchyme and are thought to induce proximal and suppress distal gene expression via Tgfβ subfamily signalling (Fuhrmann et al., 2000; Grocott et al., 2011). Exogenously supplied Tgfβ subfamily ligand (activin A) was reported to induce proximal (Wnt2b and Mitf) and inhibit distal (Pax6 and Vsx2) gene expression in explant cultures (Fuhrmann et al., 2000). In contrast to this tissue-extrinsic induction mechanism, stem cell-derived retinal organoids are reported to polarise tissue-autonomously, exemplified by the spontaneous acquisition of proximal Wnt activity (Hasegawa et al., 2016). This raises the possibility of a redundant tissue-intrinsic polarising activity. Given that distal Tgfb2 expression correlates with Pax6 (Fig. 1E,I) we asked whether Pax6 might induce Tgfb2 to activate proximal target genes tissue-autonomously. In comparison with GFP controls (1.06±0.17 fold; n=8; Fig. 3A,B), mis-expression of dnPax6 in single optic vesicles diminished Tgfb2 expression relative to contralateral control vesicles (0.79±0.54 fold; P<0.05; n=15; Fig. 3A,C). Thus, the Pax6 master controller is required for Tgfb2 expression in the distal vesicle, consistent with a report of Pax6-binding sites located within the Tgfb2 promoter (Wolf et al., 2009).
This presents a paradox, however: Tgfb2 expression (Fig. 1I) negatively correlates with its positive targets Wnt2b and Mitf (Fig. 1G,H), yet positively correlates with its negative targets Pax6 and Vsx2 (Fig. 1E,F) (Fuhrmann et al., 2000). How might Tgfβ pathway activation become inverted relative to Tgfb2 gene expression? We considered whether Pax6 might also activate Fst (Fig. 1J), a Tgfβ antagonist, to grant distal immunity from Tgfβ signalling. Compared with GFP controls (1.31±0.63 fold; n=6; Fig. 3D,E), mis-expression of dnPax6 in a single optic vesicle significantly reduced Fst expression (0.69±0.34 fold; P<0.05; n=8; Fig. 3D,F). Thus, Pax6 function is additionally required for Fst expression in the distal vesicle.
The paradoxical out-of-phase expression of distal Tgfb2 and its proximal (positive) targets might then be explained by differential diffusion of Tgfb2 and Fst gene products resulting in: (1) Tgfβ2 being locally sequestered by slow-diffusing Fst within the distal vesicle, thereby preserving distal character; and (2) fast-diffusing Tgfb2 dispersing proximally away from Fst, to induce proximal character within the neighbouring proximal vesicle.
To test whether this hypothesis is plausible, we examined a reaction-diffusion model of the interactions summarised in Fig. 4A (Model A, Fig. S1; see supplementary information) and performed numerical simulations in one dimension only to represent the anterior-posterior axis (comprising anterior-proximal, distal and posterior-proximal domains) of the optic vesicle. Simulations were performed with both zero-flux (Fig. 5) and periodic (Movies 1 and 2) boundary conditions to represent dissected optic vesicle explants and spherical organoids, respectively.
A variety of diffusion ratios for Tgfb2 dimers and Fst monomers versus Fst:Tgfb2 complexes were explored (e.g. Fig. 4B,B′; Fig. S2; Movie 1). Simulations demonstrated that local inhibition and lateral-activation of Tgfb signalling may occur if the diffusion rate of Fst:Tgfb2 complexes exceeds that of Fst monomers. Although initially counter-intuitive, there is precedent for ligand:antagonist complexes that disperse faster than their individual constituents (Esteve et al., 2011), and our subsequent simulations assume this condition is satisfied.
Pax6, Fst and Tgfb2 form a self-organising Turing network that can dynamically polarise the optic vesicle
Given that Tgfb signalling is known to disrupt Pax6 protein function (Grocott et al., 2007), such local inhibition and lateral activation of Tgfβ signalling equate to local positive feedback and lateral-inhibition of the Pax6 master control gene, respectively (Fig. 4C). This is functionally equivalent to a simple activator-inhibitor type (Fig. 1B) Turing network (Gierer and Meinhardt, 1972; Turing, 1952), which can serve as a spontaneous pattern generator: Pax6 and Fst provide a short-range auto-regulating activator; and Tgfb2 is the long-range inhibitor (compare Fig. 1B with Fig. 4C). To explore whether the network of Fig. 4C possesses spontaneous polarising activity, we simply extended Model A to include inhibition of Pax6 function by Tgfβ signalling (Model B; Fig. S3; see supplementary information). Simulations showed that an initially homogenous but noisy Pax6 distribution is readily converted into a polarised pattern, wherein Pax6 expression becomes regionally restricted (Fig. 4D) and out-of-phase with Tgfb receptor activation (Fig. 4D′; Fig. S4; Movie 2). Additionally, simulating larger tissue sizes results not in a larger Pax6-expressing distal pole, but in a greater number of Pax6-expressing distal poles of approximately equal size (Fig. 4E,E′). This hallmark feature of Turing networks is remarkably consistent with observations of retinal organoid cultures in which stem cell aggregates yielded between one and four retinas each (Eiraku et al., 2011).
