DEVELOPMENTAL DYNAMICS 239:19051918, 2010

REVIEWSA PEER REVIEWED FORUM

Molecular Haeckel
Richard P. Elinson* and Lorren Kezmoh
More than a century ago, Ernst Haeckel created embryo drawings to illustrate the morphological similarity of vertebrate early embryos. These drawings have been both widely presented and frequently criticized. At the same time that the idea of morphological similarity was recently attacked, there has been a growing realization of molecular similarities in the development of tissues and organs. We have surveyed genes expressed in vertebrate embryos, and we have used them to construct drawings that we call Molecular Haeckels. The Molecular Haeckels emphasize that, based on gene expression, there is a greater similarity among vertebrate embryos than even Haeckel might have imagined. Developmental Dynamics 239:19051918, 2010. V 2010 Wiley-Liss, Inc.
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Key words: comparative embryology; orthologs; modularity; model organisms; ectoderm; mesoderm; endoderm

Developmental Dynamics

Accepted 12 May 2010

INTRODUCTION
The most famous drawings of embryos are those that Ernst Haeckel used to illustrate his law of ontogenetic connection of systematically related forms (Elinson, 1987). Haeckel argued that vertebrate embryos at a particular point in their development look so similar that they cannot be told apart by a casual examination. Haeckels drawings have been alternatively praised and damned over the decades, with a renewal of attacks approximately 10 years ago (Richardson, 1995; Richardson et al., 1997). Despite counters to the attacks (Sander, 2002; Hopwood, 2006) and a reassessment (Richardson et al., 1998a; Richardson and Keuck, 2002), Haeckels drawings have been removed from textbooks. Compare Gilbert (1997) and Alberts et al. (1994) to Gilbert (2000) and Alberts et al. (2002).

It is ironic that the recent attacks on Haeckels concept of a common vertebrate morphological stage came in an era when developmental biologists became increasingly aware of common molecular events underlying development in a wide range of animals from fruit ies to mammals. Anteriorposterior patterning by a complex of homeodomain-containing transcription factors is a prominent example of highly conserved molecular events (Slack et al., 1993). Others are Pax6 control of eye development and the importance of tinman/NKx2.5 in producing a beating heart. Conserved gene expressions and the molecular interactions that produce particular tissues and organs have led to concepts like the molecular tool-box (Carroll et al., 2001; Canestro et al., 2007; Carroll, 2008; Holland, 2009), gene kernels and subcircuits (Hinman and

Davidson, 2007; Peter and Davidson, 2009), conserved core components (Gerhart and Kirschner, 2007), gene regulatory networks (Davidson et al., 2002; Rebeiz et al., 2005; Davidson and Levine, 2008; Ettensohn, 2009), and core gene network (Woodland and Zorn, 2008). To a taxonomist, many of these elements are symplesiomorphies or shared basal characters (Richardson et al., 2001). Haeckel sensed a commonality in early embryos. Although that sense could have biased his depiction of embryos, Haeckel was more correct than the data of his day allowed. Commonality in early embryos is more apparent from molecules than from morphology. Molecules may provide a better way to depict the common vertebrate embryonic stage that Haeckel sought, and a rst generation of that molecular depiction will be presented here.

Additional Supporting Information may be found in the online version of this article. Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania Grant 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: elinson@duq.edu DOI 10.1002/dvdy.22337 Published online 2 June 2010 in Wiley InterScience (www.interscience.wiley.com).

C V 2010 Wiley-Liss, Inc.

1906 ELINSON AND KEZMOH

CONSTRUCTING A MOLECULAR HAECKEL: DESIGNING A MORPHOLOGICAL FRAMEWORK

To create a Molecular Haeckel, we will place regulatory molecules, common among vertebrate embryos, onto a schematic morphology. The rst step is to design the morphological framework of an idealized, generic vertebrate embryo at an early developmental stage. There are a large number of common features of vertebrate early embryos that can be incorporated into the framework. This collection of common features has been used to generate concepts such as the basic or primitive vertebrate body plan (e.g., Arey, 1924, 1954), the pharyngula (Ballard, 1981), the phylotypic stage (Sander, 1983, as cited in Sander, 2002; Slack et al., 1993), and the developmental hourglass (Elinson, 1987; Duboule, 1994). This generic vertebrate embryo has three germ layers arranged in concentric tubes and a set of dorsal axial structures. In the trunk and post-anal tail, the dorsal axial structures consist of a spinal cord, underlain by a notochord and anked by segmented, mesodermal somites. Completing the mesodermal tube on the lateral and ventral sides are intermediate mesoderm and lateral plate mesoderm, split into an inner splanchnic layer and an outer somatic layer. Four limb buds arise from the somatic layer. The mesodermal tube surrounds the inner endodermal tube, which joins the outer ectoderm at the mouth and the anus. Various evaginations of the endodermal epithelium foreshadow organs such as the liver and pancreas. The anterior end of the embryo is highly modied to form the head. The neural tube is expanded to form the brain; sense organs arise from ectodermal placodes and connect to the brain, and much of the prospective craniofacial cartilage is derived from cranial neural crest. The important roles played by placodes and cranial neural crest led to Gans and Northcutts (1983; Northcutt, 2005) intriguing hypothesis that the evolution of vertebrates involved the addition to an ancestral chordate of a new head from these two embryonic cell types.

