Molecular Haeckel

a REVIEWS–A PEER REVIEWED FORUM

Molecular HaeckelRichard P. Elinson* and Lorren Kezmoh

More than a century ago, Ernst Haeckel created embryo drawings to illustrate the morphological similar-ity of vertebrate early embryos. These drawings have been both widely presented and frequentlycriticized. At the same time that the idea of morphological similarity was recently attacked, there hasbeen a growing realization of molecular similarities in the development of tissues and organs. We havesurveyed genes expressed in vertebrate embryos, and we have used them to construct drawings that wecall Molecular Haeckels. The Molecular Haeckels emphasize that, based on gene expression, there is agreater similarity among vertebrate embryos than even Haeckel might have imagined. DevelopmentalDynamics 239:1905–1918, 2010. VC 2010 Wiley-Liss, Inc.

Key words: comparative embryology; orthologs; modularity; model organisms; ectoderm; mesoderm; endoderm

Accepted 12 May 2010

INTRODUCTION

The most famous drawings of embryosare those that Ernst Haeckel used toillustrate his law of ontogenetic con-nection of systematically relatedforms (Elinson, 1987). Haeckel arguedthat vertebrate embryos at a particu-lar point in their development look sosimilar that they cannot be told apartby a casual examination. Haeckel’sdrawings have been alternativelypraised and damned over the decades,with a renewal of attacks approxi-mately 10 years ago (Richardson,1995; Richardson et al., 1997). Despitecounters to the attacks (Sander, 2002;Hopwood, 2006) and a reassessment(Richardson et al., 1998a; Richardsonand Keuck, 2002), Haeckel’s drawingshave been removed from textbooks.Compare Gilbert (1997) and Albertset al. (1994) to Gilbert (2000) andAlberts et al. (2002).

It is ironic that the recent attackson Haeckel’s concept of a common ver-tebrate morphological stage came inan era when developmental biologistsbecame increasingly aware of commonmolecular events underlying develop-ment in a wide range of animals fromfruit flies to mammals. Anterior–pos-terior patterning by a complex ofhomeodomain-containing transcrip-tion factors is a prominent example ofhighly conserved molecular events(Slack et al., 1993). Others are Pax6control of eye development and theimportance of tinman/NKx2.5 in pro-ducing a beating heart. Conservedgene expressions and the molecularinteractions that produce particulartissues and organs have led to con-cepts like the molecular tool-box (Car-roll et al., 2001; Canestro et al., 2007;Carroll, 2008; Holland, 2009), genekernels and subcircuits (Hinman and

Davidson, 2007; Peter and Davidson,2009), conserved core components(Gerhart and Kirschner, 2007), generegulatory networks (Davidson et al.,2002; Rebeiz et al., 2005; Davidsonand Levine, 2008; Ettensohn, 2009),and core gene network (Woodland andZorn, 2008). To a taxonomist, many ofthese elements are symplesiomor-phies or shared basal characters(Richardson et al., 2001).Haeckel sensed a commonality in

early embryos. Although that sensecould have biased his depiction ofembryos, Haeckel was more correctthan the data of his day allowed. Com-monality in early embryos is more appa-rent frommolecules than frommorphol-ogy. Molecules may provide a better wayto depict the common vertebrate embry-onic stage that Haeckel sought, and afirst generation of that molecular depic-tion will be presented here.

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Additional Supporting Information may be found in the online version of this article.

Department of Biological Sciences, Duquesne University, Pittsburgh, PennsylvaniaGrant sponsor: NSF; Grant number: IOB-0343403.*Correspondence to: Richard P. Elinson, Department of Biological Sciences, Duquesne University, 600 Forbes Avenue,Pittsburgh, PA 15282. E-mail: [email protected]

DOI 10.1002/dvdy.22337Published online 2 June 2010 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 239:1905–1918, 2010

VC 2010 Wiley-Liss, Inc.

CONSTRUCTING A

MOLECULAR HAECKEL:

DESIGNING A

MORPHOLOGICAL

FRAMEWORK

To create a Molecular Haeckel, we willplace regulatory molecules, commonamong vertebrate embryos, onto aschematic morphology. The first step isto design the morphological frameworkof an idealized, generic vertebrateembryo at an early developmentalstage. There are a large number of com-mon features of vertebrate earlyembryos that can be incorporated intothe framework. This collection of com-mon features has been used to generateconcepts such as the basic or primitivevertebrate body plan (e.g., Arey, 1924,1954), the pharyngula (Ballard, 1981),the phylotypic stage (Sander, 1983, ascited in Sander, 2002; Slack et al.,1993), and the developmental hour-glass (Elinson, 1987; Duboule, 1994).

This generic vertebrate embryo hasthree germ layers arranged in concen-tric tubes and a set of dorsal axialstructures. In the trunk and post-analtail, the dorsal axial structures con-sist of a spinal cord, underlain by anotochord and flanked by segmented,mesodermal somites. Completing themesodermal tube on the lateral andventral sides are intermediate meso-derm and lateral plate mesoderm,split into an inner splanchnic layerand an outer somatic layer. Four limbbuds arise from the somatic layer.The mesodermal tube surrounds theinner endodermal tube, which joinsthe outer ectoderm at the mouth andthe anus. Various evaginations of theendodermal epithelium foreshadoworgans such as the liver and pancreas.The anterior end of the embryo ishighly modified to form the head. Theneural tube is expanded to form thebrain; sense organs arise from ecto-dermal placodes and connect to thebrain, and much of the prospectivecraniofacial cartilage is derived fromcranial neural crest. The importantroles played by placodes and cranialneural crest led to Gans and North-cutt’s (1983; Northcutt, 2005) intrigu-ing hypothesis that the evolution ofvertebrates involved the addition toan ancestral chordate of a new headfrom these two embryonic cell types.

This generic embryo looks similar toactual embryos to a greater or lesserdegree. There are three major sourcesof deviations. First, different verte-brate embryos have different amountsof yolk, ranging from no yolk in pla-cental mammals to huge amounts ofyolk in birds. Furthermore, yolk mayeither be included within the embryo’sbody, as in amphibians, lungfish, tele-osts, and sturgeon, or contained in anextraembryonic yolk sac attached by astalk to the body, as in shark, coela-canth, birds, and reptiles. Whenimages of embryos are modified toremove the yolk, they look much moresimilar (Richards, 2008, p. 307).