Similarly, reducing tissue size limits the number rather than the size of pattern elements generated by a Turing network so that, for example, a single ‘spot’, half a ‘spot’ (i.e. a gradient) or no ‘spot’ is generated. When cultured as isolated explants in the absence of serum, polarised HH10 optic vesicles (e.g. Fig. 4F) collapse into compact spheroids (Fig. 4G), reducing the longest dimension of this tissue to ≤0.5 fold. To better understand how the Pax6/Fst/Tgfb2 network might respond in this situation, we performed 2D simulations of Model B on an explant-shaped domain with an initial distal-high to proximal-low Pax6 pattern (Model C; Fig. 4H; see supplementary information). For these simulations, we explored both zero-flux and fixed-boundary conditions, disregarding the latter as the former agreed more closely with experimental observations. It may be interpreted that adsorption of morphogens to extracellular matrix and cell-surface proteins within explants prevents a significant outward flux, while the absence of morphogens from the defined bathing medium prevents an inward flux.
Owing to the reduced tissue size, this proximal-distal pattern proved unstable and Pax6 expression quickly re-polarised to form a gradient along the longest axis of the explant (i.e. perpendicular to the former proximal-distal axis; Fig. 4H′; Fig. S5; Fig. S6; Movie 3). To test this model prediction, optic vesicles were dissected for explant culture, during which their distal poles were labelled with DiO (Fig. 4I). Immunostaining of a partially dissected optic vesicle verifies that DiO labelling coincides with the initial Pax6+ distal pole (Fig. 4I′). Following overnight culture, however, Pax6 expression no longer coincides with the distal DiO label but instead re-polarises along the longest axis of each explant (Fig. 4J,J′) consistent with simulations. This suggests that the Pax6/Fst/Tgfb2 network can dynamically repolarise its expression in a self-organising fashion.
Intrinsic positional information constrains Pax6/Fst/Tgfb2 self-organisation
In explant culture, optic vesicles are isolated from inductive tissue interactions and thus from extrinsic positional information. However, we questioned whether Pax6 repolarisation might be influenced by intrinsic positional information. A ventral-high to dorsal-low gradient of Sonic hedgehog (Shh) signalling activity exists within the optic vesicle, which is known to restrict the ventral extent of Pax6 expression (Ekker et al., 1995; Macdonald et al., 1995). Might Shh positional information push the Pax6+ pole towards the dorsal side of the explant? Electroporation of a GFP expression construct was targeted to the ventral optic vesicle prior to dissection and overnight explant culture. Whole-mount immunofluorescence staining showed that the Pax6+ pole negatively correlates with ventral GFP expression (100% of explants; n=5; Fig. 5A,A′), supporting this idea.
To explore how Shh positional information might interact with the Pax6/Fst/Tgfb network, we extended Model C by incorporating Shh suppression of Pax6 into the governing equations (Model D; Fig. 5B; see supplementary information) while adding a Shh positional information gradient (Fig. 5C; Fig. S7). Simulations showed that the Pax6+ pole reorientates away from the ventral-high end of the Shh gradient (Fig. 5C′; Fig. S8; Fig. S9) as was observed experimentally (Fig. 5A,A′). Moreover, inverting the Shh gradient (Fig. 5D) caused a reversal of Pax6 polarity (Fig. 5D′).
Exploring Model D, we next simulated the ability of Pax6 to repolarise in the absence of Tgfβ-mediated self-organisation and found that the Shh positional information gradient was sufficient to generate a dorsal Pax6+ pole (Fig. 5E,E′). This prediction was tested experimentally by culturing optic vesicle explants in the presence of a Smad3 inhibitor, SIS3 (Jinnin et al., 2006). As Tgfβ inhibits Pax6 protein function via its specific and direct interaction with Smad3 (Grocott et al., 2007), SIS3 should block the inhibition of Pax6 by Tgfβ2 (Fig. 5B). Following overnight culture with 10 μM SIS3, optic vesicle explants still exhibited distinct Pax6+ poles (91% of explants; n=11; Fig. 5F,F′) as predicted. These data show that Tgfβ-mediated self-organisation is not required for Pax6 polarisation in cultured explants, presumably due to the redundant action of Shh positional information.