This generic embryo looks similar to actual embryos to a greater or lesser degree. There are three major sources of deviations. First, different vertebrate embryos have different amounts of yolk, ranging from no yolk in placental mammals to huge amounts of yolk in birds. Furthermore, yolk may either be included within the embryos body, as in amphibians, lungsh, teleosts, and sturgeon, or contained in an extraembryonic yolk sac attached by a stalk to the body, as in shark, coelacanth, birds, and reptiles. When images of embryos are modied to remove the yolk, they look much more similar (Richards, 2008, p. 307). The second major source of deviation between the generic embryo and actual embryos is the relative size of parts of the embryo. The size differences can be quantitative or qualitative. The most obvious quantitative difference is the number of somites in the trunk (Richardson et al., 1998b; Woltering et al., 2009; Gomez and Pourquie, 2009). The frog X. laevis has only nine trunk somites (Tucker and Slack, 1995), whereas animals with elongated bodies, such as eels, caecilian amphibians, and snakes, can have hundreds of somites (Gomez et al., 2008; Woltering et al., 2009). Chick and mouse have 39 and 35 non-tail somites, respectively (Burke et al., 1995). An example of a qualitative difference is the size of the head relative to the body. Amniote embryos generate anterior structures, including the head and heart, precociously relative to the trunk and tail, and these structures are disproportionately large. Within amniotes, embryos of non mammalian amniotes have much larger eyes proportional to the head, than mammalian embryos (Jeffery et al., 2002). There are many examples of size differences in limb development. Marsupials have very large forelimbs early to enable the embryo to crawl to the teat. Conversely, tadpoles of frogs have practically no limb buds until they begin feeding, and after that, the limbs remain small and inconspicuous. The third major source of deviation between the generic embryo and actual embryos is the relative timing of development of one part relative to another. For example in some embryos, forelimb

buds arise before hindlimb buds, while hindlimb buds appear rst in other embryos (Bininda-Emonds et al., 2007; Richardson et al., 2009). These heterochronies are rampant (Richardson, 1995), complicating comparisons between different embryos and muddying the concept of a common phylotypic stage. Analytical schemes have been proposed not only to detect heterochronies between embryos of different animals but also to provide measures of differences when comparing different embryos (Smith, 2001; Schlosser, 2001; Jeffery et al., 2005; Maxwell and Harrison, 2009; Werneburg, 2009). Whereas heterochrony makes it difcult to compare whole embryos of different animals, the concept of modularity obviates difculties in comparisons at lower levels of organization. Modularity refers to the integrated and autonomous behavior of a molecular pathway, a group of cells in a tissue, or even a developing organ (Schlosser and Wagner, 2004). For example, many cellular and molecular interactions occur autonomously within a developing limb bud, without reference to the rest of the embryo. Similarly, a relatively small number of transcription factors serve as master regulatory genes for a tissue, like skeletal muscle, or an organ, like the pancreas. Once these master regulatory genes are turned on, a cascade of molecular and cellular events follows to generate the tissue or organ. Given modularity, we will place key regulatory molecules that operate within a tissue or organ on our morphological framework. Essentially, the embryo is depicted as a mosaic of gene expression territories, largely independent of each other. In one sense, we are simply shifting the concept of a common embryonic stage from one level of organization, the whole embryo, to a lower level of organization, the individual tissues and organs. By doing so, we suggest that molecular commonality of tissues and organs is stronger than the morphological commonality of the whole embryo at the phylotypic stage. We suggest that our molecular pictures have core truths, as Haeckel claimed for his morphological pictures.