The second major source of devia-tion between the generic embryo andactual embryos is the relative size ofparts of the embryo. The size differen-ces can be quantitative or qualitative.The most obvious quantitative differ-ence is the number of somites in thetrunk (Richardson et al., 1998b; Wol-tering et al., 2009; Gomez and Pour-quie, 2009). The frog X. laevis hasonly nine trunk somites (Tucker andSlack, 1995), whereas animals withelongated bodies, such as eels, caecil-ian amphibians, and snakes, can havehundreds of somites (Gomez et al.,2008; Woltering et al., 2009). Chickand mouse have 39 and 35 non-tailsomites, respectively (Burke et al.,1995).

An example of a qualitative differ-ence is the size of the head relative tothe body. Amniote embryos generateanterior structures, including thehead and heart, precociously relativeto the trunk and tail, and these struc-tures are disproportionately large.Within amniotes, embryos of nonmammalian amniotes have muchlarger eyes proportional to the head,than mammalian embryos (Jefferyet al., 2002). There are many exam-ples of size differences in limb devel-opment. Marsupials have very largeforelimbs early to enable the embryoto crawl to the teat. Conversely, tad-poles of frogs have practically no limbbuds until they begin feeding, and af-ter that, the limbs remain small andinconspicuous.

The third major source of deviationbetween the generic embryo and actualembryos is the relative timing of devel-opment of one part relative to another.For example in some embryos, forelimb

buds arise before hindlimb buds, whilehindlimb buds appear first in otherembryos (Bininda-Emonds et al.,2007; Richardson et al., 2009). Theseheterochronies are rampant (Richard-son, 1995), complicating comparisonsbetween different embryos and mud-dying the concept of a common phylo-typic stage.Analytical schemes have been pro-

posed not only to detect heterochro-nies between embryos of different ani-mals but also to provide measuresof differences when comparing differ-ent embryos (Smith, 2001; Schlosser,2001; Jeffery et al., 2005; Maxwelland Harrison, 2009; Werneburg,2009). Whereas heterochrony makesit difficult to compare whole embryosof different animals, the conceptof modularity obviates difficultiesin comparisons at lower levels oforganization.Modularity refers to the integrated

and autonomous behavior of a molec-ular pathway, a group of cells in atissue, or even a developing organ(Schlosser and Wagner, 2004). Forexample, many cellular and mole-cular interactions occur autono-mously within a developing limb bud,without reference to the rest of theembryo. Similarly, a relatively smallnumber of transcription factors serveas master regulatory genes for a tis-sue, like skeletal muscle, or an organ,like the pancreas. Once these masterregulatory genes are turned on, a cas-cade of molecular and cellular eventsfollows to generate the tissue ororgan.Given modularity, we will place key

regulatory molecules that operatewithin a tissue or organ on our morpho-logical framework. Essentially, theembryo is depicted as a mosaic of geneexpression territories, largely inde-pendent of each other. In one sense, weare simply shifting the concept of acommon embryonic stage from onelevel of organization, the wholeembryo, to a lower level of organiza-tion, the individual tissues and organs.By doing so, we suggest that molecularcommonality of tissues and organs isstronger than the morphological com-monality of the whole embryo at thephylotypic stage. We suggest that ourmolecular pictures have core truths, asHaeckel claimed for his morphologicalpictures.

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1906 ELINSON AND KEZMOH

TABLE

1.GenesIn

volved

inDevelopmentofVertebrate

Embryosa

Basa

lto

teleost

Teleost

zebrafish

(unless

noted

)

AnuranXen

opus

(unless

noted

)

Bird

chicken

Mammal

mou

seOthers

Epidermis

BMP

283

407

376

94

p63

215

234

426

203,204

Tfap2c/TFAP2g/AP-2g

157

134,157

141,157

Epidermalappen

dages

scale

feather

hair,tooth,etc.

reptile

scale

ectodysp

lasinpathway

(EdaEdarEdaradd)

86(s),201(m

)nr

171

259

106

Placodes

Signals

-344,379.Hypothesis:Fgf,(-)B

MP,(-)W

ntbutdata

mainly

chick,Xen

opuson

ly.

Six

1/2,Six

4/5

(TX)

24,201

344

379

344,379

Eya1/2

344,379

344

379

344,379

Nervou

sSystem

Sim

ilarAPpatternsin

thehem

ichordate

Saccog

lossus(197,230,231),theuroch

ordate,variou

sascidians(254,342),thecephaloch

ordate

Amphioxus(160,161,342),

andtheagnathanlamprey(274,342)

Foreb

rain

Nkx2-1

253

253

253

253,419

419-human

Dlx

253

253

253

253,419

Otp

335

18

18,335

Otx1/2

382-lamprey,

bichir,sk

ate

219

18(r),186

185

185,419

419-human

Tbr(T

brain

-Tbr1/

Eom

es/Tbr2)

263

253,333

253

253

Emx1

102-dog

fish

253

253

253

253,419

Arx

265

352

84

265,419

419-human

Lhx5(Lim

5)

398,419

398,419

419

Fezf1/2

358

248,358

nr

248,358

Six3

211

,438

438

438

211

,293

411

-human

Pax6

101-lamprey,

dog

fish

253

253

253

253,419

419-human

Msx1

108,307(different

msx)

383

23,323

Eye

Rx

15,15(m

),381(a)

15

15

15

Pax6

253

253

253

253,419

419-human

Lhx2

9419

239

419

Vax

387

17

347

17,419

Barh

l2325

325

325

Midbrain

Otx1/2

219

186

185

10,185

Barh

l1325

325

325

229-human

Lmx1a/b

291-Lmx1b

145-Lmx1b

8,10

8,10

En2

109,120

152

10,185

Msx

1108,307(different

msx)

383

8,10

8,10,23,323

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MOLECULAR HAECKEL 1907

TABLE

1.(C

ontinued)

Basa

lto

teleost

Teleost

zebrafish

(unless

noted

)

AnuranXen

opus

(unless

noted

)

Bird

chicken

Mammal

mou

seOthers

Midbrain/hindbrain

bou

ndary

Otx2

326

397

185

185

Gbx2

326gbx1

397

185

185

Wnt1

185

Fgf8

324

327,397

185

185

Hindbrain

Gbx2

326gbx1

397

185

185

Hox

1,2,3,4,(5)

317,342

134,301

193

342,420

Krox20

296,342

281

281

281

Kreisler(K

r/mafB

)266,342

174

240

240,342

En2

109,120

152

185

Barh

l1325

325

325

229-human

RA

133

133

133

133

(133-ex

pressionpatternsof

RALDH2,CYP26’s,RAR’s,CRABP’s.Mostex

pressed

inhindbrain

butnot

all;common

elem

ent¼

RA.)