Model D simulations lacking Shh positional information (Shh LOF; Fig. 5G) predicted that the Pax6/Fst/Tgfb2 network should suffice to generate a Pax6+ pole in the absence of Shh activity (Fig. 5G′). To test this, optic vesicle explants were cultured overnight with 2.5 μM cyclopamine: a steroidal alkaloid that inhibits the Hedgehog pathway transducer Smoothened (Chen et al., 2002). As predicted, explants still exhibited Pax6+ poles in the absence of Shh activity (82% of explants; n=11; Fig. 5H,H′). Thus, Shh positional information is not required for Pax6 polarisation in optic vesicle explants, suggesting that the Pax6/Fst/Tgfb2 network is sufficient to self-organise the Pax6+ pole. However, although still polarised in the absence of Shh positional information, Pax6 expression is subtly upregulated both in simulations (compare Fig. 5E′ with 5G′) and in experiments (compare Fig. 5F with 5H).
Further Model D simulations predicted that simultaneous loss of both Shh positional information (Fig. 5I) and Tgfβ-mediated self-organisation should prevent Pax6 polarisation in cultured explants (Fig. 5I′); instead of polarising, Pax6 was expressed uniformly throughout the simulated explant. Consistent with this, optic vesicle explants cultured with both 2.5 μM cyclopamine and 10 μM SIS3 mostly failed to exhibit Pax6 polarisation as expression was approximately uniform across their lengths (67% of explants; n=12; Fig. 5J,J′). In other words, the Pax6/Fst/Tgfb2 network appears to be both sufficient (Fig. 5H,H′) and necessary (Fig. 5J,J′) to self-organise Pax6 polarisation in the absence of positional information.
The Pax6/Fst/Tgfb2 gene network regulates distal neural retinal identity in vivo
The preceding data suggest that, although the Pax6/Fst/Tgfb2 network may freely self-organise in isolation (e.g. in retinal organoids), in vivo this network is constrained by intrinsic (e.g. Shh) and extrinsic (e.g. Bmp4) positional information to ensure correct alignment of the distal Pax6+ pole within the camera eye. Thus, functional perturbations in vivo are not expected to drive the kind of dynamic re-polarisation observed in cultured explants. How then might functional perturbation of the Pax6/Fst/Tgfb2 network impact optic vesicle patterning in vivo?
According to our model, interference with Fst gene expression should de-repress Tgfβ signalling and inhibit Pax6 protein function in the distal vesicle, via the direct Tgfβ-dependent interaction of Smad3 with Pax6 (Grocott et al., 2007). Moreover, if Pax6 auto-regulates in the distal vesicle, this should manifest as a Tgfβ-mediated reduction in Pax6 gene expression. To test this prediction, we employed morpholino oligonucleotides to suppress translation of Fst 315 and Fst 300 isoforms (Fig. 6A) within single optic vesicles. Pax6 expression was then compared between these and unperturbed contralateral vesicles. Fst morpholino (FstMO) was first shown to suppress endogenous translation of both Fst isoforms in cultured chick embryonic cells via western blotting when compared with a standard control morpholino (StdMO) that does not target Fst (Fig. 6B).
In vivo, StdMO controls had no impact on Pax6 expression in transfected optic vesicles (1.05±0.31 fold; n=20; Fig. 6C,D). In comparison, FstMO reduced Pax6 expression in transfected vesicles (0.76±0.50 fold; P<0.01; n=18; Fig. 6C,E). We were able to rescue this loss of Pax6 expression by co-transfecting FstMO together with an exogenous Fst transgene that evades FstMO and encodes the Fst 315 isoform (0.98±0.35 fold; P>0.05; n=25; Fig. 6C,F). This confirmed that loss of Pax6 was not due to a morpholino off-target effect and that Fst gene function is required for distal Pax6 expression in the optic vesicle. This is consistent with earlier reports that neural induction by way of Fst overexpression induces Pax6 in Xenopus animal cap explants (Altmann et al., 1997).
To verify that loss of Pax6 expression is indeed due to the predicted de-repression of Tgfβ signalling, we attempted an alternate rescue by co-transfecting FstMO together with a cell-autonomous Tgfβ/Activin/Nodal pathway inhibitor, Smad7. As can be seen (Fig. 6C,G), no significant loss of Pax6 expression was observed (0.91±0.31 fold; P>0.05; n=13) when Fst translation and Tgfb signalling were simultaneously suppressed.
