Embryo, Larval, and Juvenile Staging of Lytechinus Pictus from Fertilization

Home , Hemicentrotus, Lytechinus, Lytechinus pictus, Toxopneustidae

Developmental Dynamics

1 RESEARCH ARTICLE Received: Jan 30, 2020;Revised: Jun 17, 2020;Accepted: Jun 21, 2020 21, Jun 2020;Accepted: 17, Jun 2020;Revised: Jan 30, Received: Inc. Periodicals, Wiley 2020 © version. the edited advance final of issues, future for articles unedited accepted, are Articles Accepted Marine Biology Research Division, S

Embryo, larval, Key Words: NIH ES027921 and ES and ES027921 NIH Numbers: and Sponsor Grant *Corresponding Author sea urchin

and juvenile and 9500 Gilman Drive, La Jolla 92 CA

, model Katherine T. Nesbit

staging

This article isprotected bycopyright. All rightsreserved. organism, stag cripps Institution of Oceanography, University of California of University California InstitutionOceanography, cripps of ( of of [email protected] maturation Lytechinus pictus San Diego 1

, Amro Hamdoun ing , larva . - 093 from fertilization through sexual sexual through from fertilization

0202 USA 0202 030318;

; 858 temporarily published online in in online published temporarily 1*

- - 822

NSF 1840844 1840844 NSF

5839 DOI10.1002/dvdy.223 ) )

Dev Dyn Dev

amics

Developmental Dynamics

CRISPR by enabled studies newly multigenerational genetic and future the key advantage of rapid development to advantage development of key rapid the growth rate ing on individual depend 3 by metamorphose and days which subsequently give to the subsequently rise which post 45 minutes cleavages every subsequent followed by 90 minutes, maturation of genetic, transgenerationalgenetic, studies Strongylocentrotus purpuratus , have long fertilization and development early Background: Background: ABSTRACT: a a rapidly developing echinoderm species, Lytechinus pictus, - fertilization (hpf) fertilization studies in L. in studies pictus .

When grown at room temperature ( grown roomWhen at temperature Sea urchinSea . Th

embryo which , which e swimminge embryos gastrulate from

weeks. s have used a than been for more urchins but featuresofha ofattractive urchins other the many share . This article isprotected bycopyright. All rightsreserved. Results: Results: .

larval However, several of the species . Juveniles reach sexual maturity at Conclusions: Conclusions:

skeleto generation time

Here, we present an overview of the development presentofdevelopment we the Here, overview an sexual n 20°C maturation. and immun Th is )

embryos staging scheme lays foundation a for scheme staging s suboptimal themmaking suboptimal ocytes from fertilization through sexual

This This - 12-

CAS mediated gene gene mediated editing. CAS complete the first cell cycle in , inghatch at to leading century century hpf and produce the the and produce 36 hpf is used, as such . Larvae significant significant 4- 6 months in

the study of study the begin tobegin 2 at feed for for

of age, for for

9 hour cells

ve s

Developmental Dynamics

significant of these is their limited utility in multigenerational genetic studies, namely due to echinoderm used widely toutilization the contributed Hemicentrotus pulcherrimus purpuratus. pathways easily delivered into the egg bymicroinjection In addition, transparent, spawning single a played afoundational role in experimental embryology. E from marine organisms others – cnidarians biological discovery. Many marine organisms annelids including – INTRODUCTION: of of was first of these to be published S. be purpuratus to of wasfirst these The most frequently usedspeciesis urchin The most frequently platform providesrich a oceans the within function formand of animal The diversity , used lab the been in have . 3

, copepods mRNAs, Several other species including and development is rapid development andand synchronous

, fertilization occurs externally, eggs and embryos large are and relatively guide RNAs, morpholinos,proteins, and small molecule reporters can 4 , diatoms 10- species 17 .

are alsoare used where more theyare readily available Echinoderms in general,

of this species. However, species. of this there remain major 5 , echinoderms in This article isprotected bycopyright. All rightsreserved.

modern cell and developmental biology. most the Perhaps leading to a leading

Lytechinus variegatus, Paracentrotus lividus , and 6 , facilitating theal manipulation of development 6

long history of significant contributions of significant long history , oysters 18

the purple sea urchin, Strongylocentrotus sea purple the

e resulting the resourceand and theand urchinparticular in sea 7 in , sponges , ach

little more thanlittle

female releasefemale 1 8 , choanoflagellates , and tunicates , and

limitations to th a dishofa s millions 19

. has has The genome 9

among sea water.sea greatly , 2

have have of eggs ,

coming be be e e for for in in

Developmental Dynamics

diameter In molecular evidence from mitochondrial DNA and bindin Lytechinus anamesus cross , 2 at beds between kelp depths has and Arabacia diverged from S. > , and purpuratus Central California to Cedros Island,Mexico ofestablishment genetic sizes (~1 addition, as such purpuratus. of months times. long generation their the l the genetic aboratory,