Msx1

108,307(different

msx)

383

23,323

Spinalcord

-AP

Hox

4316

(134b4No)

Hox

5316

134

91,92

Hox

64(s),316

91,92

52

Hox

7316

134

92

52

Hox

8316

199

91,92

52

Hox

94(s),316

134,228

91,92

52

Hox

10

4(s),316,403

228

92,212

52

Hox

11403

228

92

52

Hox

12

403

228

52

Hox

13

403

228

52

Raldh2

nr

112(co)

91,237

237

Barh

l2325

Spinalcord

-DV

Msx1(roofplate)

108,307(different

msx)

383

222

23,74,323

Lmx1(roofplate)

nr

(247nr)

74

74

Math1

300,413

386-A

th3

74,421

74,360

(atonal/Cath1/Xath5a?)

195-X

ath1not

spinalcord

Ngn1/2

28

236

421

360

Mash

16

439-X

ash

3180,421

360,224(ra)

119-X

ash

1not

spinalcord

Tlx3(R

nx/H

ox11

L2)

213

302

225,421

319

Pax7

348,363(c)

66

181,421

103,250

Lbx1

279,289

243

255,421

177

Lhx1/5

(Lim

1/5)

399-lim

1385-Lim

1360-lim

1,421

309,360

398-Lim

5398-Lim

5Pax2

258-Pax2a

151

421

309

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TABLE

1.(C

ontinued)

Basa

lto

teleost

Teleost

zebrafish

(unless

noted

)

AnuranXen

opus

(unless

noted

)

Bird

chicken

Mammal

mou

seOthers

Dbx1/2

137

131

181,421

103,250

Lmx1b

251

145

421

69,319

Irx3

217

22

181,421

103,250

Pax6

318

153

181,421

103,250

Evx1/2

393

329-X

hox

3421

250

Nkx6.1

63

436

181,421

103,250

Olig2

218

39

421

437

Isl1/2

218

36

91,402,421

103,250

Hb9(M

nx1/H

lbx9/M

NR2)

416

338

389,421

12,103

Nkx2.2

341-N

kx2.2b

337-X

eNK-2

181,421

103,250

Shh(floorplate)

378

110

378

378

Fox

a1/2

(Hnf3a,b)(floor

plate)

378

331

330,378

103,378

ntn1(netrin)(floorplate)

378

96

378

349

Neu

ralcrest

Inductionsignals

(257)

BMP

339-lamprey

150,322

150,172,322

(172no,

150?)

160-amphioxus

Wnt

339-lamprey

150,172,322

150,172,322

150,172,322

172

FGF

150,172

Msx1(neu

ralep

idermal

bou

ndary)

160-amphioxus

108,307(different

msx)

383

74

23,74,323

Neu

ralcrestsp

ecifiers(257)

AP2a

339

172,257

150,172,257

172

150,172,257

Snail,Slug

(339-lamprey,

172,257

150,172,257

150,172

150,172,257

295-hagfish

butnot

NC

specific)

Sox

8/9/10(Sox

E)

339-lamprey,

295-hagfish

172,257

150,172,257

150,172

150,172,257

Fox

D3

339-lamprey

172,257

150,172,257

150,172

150,172

Twist

339-lamprey

130

172,257

172

172,257

Ets1

339-lamprey

172

172

172

Cra

nialneu

ralcrest

Ednra

(ETA)

276

32(cranial,trunk)

190,310

83,310

Edn1(E

T-1)

261

(32activity)

310

83,209,310

(pharyngea

larchparaxialmesoþ

epithelium)

Tru

nkneu

ralcrest

Ednrb

299

310

310

249-human

Edn3(ectod

erm)

310

249-human

Notochord

Shh

374

138,244

34,38

38

(Note404:Xen

opusShh&

Ihhbothin

Ihhfamily)

Som

itesegmen

tation

Pre-som

itic

mesod

erm

(PSM)

Notch

163

369

81,100,368

99,184

418-human

FGF

81,100,368

227

104

99

135-snake

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TABLE

1.(C

ontinued)

Basa

lto

teleost

Teleost

zebrafish

(unless

noted

)

AnuranXen

opus

(unless

noted

)

Bird

chicken

Mammal

mou

seOthers

Wnt/bcatenin

14,99

135-snake

Lfng

315,320,114(m

)(not

cyclic)

(425nr)

81,100,368

122

135-snake

Hairy(H

er/H

es/H

EY/esr)

163,114(m

)220

297

184

paraxis

(TCF15)

353

53,401

19

45

135-snake

Msgn1

135

135

135

135-snake

PSM/Som

iteborder

Mesp2(M

espb,

thylacine1

)269,340,392(m

)269,369

76

336

418-human

Tbx6

(164,282Tbx24)

155

198

61,290

Ripply

(bow

line)

188,269(f)

154,269

nr

57,100

Som

ite

Raldh2(R

A)

187,269(f)

269,270

104

81,100,368

135-snake

paraxis

(TCF15)

353

53,401

19

45

135-snake

Lfng

315,320,114(m

)(not

cyclic)

(425nr)

81,100,368

122

135-snake

Som

itepatterning

Wnt4,Wnt6,Wnt7a(ectod

erm

signal)

(374-nr)

38

38

Wnt1,Wnt3a(dorsa

lneu

raltubesignal)

(374-nr)

34,38

38

BMP4(lateralmeso)

(374-nr)