Developmental Dynamics

Developmental Dynamics
TABLE 1. Genes Involved in Development of Vertebrate Embryosa

Basal to teleost

Teleost zebrash (unless noted) Others

Anuran Xenopus (unless noted)

Bird chicken

Mammal mouse

Epidermis BMP 283 407 376 94 p63 215 234 426 203, 204 Tfap2c/TFAP2g/AP-2g 157 134, 157 141, 157 Epidermal appendages scale feather hair, tooth, etc. reptile scale ectodysplasin pathway 86 (s), 201 (m) nr 171 259 106 (Eda Edar Edaradd) Placodes Signals - 344, 379. Hypothesis: Fgf, (-)BMP, (-)Wnt but data mainly chick, Xenopus only. Six 1/2, Six 4/5 (TX) 24, 201 344 379 344, 379 Eya 1/2 344, 379 344 379 344, 379 Nervous System Similar AP patterns in the hemichordate Saccoglossus (197, 230, 231), the urochordate, various ascidians (254, 342), the cephalochordate Amphioxus (160, 161, 342), and the agnathan lamprey (274, 342) Forebrain 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, skate 219 18 (r), 186 185 185, 419 419-human Tbr (T brain - Tbr1/ 263 253, 333 253 253 Eomes/Tbr2) Emx1 102-dogsh 253 253 253 253, 419 Arx 265 352 84 265, 419 419-human Lhx5 (Lim5) 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, dogsh 253 253 253 253, 419 419-human Msx1 108, 307 (different 383 23, 323 msx) Eye Rx 15, 15(m), 381(a) 15 15 15 Pax6 253 253 253 253, 419 419-human Lhx2 9 419 239 419 Vax 387 17 347 17, 419 Barhl2 325 325 325 Midbrain Otx1/2 219 186 185 10, 185 Barhl1 325 325 325 229-human Lmx 1a/b 291 - Lmx1b 145 - Lmx1b 8, 10 8, 10 En2 109, 120 152 10, 185 Msx 1 108, 307 (different 383 8, 10 8, 10, 23, 323 msx)

MOLECULAR HAECKEL 1907

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TABLE 1. (Continued) Teleost zebrash (unless noted) 397 397 327, 397 185 185 185 185 185 185 185 (unless noted) chicken mouse Others Anuran Xenopus Bird Mammal

1908 ELINSON AND KEZMOH

Basal to teleost

397 185 185 134, 301 193 342, 420 281 281 281 174 240 240, 342 152 185 325 325 133 133 133 in hindbrain but not all; common element RA.) 383 23, 323

229-human

(134 b4 No) 134 92 92 92 92 212 134 199 134, 228 228 228 228 228 112 (co) 91, 91, 92 91, 91, 92, 92

91, 237

52 52 52 52 52 52 52 52 237 325 222 23, 74, 323 74 74, 360 360 360, 224 (ra) 319 103, 250 177 309, 360 309

Midbrain/hindbrain boundary Otx2 326 Gbx2 326 gbx1 Wnt1 Fgf8 324 Hindbrain Gbx2 326 gbx1 Hox 1,2,3,4,(5) 317, 342 Krox 20 296, 342 Kreisler (Kr/mafB) 266, 342 En2 109, 120 Barhl1 325 RA 133 (133 - expression patterns of RALDH2, CYP26s, RARs, CRABPs. Most expressed Msx1 108, 307 (different msx) Spinal cord - AP Hox4 316 Hox5 316 Hox6 4 (s), 316 Hox7 316 Hox8 316 Hox9 4 (s), 316 Hox10 4 (s), 316, 403 Hox11 403 Hox12 403 Hox13 403 Raldh2 nr Barhl2 Spinal cord - DV Msx1(roof plate) 108, 307 (different msx) Lmx1 (roof plate) nr Math1 300, 413 (atonal/Cath1/Xath5a?) Ngn1/2 28 Mash1 6 383

Tlx3 (Rnx/Hox11L2) Pax7 Lbx1 Lhx1/5 (Lim1/5)

Pax2

213 348, 363 (c) 279, 289 399-lim1 398-Lim5 258-Pax2a

(247 nr) 74 386-Ath3 74, 421 195-Xath1 not spinal cord 236 421 439-Xash3 180, 421 119 -Xash1 not spinal cord 302 225, 421 66 181, 421 243 255, 421 385-Lim1 360-lim1, 421 398-Lim5 151 421