Lytechinus pictus 20,21

S. purpuratus, L. pictus been reported to live reported been -4 23 they can be cultured at room temperature (20 be room at can cultured temperature they and the sand Dendraster dollar excentricus lines difficultprospect. a . cm , and perhaps, as long as twoyears

These urchins share most of the advantages of other urchins but, of other most urchinsThese ofadvantages share the

test diameter test is nativeis toEast Pacific the Ocean, with ageographic range spanning from L. pictus live between 7 L. pictus (aka the white or painted urchin) theor painted white (aka lines (inbred and transgenic) and (inbred lines - ). fertilization between L. between pictusfertilization and In the case of S. In case the

This This on sandy m - have relatively short This article isprotected bycopyright. All rightsreserved. development rapid 200 separated million years from sea urchins ofgenus the

300 m - bottomsand in sea grass bays, as wellas in and around - 28 9 years, and growto approximately 4 test cm 24 . Originally thought to be a distinct speciesfrom distinct thought toa be . Originally . This species

u purp

for robust reproduction ratus

generation time 25- an and smaller adult size make 27 - 24 . 22°C) and the adults the and hav 22°C) the generation timeis at least 11 L. is pictus an

attainable goal. , indicates , indicates is approximately 40 million years

an attractive alternative to L. anamesus s that these are one that are species. these of 20

abundant urchin species species urchin abundant , making the generation generation the making , 4-8 months 29 ter , and later unlike species species unlike e

the the small bodysmall 22,23

. S. S.

In In

Developmental Dynamics

envelope Early RESULTS: establishhelp comparability across species. This staging scheme work will help standardize across labs and compare our well as summaries of laterlarval development, and post developmental events in embryogenesis as such and detailed imaginga developmental of staging scheme scheme for this species smaller micromeres at intervals ( 1C) (Fig. potential ofegg the electrical changeswith the in embryogenesis and larval morphogenesis

3.75 hpf 3.75 c leavage eggs (Fig. 1A) L. pictus(Fig. eggs Prior and

(Fig. 1B) (Fig. 2.25 and and and

laid a foundation for generating a standard staging standard a of L. for generating culturing foundation work pictus a on laid two subsequent symmetric cleavages symmetric two subsequent staging scheme with the with staging scheme spatial s is the first asymmetric first is the tages

following the initiation of the cortical reaction fertilization at ofcortical the initiation the following 3 hpf 3

in early cleavage. Division alltheof cells except the micromeres occurs

and temporal map 22,23 ) . The . However, a gap in what is known is a isknown is what gapin a . However, a fourth fourth verage This article isprotected bycopyright. All rightsreserved. division timing of development

110 µm cell division,cell giving rise to four and macromeres four s

of of useful for staging embryos for staginguseful major , which in

early cleavage, blastula stages, gastrulation, as diameter 31

al eventsdevelopment (Fig. 1D (Fig. .

At 20°C, t form - metamorphic lifemetamorphic history for for s , E and form a conspicuous forma and the 16the ) ) L. pictus i in he he occur in the following the in occur S. purpuratus first cell cycle takes

- detailed cell cell .

Here we provideupdated Here ncluding key key ncluding embryo .

description of description to to (Fig. 1F) (Fig. 30 assist in in assist and coinciding coinciding and . W

fertilization 45 1.5 hours e aimedtoe , occurs -minute -minute

Developmental Dynamics

(Fig. 2A (Fig. fifth and the and regularly spaced, c shape frommore to rounded intermediate an cells The have divided to atotalof 8 cells, reside Expansion of theblast segregate stage small molecul of fluorescentsubstrates transporters e ha germ contributethe line to formationof or indirectly directly mesenchyme cells which form the larval skeleton while the small micromeres presumed are to micromeresfour and 4.5hpf at next, ve cavity of the blastocoel is ofblastocoel the cavity between between cleavages,and tenth and reduced

(Fig. 1H) (Fig. The The By 8 By -

B). B). vegetal pole the contents of the blastocoel from the external environment. external the from ofblastocoel contents the is is The openingtoblastocoel the

cavity is a is embryo hpf the efflux transportefflux activity

septate septate thisstage, at and , yielding a28

between cells of the early embryo olumnar epithelium. Nucleislightly are

cluster of small micromeres to moreresolve difficult becomes small micromeres.small The micromeres ultimately give rise to the primary ocoel

and -

cell stage embryo more pronounced and changes in the morphology of the layer of layer the morphology the of in changes and pronounced more

mature g astrulation This article isprotected bycopyright. All rightsreserved. 33

at the vegetal pole pole vegetal of embryo the at the 6.5- which can be used to selectively load these cells with with load cells selectively these to used be can which blastula cell 7.5

junctions are beginning tojunctions beginning form are sible and aclustersible of small micromeres, which hpf hpf