34

Pax1(sclerotom

e)264(m

)147

34,38

38,75

Pax9(sclerotom

e)285,264(m

)34,38

38,75

Pax3(dermom

yotom

e)374

242

34,38

38,43,75

Pax7(dermom

yotom

e)374

66

34

43

MyoD

(MRF)

374

60,166

34,38,60

38,43,60,75

Myf5

(MRF)

374

60,167

34,38,60

38,43,60,75

Sim

1350

88

34

116

Lbx1

144,279

243

34,144

144

scleraxis

-tendon

TX

98

37

89

Som

iteAP

Hox

3128

128

Hox

4134

46,129

46,129

Hox

54(s)

46,129

46,129

423-caecilian

Hox

6(lev

elof

forelimb)

316,4(s)

46

46,129

46,129

423-caecilian

Hox

7316

46,129

46,129

Hox

8316

46

46

423-caecilian

Hox

9316,4(s)

46

46

Hox

10(lev

elof

hindlimb)

316,403,4(s)

228

46

46

423-caecilian

Hox

11403

228

46

46

Hox

12(start

oftail)

403

228

46

46

Hox

13

403

228

46

423-caecilian

(423corn

snakeOK

forHox

3,4,5,10,13;order

differencesforHox

6,7,8,9)

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TABLE

1.(C

ontinued)

Basa

lto

teleost

Teleost

zebrafish

(unless

noted

)

AnuranXen

opus

(unless

noted

)

Bird

chicken

Mammal

mou

seOthers

Lim

binitiation

,ou

tgrowth,patterning

Fgf10(m

esen

chyme)

256

429

245,284

245,284

Fgf8

(distalep

idermis

-AER)

256

78

377

377

Dlx

(AER)

286(D

lx2a)

nr

118(D

lx5)

223(D

lx5,6)

223-human

Wnt7a(dorsa

lecto)

286

79(ecto,

meso)

49

49

Lmx1b(dorsa

lmeso-

amniotes)

251(?location

)nr

49

49

R-fng

320-nr

79(ecto,

meso)

49(dorsa

lecto)

268(ecto,

meso)

48(new

t,meso)

En1(ven

tralecto)

286

79

126

49

Shh(ZPA

-posterior

margin)

90-skate,sh

ark

5,206

117,377,148(co)

377

377

See

404Fig

2forShh

overview

93-paddlefish

Hox

9–13(nested

expression)

123-catshark

93-paddlefish

77,228

277

362,430

396-m

odified

inaxolotl

Lbx1

144,279

(243nr)

144

144

Forelim

b-fin/w

ing/arm

Tbx5

390-dog

fish

226,332,388

377

226,377

226,377

Raldh2(R

A,initiation

)11

2,256

112(co)

112,256

112,256

Hindlimb

Tbx4

390-dog

fish

226,332,388

377

226,377

226,377

Pitx1

354(s)

59

226

97,226

Hea

rtRaldh2(R

A)

189

85

156,364

334,364

Nkx2.5

64,294

82,139,294

1,42

1,107,294

Tbx5,Tbx20

127,312,384

40,169

41,311

,312

107,312,375

GATA

4,5,6

294,305

294,305

182

107,294

HAND

1,2

11,294,427

11,294

371

107,294

Mef2c

394

7107,294

Islet1(Isl1)Secon

dhea

rtfield(SHF)

(173neu

ronal;

hea

rtnr)

36

26,44

1,26,44,107

Kidney

Pronep

hrosis

examined

inzebrafish

andXen

opus,

whilemetanep

hrosis

gen

erallyex

amined

inch

ickandmou

se.

Thecomparisonseem

sjustified

,given

thecommon

ality

ofkey

gen

eex

pressions(105,170,183,406).

Osr1/2

391

391

179,380

178,366

Raldh2(R

A)

13,55

29

55

Lim

1399

54,58

105,400

Pax2/8

170,350

54,55

35,105

35,55,105

GDNF/Ret

170,356

210

165

87

WT1

170,356

410

51,191

105,207

191-alligator

Stomach

mesen

chyme,

musculature

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TABLE

1.(C

ontinued)

Basa

lto

teleost

Teleost

zebrafish

(unless

noted

)

AnuranXen

opus

(unless

noted

)

Bird

chicken

Mammal

mou

seOthers

Barx1

273,370

365?m

esen

194,260

Bapx1(N

kx3.2/Xbap)

262

278

280

405

Digestivetract

Nod

al

136,440-cyc/sq

t136,440

136,440

Mix

136,440

136,440

136

136,440-M

ixl1?

GATA

136,440

136,440

62

136,440

GATA5

GATA4/5/6

Sox

-F136,440

136,440-Sox

17

62-Sox

17

136,440-Sox

17

casa

nov

aFox

A136,440-Fox

a2

136,440

136,440

Stomach

Sox

2273-antgut

56

175

357

Odd1

357

Intestine

Cdx1

67,71,121-cdx1b

56-X

cad2

124-C

dxA

275

Cdx2(m

oreim

portant)

67,71,121-cdx1b

56-X

cad1

241-C

dxC

125

Zeb

rafish

Cdx2lost;cd

x1bfunctionslikeCdx2(67,71,121,272).

Shh(endo)

inducesBMP

(meso)

See

404Fig

2for

Shhov

erview

(408Shhon

lyesop

hagus;

BMPnr)

176

328

238

Hox

13(cloaca,anus)

403

228

20,428

20,202

Thyroid

gland

Titf1/N

kx2.1

(TTF-1,T/

EBP)

95,314

95

95

95

Fox

e1(TTF-2)

95,111

95

Pax2/5/8

314-Pax2a

95-Pax2

95

(Pax8(m

ammal)butrelatedPax2andPax2ain

zebrafish

,Xen

opus).