In addition to inducing Pax6 (Altmann et al., 1997), overexpression of Fst in Xenopus animal cap explants was reported to induce expression of the retinal photoreceptor marker Opsin (Hemmati-Brivanlou et al., 1994). We therefore investigated whether Vsx2, a distally expressed neural retinal marker (Liu et al., 1994) (Fig. 1F), is similarly affected upon disruption of the Pax6/Fst/Tgfb2 gene network. In comparison with StdMO controls (1.51±1.05 fold; n=7; Fig. 6H,I), FstMO significantly reduced distal Vsx2 expression in transfected optic vesicles (0.69±0.33 fold; P<0.05; n=9; Fig. 6H,J). Thus, de-repression of endogenous Tgfβ signalling in the distal vesicle is detrimental for correct proximal-distal patterning, including specification of the neural retina. These results are consistent with our general model and support the idea that Fst and Tgfb2 morphogens positively and negatively regulate Pax6 function, respectively, in order to polarise the optic vesicle.
The issue of the master control mechanism of Pax6 has now been unresolved for a quarter of a century (Cvekl and Callaerts, 2017). Here, we have shown that the vertebrate Pax6 directs expression of a pair of morphogen coding genes, Fst and Tgfb2, which modulate Pax6 function via positive and negative feedback. This Pax6/Fst/Tgfb2 gene network topology is consistent with an activator-inhibitor type Turing network and appears to exhibit a self-organising pattern-forming ability in the absence of positional information. This spontaneous pattern-forming potential could explain both the ability of Pax6 to trigger ectopic eye development across the animal kingdom (Chow et al., 1999; Halder et al., 1995; Tomarev et al., 1997) and the spontaneous development of self-organising optic cups from stem cell aggregates cultured in vitro (Eiraku et al., 2011).
Prerequisites for retinal self-organisation
Our reaction-diffusion simulations showed that the Pax6/Fst/Tgfb2 gene network may act as a self-organising Turing network, providing certain assumptions are satisfied. For example, we have assumed that larger Fst:Tgfb2 complexes diffuse more quickly than smaller Fst monomers. This is counter-intuitive as pure diffusion rate is a function of molecular mass. Yet there is a precedent for this phenomenon: for example, Sfrp:Wnt complexes have been observed to diffuse further than Wnt alone (Esteve et al., 2011). We postulate that Fst monomers disperse sub-diffusively due to binding interactions with extracellular matrix components and/or cell surface factors, e.g. heparin sulfate proteoglycans (Nakamura et al., 1991) or fibronectin (Maguer-Satta et al., 2006). In the context of Fst:Tgfb2 complexes, the relevant interaction surfaces may be shielded, enabling the larger complex to disperse further and faster than its constituents.
This assumed rapid dispersal of Fst:Tgfb2 complexes is only required if Tgfb2 sequestration by Fst is reversible, which is currently unknown. Low-affinity Fst:Bmp interactions are known to be reversible, whereas high-affinity Fst:Activin interactions are effectively irreversible (Iemura et al., 1998). If Fst:Tgfb2 associate irreversibly then spontaneous pattern formation is still possible, but it changes assumptions regarding effective diffusion rates: Fst:Tgfb2 diffusion would then become irrelevant and, instead, Tgfb2 dimers must diffuse faster than Fst monomers (Murray, 2003).
Self-organisation versus positional information in vivo
By demonstrating how Pax6 may drive self-organisation of the primary retinal axis, our findings offer the first mechanistic explanation of the long-known but poorly understood master control function of Pax6. In the embryo, we propose that this putative Turing network acts to self-organise the proximal-distal axis of the optic vesicle (as summarised in Fig. 7A,B), in concert with positional information (e.g. from previously identified inductive interactions) to ensure correct alignment with neighbouring tissues.
In Model D, we accounted for intrinsic positional information by incorporating direct suppression of Pax6 expression by a ventral-high to dorsal-low gradient of Shh activity (Fig. 5; see supplementary information) (Ekker et al., 1995; Macdonald et al., 1995). This is a convenient abstraction, however; at later stages, the ventral extent of Pax6 expression in vivo is refined via reciprocal inhibition between distal Pax6 (prospective neural retina) and ventral Pax2 (prospective optic stalk) (Schwarz et al., 2000), the expression of which is activated by ventral Shh (Ekker et al., 1995; Macdonald et al., 1995).
Regarding extrinsic positional information, Bmp signals from the overlying head ectoderm appear to activate the Pax6/Fst/Tgfb2 network and may also bias proximal-distal polarity to align the distal Pax6+ pole with the prospective lens. This would explain why Bmps from the head ectoderm have been attributed with inducing both proximal retinal pigment epithelium (Müller et al., 2007) and distal neural retina (Pandit et al., 2015) within the optic vesicle.