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TABLE 1. (Continued) Teleost zebrash (unless noted) 137 251 217 318 393 63 218 218 416 341-Nkx2.2b 378 378 378 96 378 349 131 145 22 153 329-Xhox3 436 39 36 338 337-XeNK-2 110 331 181, 421 421 181, 421 181, 421 421 181, 421 421 91, 402, 421 389, 421 181, 421 378 330, 378 103, 250 69, 319 103, 250 103, 250 250 103, 250 437 103, 250 12, 103 103, 250 378 103, 378 (unless noted) chicken mouse Others Anuran Xenopus Bird Mammal

Basal to teleost

Dbx1/2 Lmx1b Irx3 Pax6 Evx1/2 Nkx6.1 Olig2 Isl1/2 Hb9 (Mnx1/Hlbx9/MNR2) Nkx2.2 Shh (oor plate) Foxa1/2 (Hnf3a,b) (oor plate) ntn1 (netrin) (oor plate) Neural crest Induction signals (257) BMP 150, 322 150, 172, 322 108, 307 (different msx) 257 257 257 257 150, 172, 257 150, 172, 257 172, 257 172 32 (cranial, trunk) (32 activity) 150, 172, 257 150, 172, 257 74 150, 172, 322 150, 172, 322 (172 no, 150?) 172 23, 74, 323

339-lamprey 160-amphioxus 339-lamprey 150, 172, 322 150, 172 383

Wnt FGF Msx1 (neural epidermal boundary) Neural crest speciers (257) AP2a Snail, Slug 172 150, 172 150, 172 150, 172 172 172 190, 310 310

160-amphioxus

339 172, (339-lamprey, 172, 295-hagsh but not NC specic) 339-lamprey, 295-hagsh 172, 339-lamprey 172, 339-lamprey 130 339-lamprey 276 261

150, 172, 257 150, 172, 257 150, 172, 257 150, 172 172, 257 172 83, 310 83, 209, 310

Sox 8/9/10 (Sox E) FoxD3 Twist Ets1 Cranial neural crest Ednra (ETA) Edn1 (ET-1) (pharyngeal arch paraxial meso epithelium) Trunk neural crest Ednrb Edn3 (ectoderm) Notochord Shh 299 374

310 310

310

249-human 249-human 138, 244 34, 38 38 (Note 404: Xenopus Shh & Ihh both in Ihh family)

MOLECULAR HAECKEL 1909

Somite segmentation Pre-somitic mesoderm (PSM) Notch FGF

163 81, 100, 368

369 227

81, 100, 368 104

99, 184 99

418-human 135-snake

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TABLE 1. (Continued) Teleost zebrash (unless noted) (425 nr) 220 53, 401 297 19 135 76 198 nr 104 19 81, 100, 368 61, 290 57, 100 81, 100, 368 45 122 135-snake 135-snake 135-snake 336 184 45 135 315,320,114 (m) (not cyclic) 163,114 (m) 353 135 269, 340, 392(m) (164, 282 Tbx24) 188, 269(f) 187, 269(f) 353 315,320,114 (m) (not cyclic) 269, 270 53, 401 (425 nr) 155 154, 269 269, 369 81, 100, 368 14, 99 122 135-snake 135-snake (unless noted) chicken mouse Others Anuran Xenopus Bird Mammal

Basal to teleost

Wnt/bcatenin Lfng

1910 ELINSON AND KEZMOH

135-snake 135-snake 418-human

Hairy (Her/Hes/HEY/esr) paraxis (TCF15) Msgn1 PSM/Somite border Mesp2 (Mespb, thylacine1) Tbx6 Ripply (bowline) Somite Raldh2 (RA) paraxis (TCF15) Lfng

38 38 38 38 38, 60 38, 60 144

38 38

147 242 66 60, 166 60, 167 88 243 98

38 34, 34 34, 34, 34, 34 34, 34, 34 34, 37

38, 75 38, 75 38, 43, 75 43 38, 43, 60, 75 38, 43, 60, 75 116 144 89

134 46

423-caecilian 423-caecilian 423-caecilian 228 228 228 228 423-caecilian

Somite patterning Wnt4, Wnt6, Wnt7a (ectoderm signal) (374-nr) Wnt1, Wnt3a (dorsal neural tube signal) (374-nr) BMP4 (lateral meso) (374-nr) Pax1 (sclerotome) 264(m) Pax9 (sclerotome) 285, 264(m) Pax 3 (dermomyotome) 374 Pax 7 (dermomyotome) 374 MyoD (MRF) 374 Myf5 (MRF) 374 Sim1 350 Lbx1 144, 279 scleraxis - tendon TX Somite AP Hox3 Hox4 Hox5 4(s) Hox6 (level of forelimb) 316, 4(s) Hox7 316 Hox8 316 Hox9 316, 4(s) Hox10 (level of hindlimb) 316, 403, 4(s) Hox11 403 Hox12 (start of tail) 403 Hox13 403 (423 cornsnake OK for Hox 3,4,5,10,13; order differences for Hox 6,7,8,9) 128 46, 129 46, 129 46, 129 46, 129 46 46 46 46 46 46