Hhex

95,314

95

95

95

Thymus

Pax1/9

(Pax1-nr)

nr

306

27,73,409

(Pax9-thymusnr)

Fox

n1

16-catshark

346,16(m

)nr

16

27

Parathyroid

gland

Gcm

2292-dog

fish

158,292-ecto

292

140

Liver

Fgf,BMP

359

65-Fgf

433

431,435

Fox

a(H

NF3)

nr

440

nr

214

C/EBP

235

205

nr

216,355,417

HNF4

72

159

nr

216,355,415

Hex

,Prox1

288

287,440

433-H

ex47,431,435

Pancrea

sRA

373,395

298,308,372

372

246,267

Shhsignaling

repressionphase

80,149,395

149

149

149,132

Pdx1

25,149,395

168,303,424

196

132,142

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CONSTRUCTING A

MOLECULAR HAECKEL:

CHOOSING GENES

The genes that are used to constructthe pictures are regulatory genes thatare expressed in a specific organ ortissue in embryos across a range ofvertebrates.

Range of Vertebrates

Gene expression and function in de-velopment are known mainly fromembryos of four model animals: zebra-fish, Xenopus, chick, and mouse.Chick and mouse represent the mono-phyletic Amniota; amniotes plusXenopus represent the monophyleticTetrapoda, and tetrapods plus zebra-fish represent the monophyleticOsteichthyes. If a gene is expressed inboth zebrafish and mouse embryos, itsuggests that their last commonancestor had that organ specific geneexpression and that all animalswithin Osteichthyes are expected tohave that gene expression. Geneexpression in both zebrafish andmouse embryos was our usual mini-mum criterion for inclusion in the Mo-lecular Haeckels.Of course, if Xenopus and chick also

have that gene expression, the morelikely it is that it represents a commoncharacter. Some embryonic geneexpressions have been reported forother teleosts such as medaka, fugu,and stickleback, other amphibianssuch as axolotl and caecilian, and otheramniotes such as alligator, snake, andhuman. A few embryonic gene expres-sions have been reported for non-tele-ost Actinopterygians, such as bichir(Takeuchi et al., 2009) and paddlefish(Metscher et al., 2005; Davis et al.,2007), indicating that these embryosare available to strengthen any conclu-sions of commonality based on teleosts.Some gene expressions have beenreported in embryos of Chondrich-thyes, the cartilaginous fish. Theseinclude reports on catshark, dogfish,and skate (Neyt et al., 2000; Derobertet al., 2002a,b; Tanaka et al., 2002;Okabe and Graham, 2004; Dahn et al.,2007; Bajoghli et al., 2009; Suda et al.,2009). As more genes are analyzed incartilaginous fishes, the MolecularHaeckels can be drawn to include alljawed vertebrates, the Gnathostomes.

TABLE

1.(C

ontinued)

Basa

lto

teleost

Teleost

zebrafish

(unless

noted

)

AnuranXen

opus

(unless

noted

)

Bird

chicken

Mammal

mou

seOthers

PTF1a/p48

132,221,395

2,132,303

208

132

Pax4

(303-nr)

132,142

Pax6

432-Pax6.2

303

132,142

Ngn3

(412-no)

303

132,142

Zeb

rafish

ngn3not

expressed

inpancrea

s.Given

teleostgen

omic

duplication

,theremaybeother

ortholog

ues,not

yet

clon

ed(412).

Lungs/sw

imbladder

Fox

p1/2/4

70-p1

(345-p1a/2/4

nr)

143-p2

361-p1/2

zebra

finch

adult

lungbutnomen

tion

embryo

(33-p2no)

(313-p1nr)

232-p4

Irx1/2

(68,414-nr)

21

Titf1/N

kx2.1

(TTF-1,T/

EBP)

(115-nr)

162

304

30

Prd

c271

nr

233

Fox

a1/2

(Hnf3a,b)

422

31,331-anten

do

330

50

Hb9

416,422

(338-nr)

(389-nr)

(272-nr)

am,med

aka;s,

stickleback

;c,

charr;a,blindcavefish

;f,fugu;r,rana;co,coqui;ra,rat.

Item

sin

redhighlightdifferencesacrossanim

als

orabsence

ofinform

ation

(nr).

Row

sin

grayindicate

sufficien

tdifferencesor

absencesto

excludethegen

efrom

theMolecularHaeckels.

Numbersreferto

referencesin

theSupportingIn

form

ation

.

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Regulatory Genes

Genes were chosen, primarily basedon expression patterns. A more strin-gent requirement for inclusion wouldbe demonstrations of function in thedevelopment of a particular tissue ororgan. In many cases, function wasshown in one model animal by themost convenient method, i.e., mouseknockouts, zebrafish mutants, orXenopus overexpression, and expres-sion was reported in other animals.For the gene collection used here(Table 1), gene expression was a suffi-cient criterion for inclusion.

The two main classes of regulatorymolecules are transcription factorsand components of signaling path-ways, usually the ligands. It was eas-ier to include the transcription factorscompared with ligands, although thatmay be partially an artifact of namingconventions. Both types of moleculesare composed of families, whose mem-bers are often numbered. For exam-ple, there are nine Pax genes andmore than 20 fibroblast growth factor(FGF) genes (Itoh, 2007). There aremore transcription factor familiescompared with ligand families, ofwhich there are mainly Wnt, FGF,Hh, Delta-Notch, and transforminggrowth factor-beta (TGFb). The TGFbsuperfamily is further split into bonemorphogenetic proteins (BMPs),nodals, growth differentiation factors(GDFs), and others. Another impor-tant ligand is retinoic acid, which is

generated by several differentenzymes.

For many tissues and organs, activ-ities of several signaling families areinvolved at different times and in dif-ferent places. Sorting out whether aparticular ligand or a different ortho-log is used in different animals for thesame activity is a major task, even inwell-known cases. For example, Shhis expressed in the notochord, thefloor plate, and the zone of polarizingactivity of the limb, and there is con-siderable functional analysis. Compli-cating its placement in a MolecularHaeckel is the fact that the XenopusShh ortholog falls into the Ihh familyphylogenetically (Varjosalo and Tai-pale, 2008). Because of the usage ofmultiple ligands in many tissues, lessemphasis was placed on ligands thanon transcription factors.