(Fig. 1G) (Fig. (Fig. 2C) (Fig.

the embryo isin the are apparentare

expands quite dramatically . By By . The fifth cleavage gives rise to givesfour cleavage rise fifth The . with 5.75 hpf 5.75 closer tobasolateral the membrane, 32 cell . S . mall micromeres of

, the embryo is at embryo , the shapes more akin more shapes to a

the early blastulathe early stage

(Fig.white 2B, arrow) 34 , which help help , which

between the between . The . The the the L. pictus 60- s cell

Developmental Dynamics

are easilyare visible halfway through gastrulation. filopodia These extendtowards the animal pole - thismid at ofarchenteron tip the to muscle and types immune cell such as pigment cells, cells(SMCs) secondarymesenchyme throughblastocoel; the tobegun extend and has archenteron the invaginationisunderway 24 hpf,secondary around archenteron. At elongation of pronounced the the before occurs pause slight invagination,a primary ingressed hpf. micromeres cells from bottle cues by part in isassisted This plateinward. bends process vegetal invagination. Primary invagination isfollowing initiated gastrulation. mesenchymal transition ends cells population of blastula stage this stage At 2D). (Fig. blastulae 35,36 Be nding of theplate vegetal characteristic of primaryinvagination, of arrangement the and Gastrulation of the embryo occurs in two main phases - phases two in main occurs ofembryo Gastrulation the

(Fig. 2F (Fig. are ciliated at this stage and spin PMCs in PMCs 39

. The . The

cells, the embryo , white arrow to majority ofmajority an ordered ordered an

the cellsat the vegetal pole will begin to thicken, forming amesenchyme (Fig. 2E) by 12

primary mesenchyme cells ), ), the blastula phases and phases mark blastula the

PMC ingression PMC hpf in 15 hpf by is evident ring gastrula phase (Fig. 3C). SMCs The have long filopodia which This article isprotected bycopyright. All rightsreserved. begins at 18 hpf and is complete by iscomplete and (Fig.hpf 3B)18 at begins hpf. Signs of delamination and the ingression of a a ingressionof the and delamination of hpf. Signs within the envelope the within

the vegetal pole (Fig. 3A)is completed

(PMCs L. pictus

when when ingression, PMC e evident in the blastocoel and at the at and blastocoel the are in evident ) which give which ) rise to skeletogenic s the subsequent onset of onset of subsequent the . This epithelial classic - , primary and secondary eventually hatching by hatching eventually , which give rise the thickened thickened the

20 37,38 hpf. After 9 hpf and by by 17

Developmental Dynamics

esophagus, post Larval differentiates it gastrulation, though morphologically the gut is still very simple whe until development laterin Compartmentalization gut isongoingthroughout patterning oflarval the and functional ready to mouth. ectoderm formthe tothe with fuse isnearly indicatingit animal the , while rods isapparent stage (Fig. 3F, arrows) white to origins skeletogenic cells are clear and they have begun to form skeletal rods 30 also distinct arrangements of PMCs also distinct are ectoderm. embedintoThere the to blastocoel later migrating through cellsare the pigment and can interact with surrounding with interact can cells and migrate to the left and right coelomic pouches coelomic right and toleft the migrate hpf, the archenteron has crossed the space of the blastocoel and the arrangements the and of ofblastocoel the space the hascrossed archenteron hpf, the - ) (dpf fertilization d of triradiate the The first larval stage of By By evelopment 38 hpf stomach, and intestine (corresponding fore to former the intestine and stomach,

further intofurther the embryos are at are embryos

. At this time the larvae have three distinct have gut compartments larvae three the thistime At .

the archenteron has a more pronounced bend towards the oral side of side oral towards the bend pronounced the archenteron more hasa (Fig. 3D, arrow). white The primordial germ cells (PGCs) presumed are .

L. L.

tripartite the the pictus This article isprotected bycopyright. All rightsreserved.

into the triradiate skeleton. lategastrulation By triradiate into the (Fig. 3 late 40 is is . The

prism stage (Fig. 3F) m and the plute the fore

-, subset of SMCs that will further differentiateinto further will that of SMCs subset mid

during later gastrulation and into the prism prism into and the latergastrulation during us stage us -, and hindgut.and

(Fig. 3F ) )

which occurs 2which by days ineralizationskeletal of -

branching out from the from the out branching mid - and hindgut) and , the the 41 D), at .