We did not investigate the role of Wnt in establishing proximal identity within the optic vesicle, except to test for direct synergism between Wnt and Bmp, as previously proposed (Steinfeld et al., 2013). In the absence of such synergism, we suggest that Wnt acts downstream of the Pax6/Fst/Tgfb2 gene network, as: (1) Wnt2b is a Tgfβ target gene (Fuhrmann et al., 2000) restricted to the proximal optic vesicle (Fig. 1G); and (2) expression of Wnt2b is absent from the peri-ocular surface ectoderm until stage HH11 (Grocott et al., 2011), prior to which polarised Wnt2b expression is already established within the optic vesicle itself (Fig. 1G).
In addition to the loss of inductive signals, ablation of the overlying lens ectoderm (Steinfeld et al., 2013) may permit periocular Tgfβ proteins from the surrounding neural crest mesenchyme (Fuhrmann et al., 2000; Grocott et al., 2011) to overwhelm the autonomous polarising activity of the Pax6/Fst/Tgfb2 network. In turn, it has not escaped our attention that distal Fst may mediate classical lens induction (Spemann, 1901) by opposing these same lens-inhibitory Tgfb signals (Grocott et al., 2011); indeed, Fst overexpression induces lens crystallin expression in Xenopus animal cap explants (Altmann et al., 1997).
Retinal organoids and self-organisation in vitro
During retinal organoid development in vitro, we propose that the Pax6/Fst/Tgfb2 network may suffice to self-organise the primary axis of the retina in the absence of the well-organised positional information normally present in vivo. For example, ventral optic vesicle structures are absent in self-organising retinal organoids (Eiraku et al., 2011), which suggests an absence of intrinsic Shh positional information.
The comparatively chaotic nature of organoids makes them an ideal counterpart to embryonic models of development as they can unmask cryptic self-organising mechanisms and test them to breaking point; contrast the straightforward elaboration of an existing pre-pattern (Fig. 4B,B′; analogous to localised Pax6 induction by neighbouring Bmps in vivo) with the more turbulent emergence of order from disorder (Fig. 4D,D′; analogous to spontaneous Pax6 activation in retinal organoids).
In simulations of de novo pattern formation, the Pax6/Fst/Tgfb2 network is observed to oscillate (Fig. 4D,D′; Movie 2). This potential for oscillation derives from the Eigenvalues associated with the Turing condition and thus from the models' governing equations and parameter choices. For example, in Model B, the tendency to oscillate may be suppressed by increasing the negative feedback that Tgfb2 exerts on Pax6. Whether or not oscillations manifest in a given simulation is further influenced by the choice of initial conditions. For example, Model B is observed to oscillate during de novo pattern formation (Fig. 4D,D′; Movie 2), but not when elaborating an existing pre-pattern (equivalent to the Model A simulation in Fig. 4B,B′; Movie 1). For this reason, we might expect that oscillations are more likely to arise during de novo pattern formation in retinal organoid cultures and less so in the embryo, where the wealth of positional information constrains the Pax6/Fst/Tgfb2 network. Whether or not this gene network oscillates in vitro or in vivo, and the potential impact on robustness and reproducibility of organoid cultures, is yet to be investigated.
A future challenge will be to develop a full 3D model of optic vesicle patterning, incorporating the Pax6/Fst/Tgfb2 Turing network together with all sources of constraining positional information. A multi-scale approach, in which the feedback between tissue patterning (via the reaction-diffusion formalism used here) and cell dynamics (e.g. via Cellular Potts, vertex or finite element approaches) could further illuminate the feedback between tissue patterning and morphogenesis. A 2D vertex model of optic cup morphogenesis has been previously reported (Eiraku et al., 2012), but a multi-scale approach will be required to fully grasp how genes determine geometry and to identify causal links between genetic and anatomical aberrations.
The identification of defined, animal-free substrates for organoid cultures is a prerequisite for clinical applications. This, and enhanced reproducibility, strongly motivate the search for alternatives to incompletely defined and animal-derived Matrigel, which has superseded laminin as the substrate of choice for in vitro retinogenesis (Capowski et al., 2019; Eiraku et al., 2011; Meyer et al., 2009). Interestingly, Matrigel's sixth most abundant ECM component, fibronectin (Rijal and Li, 2017), is enriched within the basement membrane of the optic vesicle in vivo (Krotoski et al., 1986; Kurkinen et al., 1979) and binds Fst (Maguer-Satta et al., 2006). Could ECM components such as fibronectin support self-organisation by limiting Fst diffusion relative to Tgfb2 or the Fst:Tgfb2 complex? Further studies are needed to characterise diffusion of these morphogens both in vivo and in vitro, and to clarify the role of ECM composition in supporting their differential diffusion.