Not only are there more transcrip-tion factor families, the conservedexpression patterns of differentlynumbered family members are oftenwell-documented. One example is theexpression of the Hox genes in thespinal cord, the somites, and the limb.A second example is the tissue andorgan specific expression of Pax andTbx genes. There are interestingexceptions, however. For example,Pax8 is important for mouse thyroiddevelopment, but the related Pax2and Pax2a are used in Xenopus andzebrafish respectively (Table 2). Dif-ferent Msx and Gbx members are

expressed in zebrafish brain develop-ment compared with the tetrapods(Table 2). Other cases of differences ofortholog usage between zebrafish andtetrapods are Tbx in somite segmen-tation and Cdx in intestine (Table 2).For the most part, this type of ortho-log difference has been ignored. Afuture criterion for inclusion would bewhether the differently numberedorthologs have the same activity; thatis, can the gene from one animal sub-stitute for a family member in a dif-ferent species?

THE MOLECULAR

HAECKELS

Our drawings are shown in Figu-res 1–7. Traditional colors have beenused with blue for epidermis, greenfor central nervous system, lightgreen for neural crest, red for meso-derm, and yellow/orange for endo-derm. Each tissue or organ is con-structed using the acronyms of theimportant gene expressions in the de-velopment of the tissue or organ. Forsome tissues, there are many geneexpressions known, so the whole tis-sue texture is composed of acronyms.This is the case for the central nerv-ous system, neural crest, somites,limb buds, heart, liver, and pancreas.For other tissues, only a few keyexpressions have been identified.These tissues are represented by apattern with interspersed acronyms.

TABLE 2. Ortholog Differences in Transcription Factor Gene Expression

Gene Speciesa Tissue Difference

Pax8 m Thyroid Xenopus Pax2, zebrafish Pax2aCdx1 m Intestine Xenopus Xcad2, zebrafish cdx1bCdx2 (major) m Intestine Xenopus Xcad1, zebrafish cdx1bTbx6 m, x, c PSM/somite border Zebrafish Tbx24Msx1 m, x, c Brain, spinal cord roof plate Zebrafish msxB,C,EMath1 m, c Spinal cord Xenopus Ath3Mash1 m, c, z Spinal cord Xenopus Xash3Gbx2 m, x, c Brain Zebrafish gbx1Tbx4 m, x, c, z Hindlimb Newt Tbx5b

Tbx5 m, x, c, z Forelimb Newt Tbx4b

Hox6–9 m Thoracic somites Snake–different order

See Table 1 for references.am, mouse; c, chicken; x, Xenopus; z, zebrafish.bKhan et al. (2002) report differences in Tbx expression in newt limbs. There are other differences reported for limbs of other uro-dele amphibians. These include Fgf-8 expression in the mesenchyme of axolotl limb buds, rather than the apical ectodermalridge (Han et al., 2001), and differences in Shh expression (Stopper & Wagner, 2005).

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For some tissues, not only is geneexpression conserved but also the spa-tial location within the tissue of the cellsexpressing that gene is conserved. Hoxgenes are expressed with anterior–pos-terior polarity in the neural tube and

the somites and with proximal–distalpolarity in the limb buds. Other exam-ples are the dorsal–ventral pattern ofgene expressions in the neural tube, theregional expressions within the somite,the posterior expression of Shh in the

limb bud, and the expressions of thelimb bud ectoderm. In these cases, theplacement of acronyms reflected theconserved spatial locations.When viewed as reduced drawings

(Figs. 1–4), the acronyms are not

Fig. 1. Ectoderm. The skin has been removed from the embryo to reveal the brain and spinal cord (green), the midbrain–hindbrain boundary (tur-quoise) and the cranial and trunk neural crest (light green).

Fig. 2. Mesoderm. The skin has been removed to reveal the mesoderm. The anterior somites have formed, while somitogenesis continues poste-riorly. The somites, intermediate mesoderm, heart, and limb buds are composed of gene acronyms, while the lateral plate mesoderm and the mes-enchyme of the head and tail are represented by textures.

Fig. 3. Endoderm. The tube of endoderm has buds for the thymus and parathyroid gland, thyroid gland, lungs, liver, and pancreas, and theseorgans are well-represented by gene acronyms. There are some common gene expressions within the endoderm, as well as some specific onesfor stomach, intestine and cloaca.

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legible. The acronyms can be seen inthe full-sized drawings, and examplesof the eye, heart, liver, and pancreasare presented (Figs. 5–7). The com-plete full-sized drawings are in theSupplementary Material (Supp. Figs.S1–4, which are available online).

FUTURE MOLECULAR

HAECKELS

Constructing the Molecular Haeckelsprovides a glimpse into the difficultiesthat Haeckel must have encounteredin drawing his pictures. Which detailsshould be emphasized and which dif-ferences should be ignored? Geneswere included if they were expressedduring development of the same tis-sue or organ in a range of animals. Amore stringent requirement for inclu-sion would be a demonstration of animportant developmental function ofthe gene in both a teleost and anamniote. A second example is the de-cision to included different orthologswithin a family of transcription fac-tors, mentioned earlier (Table 2).The molecular database is much

richer than the morphological oneused by Haeckel. We have identifiedmore than 160 conserved associationsof regulatory genes involved in theearly development of specific tissues.Many of the genes are expressed inconserved locations within a tissue,adding to the number of molecularcharacters than can be used.Nonetheless, there may be a bias in

selecting the genes. Many of theincluded genes were discovered becausethey have a strong effect on

Fig. 4.

Fig. 5.

Fig. 7.

Fig. 6.

Fig. 7. Liver and pancreas. The full-sizeendoderm picture, from which this figure wastaken, is Supp. Fig. S3.

Fig. 5. Eye. The eye region is presented atfull-size to show specific eye expressions aswell as surrounding brain expressions. Thefull-size ectoderm picture, from which this fig-ure was taken, is Supp. Fig. S1.

Fig. 6. Heart. The full-size mesoderm picture,from which this figure was taken, is Supp.Fig. S2.

Fig. 4. Cross-section at the forelimb level.Dorsoventral and other spatially restrictedgene expressions are depicted for the spinalcord, somite, and both the ectoderm andmesoderm of the limb bud.

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development in one species. Once thatwas known, its ortholog was cloned andanalyzed in other species. This searchprocess is biased toward commonality,the very feature highlighted by the Mo-lecular Haeckels. Future data, gener-ated by microarrays, should giveunbiased pictures of genes expressed inparticular developmental events in dif-ferent animals. The problem thenbecomes identifying gene expressiondifferences that have significant func-tional differences between animals.