Developmental Dynamics

hood Stage I and Stage II 4A (Fig. - feeding structures features organizationof tissue and juvenile skeletal toattention later larval development, focus primarilyon the maturation of the rudiment with special structures rudiment and ofvestibule, the elaboration the and epaulettes formationof coeloms, right and left and ofextension differentiation the progressionof morphological key features, such as the acquisition of additional pairs of arms, insets). pigment(Fig. echinochrome pigment 3F, cellsgranules contain ofautofluorescent the which water surrounding Larvae at 2 days of arms, and evidence of of vestibule the evidence invagination of and arms, rods posterodorsal thatthe pair skeletal support extended. ismineralization offully There the to overhang the extends arrow The extension of the

(white arrow, Fig.4A- ) ) is is In In on the stagesbased is urchins upinto developmentsea divided in larval Subsequent

is apparent apparent

L. pictus, evidenced by the red

will begin to filt . at the the The larvae also have apopulation of conspicuous immunocytes termed

7 dpf. left and right posterodorsal right and left majority of

mouth. The tissue that forms the left and right left forms that the The tissue preoralmouth. arms is not yet II) II) In In . , there is thickening of the tissue that eventually form eventually that oftissue the isthickening , there there is further elaboration of the oral hood and it isfurtherit elaborationand hood L. oforal there the pictus The left and right coeloms extend along the stomach oflarv stomach the along extend the coeloms right and The left digestive remnants of Rhodamonas er feed phytoplankton asRhodamonas such This article isprotected bycopyright. All rightsreserved. larvae are at Stage I

arms 42

on the left side oflarva side the on left the

during Stage III (4A- larvae . Additional staging schemes detailing (Fig 4 A-I) . 3dpf at Between stomach. Between the in During During 43 . lens

this stage this

III, yellow from the

(Fig. 4A (Fig. s the oral -

, III a.

Developmental Dynamics

acquires ascaled or textured appearance the larva e towhen gut, sitsand adjacent the rudiment body. within The larval tuckedthe away are that (StageVII)stage the known state ascompetency precedeand the metamorphosis 4A V(Fig. Stage into develops organizes and 4A (Fig. metamorphosis. at animal ofjuvenile into the which matures body the torudiment, the contribute line dashed rudiment appears as acrescent r line dashed 4B (Figure oflarva side the structures onleft the the coelomic meet eventually white arrow udiment structures occurs udiment rudiment As the of development of the initiation to extend, posterodorsalcontinue As the arms -V , yellow arrow, yellow are ). Cellsthat originallymigrated to the left coelomic pouch durin ). )

The tissue which forms the vestibule folds inward towards the gut, where it will will it gut, towardswhere foldsthe inward vestibule forms the which The tissue folds of tissuefolds of ready to undergo metamorphosis the gut turns a greenish color and the tissue the colorand gutgreenish turnsa the toready undergometamorphosis

(Fig. 4A (Fig. The The

larva containlarva completion of extension of the preoral is ofpreoral arms evident the extension in aStage of V larva completion - ) ) matures, V, greyarrows) etween 10 occurswhich between , marking aStage IV (Fig. larva 4A-

into a pentagonal disc into pentagonal a -V - , white arrow, white shaped structure adjacent to the gut (Figure 4B togut (Figure the shaped structureadjacent s three pedicellariae are formed which will be carried This article isprotected bycopyright. All rightsreserved. a mature rudiment with 5 tube feet and mineralized spines spines and 5feet mineralized rudimentwith tube mature a . These . Tube feet within the rudiment will extend and theextend within rudimentwill Tube feet

). ). at 3 weeks post 3at weeks -

structures structures -

12 d 12 (Fig. 4B (Fig. pf persist in in -V L. L. , yellow dashed as line) the larva IV, white arrow fertilization (wpf) fertilization . The rudiment elaborates The pictus.

a Stage VI larva (Fig. 4A- g embryogenesis g embryogenesis ). ). The early - . At the final final . the At , yellow III, yellow - IV, yellow through VI) VI)

Developmental Dynamics

spines during their post- their during spines noare orange to anal plate becomes more apparent walking and spines feet 4 between juvenile animals, from the larva. The from pedicellariae the larva also are section, and the overall main pigmented and feet Juvenile resorption larval of arm tissue tissue reorganizationextensive occurs larva tofromthe and emerge an juvenile bodyof the allowing metamorphosis,during emerge from attach and the larvaltoside arms the the bend body. Thelarvae

longer visible. At s d there are also 20 continue to grow, they feed on diatoms and biofilms after formation of the teeth teeth formationof the after biofilms and tofeed on grow,continue diatoms they