Further exploration of the Pax6/Fst/Tgfb2 network may drive future developments in retinal organoid technology and help underpin applications in disease modelling, drug discovery and regenerative therapies. Given the deeply homologous nature of the Pax6 master control function, we would predict that Pax6 orthologues participate in functionally homologous Turing networks in non-vertebrates, which may comprise the same or different morphogens.
MATERIALS AND METHODS
Fertile brown hen's eggs (Henry Stewart) were incubated at 38°C in a humidified incubator until the required stage of development: HH8 for in ovo electroporation experiments; HH10 for in vitro explant experiments. The study was approved by the Animal Welfare & Ethical Review Board, School of Biological Sciences of the University of East Anglia, and all procedures were performed in accordance with the relevant guidelines and regulations.
HH10 embryos were incubated with 0.25% Trypsin-EDTA at 38°C for 7 min. Trypsin was then de-activated by transferring into 20% chick serum on ice for 5 min. Embryos were then washed with Tyrode's solution and pinned onto Sylgard-coated dissection dishes. Head surface ectoderm and peri-ocular mesenchyme were carefully removed using 30 gauge syringe needles from both dorsal and ventral sides. Once cleaned, both optic vesicles were removed and held in Tyrode's solution on ice. Left and right optic vesicles were separately pooled from at least five embryos, yielding two match-paired pools for use as treated and control samples. Pooled vesicles were cultured in polyHEMA (Sigma)-coated culture wells to prevent adhesion, with DMEM-F12 media (Invitrogen) supplemented with 1× N2 (Invitrogen), 1× L-glutamate and 1× penicillin/streptomycin at 37°C and 5% CO2 for 16 h. Culture media for treated samples was supplemented with the following factors as required: 35 ng/ml Bmp4 (R&D Systems), 0.5 µM BIO (Sigma) with 0.1% DMSO (Sigma), 10 µM SIS3 with 0.1% DMSO (Sigma), or 2.5 µM cyclopamine (Sigma) with 0.1% 2-hydroxypropyl-β-cyclodextrin (HBC; Sigma).
Whole-mount immunofluorescence staining of explants
Cultured explants were fixed in 4% PFA at 4°C for 90 min, dehydrated and rehydrated through methanol series. After blocking overnight at 4°C in PBTS (BSA, Triton X-100 and goat serum), explants were incubated in mouse anti-Pax6 primary antibody (diluted 1:50 in PBTS; Developmental Studies Hybridoma Bank #PAX6) for 3 days then washed in PBS-Tween. Explants were then incubated in goat anti-mouse Alexa568-conjugated secondary antibody (diluted 1:1000 in PBTS; Life Technologies A-11004) and DAPI for 3 days at 4°C, then washed in PBS-Tween. Stained explants were mounted in AF1 mounting medium (Citifluor) and z-stack images were generated using a Zeiss LSM980 confocal instrument. Relative quantification of nuclear Pax6 fluorescence was performed by normalising to DAPI using the Atlas Toolkit plug-in for FIJI/ImageJ (Grocott et al., 2016) as described.
Explant samples were lysed in 1 ml Trizol (Ambion) and processed for total RNA extraction. RNA samples were digested with DNase I (Ambion) and re-extracted by acidic phenol/chloroform. RNA concentrations were determined by NanoDrop ND-1000 Spectrophotometer. For each experiment, equal quantities of treated and control sample RNA (typically between 0.1–0.6 µg) were used as a template for first strand cDNA synthesis using Superscript II reverse transcriptase (Invitrogen) and random hexamers. cDNAs were diluted 1:20 before relative quantitation of transcript levels by real-time PCR using SYBR Green master mix (Applied Biosystems) and target-specific primers (Table S1). Relative transcript quantification was via the standard curve method, and target gene expression was normalised to the reference gene β-Actin. Fold changes were calculated for each matched-pair (treated/control) then log-transformed to bring data closer to a normal distribution (verified by a Shapiro–Wilk test) prior to plotting and null hypothesis significance testing. These were plotted as mean±s.d. Student's paired t-test was used to calculate the probability of the observed (or more extreme) differences between match-paired (treated and control) sample means, assuming that the null hypothesis is true.