There are several ways to advancethe Molecular Haeckels. One way isto design them using the enlargingdata base of gene regulatory networks(GRN). Rather than individual geneexpressions, tissues would be con-structed with conserved pathwaysand molecular interactions. Giudiceand Onorato (2003) provided a proto-type for this type of image. A secondway to advance is to include timingand the order of gene expression.That would require computer anima-tions of Molecular Haeckels.

Haeckel’s pictures have not beenforgotten after a century, becausethey contain a core truth of common-ality among vertebrate embryos.These commonalities are more appa-rent than ever.

ACKNOWLEDGMENTThe production of the drawings wassupported by aNSF grant to R.P.E.

REFERENCES

Alberts B, Bray D, Lewis J, Raff M, RobertsK, Watson JD. 1994. Molecular biology ofthe cell. New York: Garland Publishing,Inc. 1294 p.

Alberts B, Johnson A, Lewis J, Raff M, Rob-erts K, Walter P. 2002. Molecular biologyof the cell. New York: Garland Science.1463 p.

Arey LB. 1924. Developmental anatomy.Philadelphia: WB Saunders. 433 p.

Arey LB. 1954. Developmental anatomy.Philadelphia: WB Saunders. 680 p.

Bajoghli B, Aghaallaei N, Hess I, Rode I,Netuschil N, Tay BH, Venkatesh B, YuJK, Kaltenbach SL, Holland ND, Die-khoff D, Happe C, Schorpp M, BoehmT. 2009. Evolution of genetic networksunderlying the emergence of thymopoie-sis in vertebrates. Cell 138:186–197.

Ballard WW. 1981. Morphogenetic move-ments and fate maps of vertebrates. AmZool 21:391–399.

Bininda-Emonds OR, Jeffery JE, Sanchez-Villagra MR, Hanken J, Colbert M,Pieau C, Selwood L, Ten Cate C, Ray-

naud A, Osabutey CK, Richardson MK.2007. Forelimb-hindlimb developmentaltiming changes across tetrapod phylog-eny. BMC Evol Biol 7:182.

Burke AC, Nelson CE, Morgan BA, TabinC. 1995. Hox genes and the evolution ofvertebrate axial morphology. Develop-ment 121:333–346.

Canestro C, Yokoi H, Postlethwait JH. 2007.Evolutionary developmental biology andgenomics. Nat Rev Genet 8:932–942.

Carroll SB. 2008. Evo-devo and an expand-ing evolutionary synthesis: a genetictheory of morphological evolution. Cell134:25–36.

Carroll SB, Grenier JK, Weatherbee SD.2001. From DNA to diversity. MaldenMA: Blackwell Science. 214 p.

Dahn RD, Davis MC, Pappano WN, ShubinNH. 2007. Sonic hedgehog function in chon-drichthyanfins and the evolution of append-age patterning. Nature 445:311–314.

Davidson EH, Levine MS. 2008. Proper-ties of developmental gene regulatorynetworks. Proc Natl Acad Sci USA 105:20063–20066.

Davidson EH, Rast JP, Oliveri P, Ransick A,Calestani C, Yuh CH, Minokawa T, AmoreG, Hinman V, Arenas-Mena C, Otim O,Brown CT, Livi CB, Lee PY, Revilla R,Rust AG, Pan Z, Schilstra MJ, Clarke PJ,Arnone MI, Rowen L, Cameron RA,McClay DR, Hood L, Bolouri H. 2002. Agenomic regulatory network for develop-ment. Science 295:1669–1678.

Davis MC, Dahn RD, Shubin NH. 2007.An autopodial-like pattern of Hoxexpression in the fins of a basal actino-pterygian fish. Nature 447:473–476.

Derobert Y, Baratte B, Lepage M, MazanS. 2002a. Pax6 expression patterns inLampetra fluviatilis and Scyliorhinuscanicula embryos suggest highly con-served roles in the early regionalizationof the vertebrate brain. Brain Res Bull57:277–280.

Derobert Y, Plouhinec JL, Sauka-SpenglerT, Le Mentec C, Baratte B, Jaillard D,Mazan S. 2002b. Structure and expres-sion of three Emx genes in the dogfishScyliorhinus canicula: functional andevolutionary implications. Dev Biol 247:390–404.

Duboule D. 1994. Temporal colinearity andthe phylotypic progression: a basis forthe stability of a vertebrate Bauplan andthe evolution of morphologies throughheterochrony. Dev Suppl:135–142.

Elinson RP. 1987. Change in developmentalpatterns: embryos of amphibians withlarge eggs. In: Raff RA, Raff EC, editors.Development as an evolutionary process.New York: Alan R. Liss, Inc. p 1–21.

Ettensohn CA. 2009. Lessons from a generegulatory network: echinoderm skele-togenesis provides insights into evolu-tion, plasticity and morphogenesis.Development 136:11–21.

Gans C, Northcutt RG. 1983. Neural crestand the origin of vertebrates: a newhead. Science 220:268–273.

Gerhart J, Kirschner M. 2007. The theoryof facilitated variation. Proc Natl AcadSci USA 104(suppl 1):8582–8589.

Gilbert SF. 1997. Developmental biology.Sunderland MA: Sinauer Associates,Inc. 918 p.

Gilbert SF. 2000. Developmental biology.Sunderland MA: Sinauer Associates,Inc. 749 p.

Giudice G, Onorato K. 2003. Developmen-tal biology for undergraduate studentsat the University of Palermo, Italy. IntJ Dev Biol 47:123–133.

Gomez C, Pourquie O. 2009. Developmen-tal control of segment numbers in ver-tebrates. J Exp Zool B Mol Dev Evol312:533–544.

Gomez C, Ozbudak EM, Wunderlich J,Baumann D, Lewis J, Pourquie O. 2008.Control of segment number in verte-brate embryos. Nature 454:335–339.

Han MJ, An JY, Kim WS. 2001. Expres-sion patterns of Fgf-8 during develop-ment and limb regeneration of theaxolotl. Dev Dyn 220:40–48.

Hinman VF, Davidson EH. 2007. Evolu-tionary plasticity of developmental generegulatory network architecture. ProcNatl Acad Sci USA 104:19404–19409.