- evelopment to a 24 hours post 24 hours ish appearanceish - 5 d swirl, and sometimes remnants of tis larval walking walking ays ays post spines. The body of the animal typically contains a a typicallycontains ofanimal the spines. Thebody

The animals eventually acquire a purple pigmentation at the base of base the the at purple a pigmentation acquire eventually The animals -m 10 additionaljuvenile spines that , - metamorphic growthmetamorphic period. the the metamorphosis (hpm) the newly settled juvenile newlysettled the (hpm) metamorphosis etamorphosis (dpm) he pigment cells that were once once thatobserved following were cells pigment metamorphosis. The dulthood plates that willof test startplates the formthe tostructure the and fuse, body of the juvenile the body of .

This article isprotected bycopyright. All rightsreserved.

. By . By 44,45 4-months

which includes shedding of skeletal rods and shedding of skeletal includes which . They will continue to develop additional tube tube to additional develop continue will They . post

is freckled with red pigment cells retained cellsretained pigment is freckled red with

- sue can be observed metamorphosis, animal has the . In newly metamorphosed. retained In newly are locatedare on the aboral side. As pale yellow

(Fig. 5 ed ed pigment . This to benthos the A) A) ish has 5 tube has 5 tube - green

a white a

Developmental Dynamics

Perhaps the most the well Perhaps embryoprovidea and the manipulate conditions identical under gastrulatio vertebrates sch staging development, regeneration, process specific for accessibility studying zebrafish to be populations isessential cell important of differentiation and specification Towards aunified echinoderm development staging scheme. DISCUSSION: previous to most been have able we diameter, At This paper provides an initial staging schemefor L. pictus.

49 report the the four months, with n to name a few. a n to name , and chick and , emes . In the case of In case the

growth rate postgrowth- rate s

23 to include invertebrates to include . . 50 - . recognized standardized staging schemes come fromXenopus staging schemescome recognized standardized Each of these vertebrate models has unique advantages and improved improved and advantages unique vertebrate of has these models Each . sea urchin Although sexual maturation appears a function of both age and size and function a both maturationage of appears sexual Although

reliably range of or formation ofbuds. limb metamorphosis This article isprotected bycopyright. All rightsreserved.

spawn animals that 9-10 are m

body size s these include s these common language for discussion of development for. discussion of common language es

helps to helps

in development such as nervous system asnervous such system development in s for animals in our being being

query processes less easily accessed in

fertilization, cell early divisions Expanding Expanding variable

, even Knowing the precise timingprecise of the Knowing hands m diameter, with consistent diameter, m onavailable standardized the among among is

between 1 between siblings reared ing able to able and and - 46- 15 48 mm in , ,

Developmental Dynamics

fertilization envelope when cultured at 12°C at cultured when envelope fertilization the micromeres, from the andto the hatch division first27 forming reach asymmetric hours hours micromeres structuresprocesses.Thedevelopmental and asymmetric first cleavage in comparison to pictus here onepresented asthe such life cycle. Thus existing the ofgenera the members including observed in S.observed in purpuratus, development and of events core morphological progressionthe of major structures that is L. in theday pictus in of single course a . completed be development can later larval development from feeding stages through metamorphosis S. descriptions of most purpuratus the detailed species with isarguablythe . occur rapidly and synchronously. and rapidly occur By comparison,By it takes S. purpuratus 2- Here we have shown that, like other echinoderms, the echinoderms, other have like we Here shown that, There Progressionthrough larval period the for L. also pictus more occurs rapidly and follows , descriptions descriptions occurs by 3.5 hpf, by occurs have been numerous been have , the , the key divergence focuses on timing of important important S., the purpuratus focusesonof divergence timing key re is re

need for need are fragmentaryare but in half the time. the half in small micromeressmall form by5.75 hpf .

standardized staging scheme standardized Arabacia This article isprotected bycopyright. All rightsreserved. descriptions of the morphologies of other echinoderms echinoderms of other morphologies the of descriptions There are limited morphological of diL.fferences pictus , and do , and 51

, 56 Echinus . 2.5 hours for the first cell division, 6.5 hours to to hours 6.5 division, cell first the for 2.5 hours

For example, feeding for L. pictus2 by begins Thus, experiments pertaining to early pertainingtoThus, early experiments not capture not capture 52 , and , and Strongylocentrotus

all of development early stagesearly of development in L. s span , and hatching happens in 9 in happens hatching and ning 42,43 , forming the

the entire life cycle, . However, manyof early cleavages,early and 53-

55 through the .