128 46, 129 46, 129 46, 129 46, 129 46 46 46 46 46

423-caecilian

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TABLE 1. (Continued)

Basal to teleost 245, 284 377 223 (Dlx5,6) 49 49 268 (ecto, meso) 49 377 223-human

Teleost zebrash (unless noted) Others

Anuran Xenopus (unless noted)

Bird chicken

Mammal mouse

48 (newt, meso)

362, 430 144 226, 377 112, 256 226, 377 97, 226 334, 364 1, 107, 294 107, 312, 375 107, 294 107, 294 107, 294 1, 26, 44, 107

396-modied in axolotl

Limb initiation, outgrowth, patterning Fgf10 (mesenchyme) 256 429 245, 284 Fgf8 (distal epidermis 256 78 377 AER) Dlx (AER) 286 (Dlx2a) nr 118 (Dlx5) Wnt7a (dorsal ecto) 286 79 (ecto, meso) 49 Lmx1b (dorsal meso 251 (?location) nr 49 amniotes) R-fng 320-nr 79 (ecto, meso) 49 (dorsal ecto) En1 (ventral ecto) 286 79 126 Shh (ZPA - posterior 90-skate, shark 5, 206 117,377,148 (co) 377 margin) See 404 Fig 2 for Shh 93-paddlesh overview Hox 913 (nested 123-catshark 77, 228 277 expression) 93-paddlesh Lbx1 144, 279 (243 nr) 144 Forelimb - n/wing/arm Tbx5 390-dogsh 226, 332, 388 377 226, 377 Raldh2 (RA, initiation) 112, 256 112 (co) 112, 256 Hind limb Tbx4 390-dogsh 226, 332, 388 377 226, 377 Pitx1 354 (s) 59 226 Heart Raldh2 (RA) 189 85 156, 364 Nkx2.5 64, 294 82, 139, 294 1, 42 Tbx5, Tbx20 127, 312, 384 40, 169 41, 311, 312 GATA 4,5,6 294, 305 294, 305 182 HAND 1,2 11, 294, 427 11, 294 371 Mef2c 394 7 Islet 1 (Isl1) Second heart (173 neuronal; 36 26, 44 eld (SHF) heart nr) Kidney Pronephros is examined in zebrash and Xenopus, while metanephros is generally examined in chick and mouse. The comparison seems justied, given the commonality of key gene expressions (105, 170, 183, 406). Osr1/2 391 391 179, 380 Raldh2 (RA) 13, 55 29 Lim1 399 54, 58 Pax2/8 170, 350 54, 55 35, 105 GDNF/Ret 170, 356 210 165 WT1 170, 356 410 51, 191 Stomach mesenchyme, musculature 178, 366 55 105, 400 35, 55, 105 87 105, 207

MOLECULAR HAECKEL 1911

191-alligator

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TABLE 1. (Continued) Teleost zebrash (unless noted) 273, 370 262 278 365 ?mesen 280 194, 260 405 (unless noted) chicken mouse Others Anuran Xenopus Bird Mammal

Basal to teleost

1912 ELINSON AND KEZMOH

Barx1 Bapx1 (Nkx3.2/Xbap) Digestive tract Nodal Mix GATA 136 62 62-Sox17 136, 440 175 357 357 275 125 238 136, 440-Sox17 136, 440 136, 440-Mixl1? 136, 440 136, 440-cyc/sqt 136, 440 136, 440 GATA5 136, 440 casanova 136, 440-Foxa2 136, 440 56 273-ant gut 136, 440 136, 440 136, 440 GATA4/5/6 136, 440-Sox17

Sox-F

FoxA Stomach Sox2 Odd1 Intestine Cdx1 Cdx2 (more important) 67,71,121-cdx1b 56-Xcad2 124-CdxA 67,71,121-cdx1b 56-Xcad1 241-CdxC Zebrash Cdx2 lost; cdx1b functions like Cdx2 (67, 71, 121, 272). (408 Shh only 176 328 esophagus; BMP nr) 403 228 20, 428 95 95, 111 95-Pax2 95 nr nr 95 306 16 292 65-Fgf 440 205 159 287, 440 298, 308, 372 149 168, 303, 424 433 nr nr nr 433-Hex 372 149 196 95