Holland LZ. 2009. Chordate roots of thevertebrate nervous system: expandingthe molecular toolkit. Nat Rev Neurosci10:736–746.

Hopwood N. 2006. Pictures of evolutionand charges of fraud: Ernst Haeckel’sembryological illustrations. Isis 97:260–301.

Itoh N. 2007. The Fgf families in humans,mice, and zebrafish: their evolutionalprocesses and roles in development, me-tabolism, and disease. Biol Pharm Bull30:1819–1825.

Jeffery JE, Bininda-Emonds OR, CoatesMI, Richardson MK. 2002. Analyzing ev-olutionary patterns in amniote embry-onic development. Evol Dev 4:292–302.

Jeffery JE, Bininda-Emonds OR, CoatesMI, Richardson MK. 2005. A new tech-nique for identifying sequence hetero-chrony. Syst Biol 54:230–240.

Khan P, Linkhart B, Simon HG. 2002. Dif-ferent regulation of T-box genes Tbx4 andTbx5 during limb development and limbregeneration. Dev Biol 250:383–392.

Maxwell EE, Harrison LB. 2009. Methodsfor the analysis of developmentalsequence data. Evol Dev 11:109–119.

Metscher BD, Takahashi K, Crow K,Amemiya C, Nonaka DF, Wagner GP.2005. Expression of Hoxa-11 and Hoxa-13 in the pectoral fin of a basal ray-finned fish, Polyodon spathula: implica-tions for the origin of tetrapod limbs.Evol Dev 7:186–195.

Neyt C, Jagla K, Thisse C, Thisse B,Haines L, Currie PD. 2000. Evolution-ary origins of vertebrate appendicularmuscle. Nature 408:82–86.

Northcutt RG. 2005. The new head hy-pothesis revisited. J Exp Zool B MolDev Evol 304B:274–297.

Okabe M, Graham A. 2004. The origin ofthe parathyroid gland. Proc Natl AcadSci USA 101:17716–17719.

Peter IS, Davidson EH. 2009. Genomiccontrol of patterning. Int J Dev Biol 53:707–716.

Dev

elop

men

tal D

ynam

ics


Rebeiz M, Stone T, Posakony JW. 2005.An ancient transcriptional regulatorylinkage. Dev Biol 281:299–308.

Richards RJ. 2008. The tragic sense of life,Ernst Haeckel and the struggle over evo-lutionary thought. Chicago: Universityof Chicago Press. 551 p.

Richardson MK. 1995. Heterochrony andthe phylotypic period. Dev Biol 172:412–421.

Richardson MK, Hanken J, GooneratneML, Pieau C, Raynaud A, Selwood L,Wright GM. 1997. There is no highlyconserved embryonic stage in the verte-brates: implications for current theoriesof evolution and development. AnatEmbryol (Berl) 196:91–106.

Richardson MK, Hanken J, Selwood L,Wright GM, Richards RJ, Pieau C, Ray-naud A. 1998a. Letter. Science 280:983.

Richardson MK, Allen SP, Wright GM,Raynaud A, Hanken J. 1998b. Somitenumber and vertebrate evolution. De-velopment 125:151–160.

Richardson MK, Jeffery JE, Coates MI,Bininda-Emonds OR. 2001. Compara-tive methods in developmental biology.Zoology (Jena) 104:278–283.

Richardson MK, Keuck G. 2002. Haeckel’sABC of evolution and development. BiolRev Camb Philos Soc 77:495–528.

Richardson MK, Gobes SM, van LeeuwenAC, Polman JA, Pieau C, Sanchez-Vil-lagra MR. 2009. Heterochrony in limbevolution: developmental mechanismsand natural selection. J Exp Zool B MolDev Evol 312:639–664.

Sander K. 2002. Ernst Haeckel’s ontoge-netic recapitulation: irritation and incen-tive from 1866 to our time. Ann Anat184:523–533.

Schlosser G. 2001. Using heterochronyplots to detect the dissociated coevolutionof characters. J Exp Zool 291:282–304.

Schlosser G, Wagner GP, editors. 2004.Modularity in development and evolu-tion. Chicago: The University of Chi-cago Press. 600 p.

Slack JM, Holland PW, Graham CF. 1993.The zootype and the phylotypic stage.Nature 361:490–492.

Smith KK. 2001. Heterochrony revisited:the evolution of developmental sequen-ces. Biol J Linn Soc Lond 73:169–186.

Stopper GF, Wagner GP. 2005. Of chickenwings and frog legs: a smorgasbord ofevolutionary variation in mechanismsof tetrapod limb development. Dev Biol288:21–39.

Suda Y, Kurokawa D, Takeuchi M, Kaji-kawa E, Kuratani S, Amemiya C, AizawaS. 2009. Evolution of Otx paralogue

usages in early patterning of the verte-brate head. Dev Biol 325:282–295.

Takeuchi M, Takahashi M, OkabeM, AizawaS. 2009. Germ layer patterning in bichirand lamprey; an insight into its evolutionin vertebrates. Dev Biol 332:90–102.

Tanaka M, Munsterberg A, AndersonWG, Prescott AR, Hazon N, Tickle C.2002. Fin development in a cartilagi-nous fish and the origin of vertebratelimbs. Nature 416:527–531.

Tucker AS, Slack JM. 1995. Tail bud deter-mination in the vertebrate embryo. CurrBiol 5:807–813.

Varjosalo M, Taipale J. 2008. Hedgehog:functions and mechanisms. Genes Dev22:2454–2472.

Werneburg I. 2009. A standard system tostudy vertebrate embryos. PLoS ONE4:e5887.

Woltering JM, Vonk FJ, Muller H, BardineN, Tuduce IL, de Bakker MA, Knochel W,Sirbu IO, Durston AJ, Richardson MK.2009. Axial patterning in snakes and caecil-ians: evidence for an alternative interpreta-tion of the Hox code. Dev Biol 332:82–89.

Woodland HR, Zorn AM. 2008. The coreendodermal gene network of vertebrates:combining developmental precision withevolutionary flexibility. Bioessays 30:757–765.

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elop

men

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Molecular Haeckel