Of these, Of these,

Developmental Dynamics

lividus conditions S. in stage take purpuratus equivalent of the preoral arms is evident in aStage V larva between 10 eggs, prevalence generation times pictus. of 40 comparedtodays, between aslong days as28 take can at 18 earliest the dpf,but achieved posterodorsal arms during Stage III is apparent larvae at 7 dpf. In of whereasfeeding days, notably in the study of fertilization and embryogenesis. early For example, activation of pictus L. Conclusions: to L. variegatus preference the the have have Temnopleurus reevesii Echinoderms wi Echinoderms There have been already anumber of contributions important ability to culture at room temperature (20°C), , making, its generation timesimilar of of started to compile similar schemes staging Historical and futurecontributions from research inL. pictus L. pictus for this species in our hands our in for thisspecies which

of approximately 5 months to earliest gamete production 5 monthsof to approximately earliest

the the States, United Coastof West the the on have generation times th similar developmental tempos include S. purpuratus can also achieve maturity between 6 between can alsomaturity achieve - 80 days in S. in purpuratus 80 days This article isprotected bycopyright. All rightsreserved. s 25- occurs at 4 at days occurs . . 35 days 35

on the order of order 6 the on to to L. pictus 42 . Metamorphosis of Metamorphosis . 59 and the and the 42 42,56 and . . T Those working with Paracentrotus with working Those he extension of the left and right right and extension ofleft the he

- . . To reach an an pictus L. in 12. Toreach dpf

it 42

- may alsomay have 8 months . The completion of extension of extension The completion . smaller adult body all sizes

the Panamanian populationsthe S. S. - optical transparency of the transparency optical months 10 purpuratus purpuratus from occurs at 21 at L. pictus occurs 57 , comparable to L. 28

. However, the L. pictus

comparable 58 this stage is is stage this

under optimalunder , most lend lend ir

Developmental Dynamics

and studies early translation the of in used mRNA reticulum (ER) membrane L. pictus resources for working with of community collection Thegrowing backgrounds for manipulation. genetic stable and variability reduced lines with ofinbred animals to create opportunities increasing later life developmental impacts resulting from events early of in embryogenesisbe to is going envelop the ofnuclear duringbreakdown the migrationconcentration ofpronuclei, calcium the embryo m the This included of metallothioneinupstream the urchinsmetal sea were in promotersstudied first the on dependent and ions of extracellular evidencewasindependent eggactivation that provided synthesis protein and 73 . RNAs stored in the egg the storedin RNAs of pictus L.

During this “CRISPR this During era” eggs with ionophores, and the resulting observations of respiration and and respiration S. of observations eggsresulting purpuratus with the and ionophores, Gene editing has now widespread become in marineorganisms 67 e also maketargeted molecular and genetic studies achievable. This includes a , during the transition between metaphase and anaphase, as well as during cleavage asduring aswell anaphase, and metaphase transition the , during between . L. pictusL. importance early early the release of intracellular calcium were were . work The comparatively short generation timeof L. enables pictus also

at fertilization was first described described fertilizationat wasfirst 74- of Nemer andof colleagues Nemer demonstrating adiverse that array of

( used in landmark studies on the cell cycle showingi changes oncell studies the landmark in used ) in L. in pictus ) MT1 76 he ability to the rear juveniles This article isprotected bycopyright. All rightsreserved. encode the newly synthesized proteins of the early synthesizedproteins the ofearly newly encode the 62,63 . protein synthesis in early development synthesis protein Sea urchins,Sea including L. 31,60 - . responsive regions and elements The dynamics of the endoplasmic ofendoplasmic dynamics the The , generateline in the eggs of the in L. pictus , including sea urchins sea including s 58 , were widelypictus, were and investigate investigate 61 . Some of of Some 64- n 66 69- . 68 .

Developmental Dynamics

that and evolutionary development of L. evolutionary pictusand ofenvironment consequences the cell or to longer types, study specific in ubiquitous togenes target pathway provide a transcriptome and fully sequenced genome that willpublicly soonavailable be control control development across echinoderms

This article isprotected bycopyright. All rightsreserved. . development through on action could .

processes processes of ourthe understanding strengthen Understanding of ecological 77 This . This - term would