Morpholino knockdown validation
Fst-expressing somite tissue from wild-type chick embryos was dissected and cultured in Dulbecco's modified Eagle medium, 10% foetal bovine serum and 1% penicillin/streptomycin for 4 h before transfecting with 1 mM translation-blocking FstMO (Gene Tools; sequence 5′-GATCCTCTGATTTAACATCCTCAGC-3′) or 1 mM StdMO negative control (Gene Tools; sequence 5′-CCTCTTACCTCAGTTACAATTTATA-3′) using Endoporter PEG (Gene Tools). Protein was extracted after 48 h. Protein lysate (30 μg) was run on pre-cast 4-15% polyacrylamide gels (Bio-Rad) and blotted onto polyvinylidene fluoride membrane (Bio-Rad). Primary antibody against Fst (Abcam ab47941; 1:2000) was applied at 4°C overnight and secondary polyclonal goat anti-rabbit-HRP (Cell Signaling Technology, 7074; 1:2000) was applied for 1 h at room temperature. Primary antibody against HSC70 (Santa Cruz, sc-7298; 1:2500) was applied at 4°C overnight and secondary polyclonal goat anti-mouse-HRP (Agilent, P0447; 1:1000) was applied for 1 h at room temperature. The blots were treated with an ECL substrate kit and imaged.
In ovo embryo electroporation
Over-expression constructs encoding dnPax6 and Fst were constructed in pCIG (see Table S3 for PCR primers). Plasmid DNA (2-5 µg/μl) or plasmid DNA and FITC-labelled morpholino oligonucleotides (2 µg/μl and 0.5 mM, respectively) were injected into the open neural tube of stage HH8 chick embryos in ovo (Fig. 2A). A pair of platinum electrodes connected to an Ovodyne electroporator and current amplifier (Intracel) were then used to electroporate the DNA or DNA+morpholino into either the left or right side of the anterior neural tube via four pulses of 22 V and 50 ms duration at 1 s intervals. Once electroporated, embryos were sealed with adhesive tape and incubated for 10-12 h at 38°C until embryos had reached stage HH10.
Whole-mount in situ hybridisation and immunofluorescence on sections
Embryos were fixed in 4% PFA overnight at 4°C, then dehydrated by methanol series and stored at −20°C. Following re-hydration, embryos were processed for whole-mount in situ hybridisation using 1 µg/ml DIG-labelled antisense probes for Pax6 N-term (Goulding et al., 1993), Pax6 C-term, Vsx2, Mitf, Fst (see Table S2 for PCR primers), Tgfb2 (EST clone ChEST262a17) (Boardman et al., 2002), Wnt2b (a gift from Susan Chapman, Clemson University, SC, USA) and Bmp4 (a gift from Elisa Martí, Institut de Biologia Molecular de Barcelona, Spain). Probes were hybridized at 65°C for up to 72 h. After incubation with 1:5000 anti-DIG antibody (Roche) and washing, 4.5 μl nitroblue tetrazolium (50 mg/ml) and 3.5 μl 5-bromo-4-chloro-3-indolyl phosphate (50 mg/ml) per 1.5 ml developing solution were used for colour development. Embryos were embedded in 7.5% gelatin, 15% sucrose and cryosectioned at 15 µm. Differences in morphology of sections are due to: (1) slight differences in staging of embryos between HH10− and HH10+; and (2) slight obliqueness and variation in the dorsal-ventral level of the horizontal sections. Following de-gelatinisation, sections were blocked in PBTS buffer (PBS with 2% BSA, 0.1% Triton X-100 and 10% goat serum) for 1 h at room temperature. EGFP transgene expression was then detected using rabbit anti-GFP primary antibody (Abcam, ab290; 1:500 dilution) and Alexa568 goat anti-rabbit secondary antibody (Invitrogen, A11036; 1:1000 dilution). Morpholino FITC fluorescence was observed directly. Labelled sections were imaged using a 20× objective on an Axioplan widefield fluorescence microscope with Axiocam HRc camera and Axiovision software (Carl Zeiss).
Relative quantification of in situ hybridisation staining
Assuming that average cell size is invariant between left and right optic vesicles of the same embryo, then the relative area of staining is proportional to the relative number of cells exceeding a common detection threshold. To quantify this, bright-field micrographs were converted to greyscale, inverted then thresholded and the area of optic vesicle staining measured in FIJI (Schindelin et al., 2012). Transfected and contralateral controls from the same embryo were processed simultaneously to ensure identical treatment. Staining area in transfected vesicles was then normalised to internal contralateral controls, yielding a fold-change in gene expression area. Fold changes were log-transformed to bring data closer to a normal distribution (verified by Shapiro–Wilk test) prior to plotting and null hypothesis significance testing. Box plots showing mean Log10(fold change)±s.d. were generated in R with the package ‘Beeswarm’. A Welch's two-sample t-test (for pairwise comparisons) or one-way ANOVA with Tukey's post-hoc test (for groupwise comparisons) were used to calculate the probability of the observed (or more extreme) differences between sample means assuming that the null hypothesis is true.