Developmental Dynamics

in FSW, followed by 6 washes with FSW. Competent larvae were left to recover from the KCl to KCl recover wereleft fromthe larvae 6 FSW.Competent with by washes followed FSW, in 60 a was inducedby below. Metamorphosis as described skeletal rods, prolapse of the gut), discarded. were Healthy larvae were observed and imaged Cultures with >10% larvae of displaying signsof stress or poor health (i.e. asymmetry, exposed frequentl (more throughout visually development waschecked reversefiltration.through health gentle Larval lens 3. mL per Larvae1 embryo wereconcentration of fed the red flagellated algae Rhodamonas further diluted to embryos a were hatching, Upon envelope. fertilization fromthe hatching described were fertilization batches only of eggs>98% with successful where forsuccess, fertilization checked using 2 fertilized 6 washed were use. Eggs undilutedkept and 15°C wascollected at until sperm spawning,and during peristomal membrane. Females were inverted and kept submerged in filtered sea water (FSW) of LarvaeCulturing EXPERIMENTAL PROCEDURES : starting- 2post at days used - 100 spawned injectionof by animals were Adult - 77 10 times with FSW and visual and FSW with times 10 . . Briefly, embryos grown were at aconcentration in FSW of 1per embryo mL until Embryos were grown with agitation at room temperature ( roomat temperature weregrown agitation with Embryos y duringy embryonic stages, and on adailybasis during larval development). - L semen into 40 mL FSW). Eggs were Eggs were FSW). intomL 40 (2µLsemen dilution sperm of fresh a drops 3

fertilization (dpf) and received water changes 3 water received and (dpf) fertilization This article isprotected bycopyright. All rightsreserved. ly examinedly for quality before fertilization. Eggswere L of 0.55 M KCl through the the 0.55 through of MKCl µL 150 - minute exposure to 50excess mM KCl 20 °C) °C) - 4 times a week week a 4 times as previously

Developmental Dynamics

the 8 the Health, Bethesda, MD, USA). Composite DIC images rendered were from z Institutes of added (National using ImageJ were micrograph and measurements suite, software Zen using captured the (DIC). contrast Imageswere interference using differential objective pluteusearly on stage a imagedthrough fromfertilization the under 20°C.were temperaturecontrolat timed Embryos Observation andLive Imaging ofDevelopment observedwere dailyfor changes in morphology growth. and Nitzschia diatom andthe dailyfor albajuveniles, they growth changes and . occurred Water biofilm natural with dishes to petri usingpipette transferred transfer trimmed a carefully were vesselsovernight. juveniles Metamorphosed 20°Cno at with culture agitation in exposure morphological features. defined by stage where to progressed developmental had a larvae, >85% of individuals we defined a developmental milestone as atimerange averag onset of Therefore, the at feeding. of development isreductionsynchrony in There structures. ofpresent,the and scoredarms and number for the daily the la stage, pluteus Soft Ltd.). reaching Upon (v6.8.0,Pro Unlimited Helicon - cell stage,cell at late gastrulation, and at the prism and pluteus stages using Helicon Focus Embryonic sea urchin development was observed, and successive divisions cell were

Zeiss LSM 700 confocal microscope (Jena, Germany) with a 20x This article isprotected bycopyright. All rightsreserved.

development of the coelomic ofcoelomic the development

ed across multiple batches of acrossmultiple ed - stacks animals of at rvae were imaged

Developmental Dynamics

response innate adult and populationsoflarval the cell aspart later differentiateinto immune which to whichskeletogenesis contribute cells mesenchyme including ourfocused magnification imaged. 1 with scale standard using M165F aLeica magnification high stereomicroscope with aCanon EOS 60D camera. A 700 focus-stacked. Helicon Focus ProUnlimited (v6.8.0, Helicon Soft Ltd.). Larval Stages IV microscope with an AxioCam ERc 5s. microscope 10x objective on a Zeiss Stemi a on Images 2000 oflarvae were the capturedobjective using a10x 82,83 the small micromeres which latergive rise to primordial germ cells observations .

Imaging of the progression of internal larval structures was taken on a Zeiss LSM wasLSM on Zeiss structures a taken larval of internal progression the of Imaging

using using mm increments was used to measure topost measure increments- mm wasused 20x 20x

The adultThe animals imaged were with aCanon EOS 60D camera. We

on populations cell of particular interest for developmental biologists 0.8 NA plan NA 0.8

This article isprotected bycopyright. All rightsreserved. Z- - with DICoptics. with Juvenile objective apo stacks of alllarval stages focus were stacked using 35,36,80 , and and metamorphic animals at each secondary mesenchyme cells - VI tiled, were as wellas 32,33,78,79 s were imaged live , primary - C 81

Developmental Dynamics

awarded Supplement animal husbandry gratefully Vacquier Vacquier rearing, ACKNOWLEDGEMENTS: The authors thank Patrickthank Leahy The authors and Andy to AH. AH. to Lab ofHamdoun the and members fellow

acknowledge Kathy for

KTN NIH ES through

Cameron dy stu throughout this

staging of on staging for insight

Le, andLe, Kasey This article isprotected bycopyright. All rightsreserved. 027921 as 027921

for his suggestions on culturing optimization and larval larval and optimization culturing for on hissuggestions .