Partial differential equations were coded in R using the function tran.1d() and tran.2d() from package ‘ReacTran’ to handle diffusion terms. 1D and 2D numerical simulations used the functions ode.1d() and ode.2d(), respectively, from package ‘deSolve’ and the default integrator. Parameter sweeps were performed to identify suitable diffusion rates (see Movies 1 and 2). 1D simulations were run with both periodic and zero-flux boundary conditions, with comparable results. 2D simulations were performed with zero-flux boundary conditions on explant-shaped domains, which best reflected experimental observations. See supplementary information for model code and narrative text. The model code is explained in the supplementary information, is available via our GitHub repository (https://github.com/GrocottLab/) and is accessible as an interactive Jupyter Notebook (https://mybinder.org/v2/gh/GrocottLab/Pax6-Fst-Tgfb2_Reaction_Diffusion_Models/master).
We thank Paul Thomas of the Henry Wellcome Laboratory for Cell Imaging for assistance with microscopy, Peep Periera Marques Leal for technical assistance and colleagues in the laboratories of Grant Wheeler and Andrea Münsterberg for valuable discussions. We thank Andrea Streit, Elisa Martí and Susan Chapman for sharing plasmids and antibodies.
Conceptualization: T.G.; Methodology: T.G.; Software: T.G.; Formal analysis: T.G.; Investigation: T.G., E.L.-V., G.F.M.; Data curation: T.G.; Writing - original draft: T.G.; Writing - review & editing: T.G., A.E.M.; Visualization: T.G.; Supervision: T.G., A.E.M.; Project administration: T.G.; Funding acquisition: T.G., A.E.M.
This work was supported by a Fight for Sight UK Early Career Investigator Award to T.G. (1365/66), a Biotechnology and Biological Sciences Research Council project grant (BB/N007034/1) to A.E.M., and by a H2020 Marie Skłodowska-Curie Actions Individual Fellowship (705089) to E.L.-V. Deposited in PMC for immediate release.
The model code is explained in the supplementary information, is available via our GitHub repository (https://github.com/GrocottLab/) and is accessible as an interactive Jupyter Notebook (https://mybinder.org/v2/gh/GrocottLab/Pax6-Fst-Tgfb2_Reaction_Diffusion_Models/master).
Peer review history
The peer review history is available online at https://dev.biologists.org/lookup/doi/10.1242/dev.185827.reviewer-comments.pdf
The authors declare no competing or financial interests.

domain: literature


text: On 14th Aug 2014, Mahapanya Vidayalai’s (MPV) Director and staffs lead students to visit an Islamic mosque (Ban Nua Mosque). Since students have been engaged to studying the subject “Religions” where they have been introducing some of leading religions. An Islam Religion is one of those leading religions, and it was an opportunity for our student to have a close look to the Islamic Mosque and observed the things how they have been serving the community people around. This mosque was one of the biggest and an important learning center for Islamic community as well as center for welfare of society existed in southern Thailand. We had a very pleasant time. We are very thankful to Imam of Mosque who delightfully welcomed us and gave a very beautiful short speech about basic Islamic Religion.


text:
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domain: economics


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Big J and DJ Logikal – Fuck This Mixtape | http://bit.ly/pPcOQR
Here is Ohio Hip-Hop artist Big J speaking with Hip-Hop Kings following the release of his free mixtape “Black Van Music”. Inspired from Jake One’s “White Van Music, Big J discusses his favourite tracks on the album, the guest producers and artists, and who he’d like to collaborate with in the future. Big J is currently working on his forthcoming project which will be fully supported by Hip-Hop Kings. Big J has also recently released a free collaborative mixtape with DJ Logikal titled “Fuck This Mixtape” which you can download from the link above. You can watch the Big J Interview with Hip-Hop Kings on the video player below.
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text:
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domain: law


text:
1 Day trip in Italy
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domain: travel


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text:
Monthly Events to build community and social connection for area seniors are planned for the third or fourth Thursday of each month from 1 – 3 pm at the First Congregational Church of Woodstock.
June 18 will feature a presentation of local interest by The Last Green Valley.
The program will offer opportunities for social interaction, recreation, and interconnection within the community through activities such as games, crafts, informational presentations, and more. The activities are free and open to anyone who wishes to attend, regardless of age, residency, or other affiliation.
“The pandemic increased isolation and stress among many in our community, young and old. We are beginning with monthly activities to build community and connect among older adults and plan to offer other events and activities to address the practical need for positive social connection, support and meaningful community for others in the community as well.” commented Rev. Dr. Kevin Downer, pastor.
The church has served as the Monday location for Community Kitchens of Northeast Connecticut for the past eight years, offering free hot meals every week. The meal program provides an important social outlet. This new program seeks to build upon this ministry by offering a variety of daytime social activities. It is hoped that the center will nurture relationships and connections between residents of Woodstock and the surrounding communities.
For more information email our Senior Ministry Team.