This work was supported through a Diversity through Diversity a wassupported This work

Mitchell forMitchell

ES030318 and the NSF 1840844 1840844 NSF the and as NIHwell ES030318 for discussion for

larvae their . We would also like to thank V to wouldthank alsoWe like assistance with larval and care

of this manuscript. We We manuscript. ic

Developmental Dynamics

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Figure 1. Early cleavages, A-V axis determination, and formation of the micromeres. A) Unfertilized egg. B) Zygote. C) 2-cell embryo. D) 4-cell embryo. E) 8-cell embryo. F) 16-cell embryo, white arrow points to the micromeres. G) 28-cell embryo. H) 60-cell embryo, white arrow points to the small micromeres. For all panels, scale = 50 µm. Time points listed in hours post-fertilization (hpf). All are

Article oriented with the vegetal pole, where discernable (from the 16-60 cell stage), pointing down. Accepted

Article

Figure 2. Blastula stages, hatching, and early ingression of PMCs in L. pictus. A) Early blastula stage. B) Embryos begin to demonstrate cell shape changes, and the small micromeres (white arrow) are visible at the vegetal pole. C) Blastula pre-hatching. D) Hatched blastula. E) White arrow points at the thickening at the vegetal plate. F) White arrow points at PMCs ingressing into the blastocoel, which are visible at the vegetal pole. For all panels, scale = 50 µm. Times listed in hours post-fertilization (hpf). Accepted

For Peer Review Article

Figure 3. Gastrulation, SMC differentiation, and skeletal rod formation in L. pictus. A) First signs of invagination of the vegetal pole are apparent after ingression of PMCs. B) Primary gastrulation completes and the vegetal pole is turned inward as indicated by the white arrow. A population of SMCs ingress into the blastocoel and the PMCs begin to arrange around the developing archenteron. C) During mid-gastrulation the archenteron moves through the blastocoel and pigment cell precursors will migrate through the blastocoel to embed into the ectoderm during mid-late gastrulation. D) Late gastrulae have clear arrangement of PMCs and triradiate spicules which will further develop into the larval skeleton (white arrow). The archenteron has nearly reached the oral side of the animal. E) Evidence of the forming coelomic pouches (white arrows) on either side of the archenteron preclude the fusion of the mouth with the oral ectoderm during the prism larval stage, and the arms begin to bud out from the larval body as the skeletal supports (yellow arrow) are further elaborated. F) Composite stack of early pluteus larva in abanal view. The gut has differentiated into three parts. A yellow box surrounds the region of the larva shown in the inset. Inset shows DIC, fluorescence, and overlay of pigment cells which contain the autofluorescent pigment echinochrome A and are embedded into the ectoderm of the larva. For all panels, scale = 50 µm. Inset panels scale = 20 µm. Times listed in hours post-fertilization (hpf). Accepted

For Peer Review Article

Figure 4. Larval staging of Lytechinus pictus. A) Scale = 250 microns. Stages I-VI of L. pictus. Blue arrow in 4A-II points to the oral hood tissue. White arrow in 4A-III marks the vestibular invagination. Yellow arrow in 4A-III marks the right posterodorsal arm. White arrow in 4A-IV marks the rudiment initiation adjacent to the gut. White arrow in 4A-V marks the pentagonal disc, while grey arrows denote the three pedicellariae, and the yellow arrow marks the fully formed left posterodorsal arm. White arrow in 4A-VI marks the fully formed rudiment, the green arrow marks the gut which now has a more textured appearance, and the grey arrows mark the two pedicellariae that are in view out of three. B) Scale = 50 microns. High magnification DIC imaging of the progression of development of coelomic structures during larval Stage III, IV, and V (from left to right). The dashed yellow lines highlight the vestibular invagination (left panel), the crescent-shaped initiation of the rudiment (middle), and the more elaborated organization

Accepted of the rudiment tissues into the pentagonal disc (right panel).

Figure 5. Post-metamorphic maturation of L. pictus. A) Juvenile at 24 hours post-metamorphosis (hpm). There are five tube feet (white arrow) as well as 20 walking spines (yellow arrow) and 10 juvenile spines (grey arrow). B) 4 months post-metamorphosis (mpm); C) Sexually mature adult. For all panels, scale = 0.5 mm. Article Accepted

For Peer Review Article

Figure 6. Summary of L. pictus development. A) Synopsis of major phases of development in the sea urchin L. pictus. B) Schematic of the L. pictus life cycle, illustrations are not to scale and time points are listed as the average time for >85% of individuals in a batch to reach a particular developmental stage. Accepted