20, 202 95 95 95 95 27, 73, 409 27 140 431, 435 214 216, 355, 417 216, 355, 415 47, 431, 435 246, 267 149, 132 132, 142

Shh (endo) induces BMP (meso) See 404 Fig 2 for Shh overview Hox13 (cloaca, anus) Thyroid gland Titf1/Nkx2.1 (TTF-1, T/ 95, 314 EBP) Foxe1 (TTF-2) Pax2/5/8 314-Pax2a (Pax8 (mammal) but related Pax2 and Pax2a in zebrash, Xenopus). Hhex 95, 314 Thymus Pax1/9 (Pax1-nr) (Pax9-thymus nr) Foxn1 16-catshark 346, 16(m) Parathyroid gland Gcm2 292-dogsh 158, 292-ecto Liver Fgf, BMP 359 Foxa (HNF3) nr C/EBP 235 HNF4 72 Hex, Prox 1 288 Pancreas RA 373, 395 Shh signaling 80, 149, 395 repression phase Pdx1 25, 149, 395

MOLECULAR HAECKEL 1913

CONSTRUCTING A MOLECULAR HAECKEL: CHOOSING GENES

The genes that are used to construct the pictures are regulatory genes that are expressed in a specic organ or tissue in embryos across a range of vertebrates.
Others

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 Zebrash ngn3 not expressed in pancreas. Given teleost genomic duplication, there may be other orthologues, not yet cloned (412). Lungs/swim bladder Foxp1/2/4 70-p1 (345-p1a/2/4 nr) 143-p2 361-p1/2 zebra nch adult lung but no mention embryo (33-p2 no) (313-p1 nr) 232-p4 Irx1/2 (68, 414-nr) 21 Titf1/Nkx2.1 (TTF-1, T/ (115-nr) 162 304 30 EBP) Prdc 271 nr 233 Foxa1/2 (Hnf3a,b) 422 31, 331-ant endo 330 50 Hb9 416, 422 (338-nr) (389-nr) (272-nr)

Range of Vertebrates
Gene expression and function in development are known mainly from embryos of four model animals: zebrash, Xenopus, chick, and mouse. Chick and mouse represent the monophyletic Amniota; amniotes plus Xenopus represent the monophyletic Tetrapoda, and tetrapods plus zebrash represent the monophyletic Osteichthyes. If a gene is expressed in both zebrash and mouse embryos, it suggests that their last common ancestor had that organ specic gene expression and that all animals within Osteichthyes are expected to have that gene expression. Gene expression in both zebrash and mouse embryos was our usual minimum criterion for inclusion in the Molecular Haeckels. Of course, if Xenopus and chick also have that gene expression, the more likely it is that it represents a common character. Some embryonic gene expressions have been reported for other teleosts such as medaka, fugu, and stickleback, other amphibians such as axolotl and caecilian, and other amniotes such as alligator, snake, and human. A few embryonic gene expressions have been reported for non-teleost Actinopterygians, such as bichir (Takeuchi et al., 2009) and paddlesh (Metscher et al., 2005; Davis et al., 2007), indicating that these embryos are available to strengthen any conclusions of commonality based on teleosts. Some gene expressions have been reported in embryos of Chondrichthyes, the cartilaginous sh. These include reports on catshark, dogsh, and skate (Neyt et al., 2000; Derobert et 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 in cartilaginous shes, the Molecular Haeckels can be drawn to include all jawed vertebrates, the Gnathostomes.

Mammal

Developmental Dynamics

chicken

Bird

mouse

m, medaka; s, stickleback; c, charr; a, blind cave sh; f, fugu; r, rana; co, coqui; ra, rat. Items in red highlight differences across animals or absence of information (nr). Rows in gray indicate sufcient differences or absences to exclude the gene from the Molecular Haeckels. Numbers refer to references in the Supporting Information.

Anuran Xenopus Teleost zebrash

TABLE 1. (Continued)

Basal to teleost

(unless noted)

(unless noted)

1914 ELINSON AND KEZMOH

TABLE 2. Ortholog Differences in Transcription Factor Gene Expression Gene Pax8 Cdx1 Cdx2 (major) Tbx6 Msx1 Math1 Mash1 Gbx2 Tbx4 Tbx5 Hox69 Speciesa m m m m, m, m, m, m, m, m, m Tissue Thyroid Intestine Intestine PSM/somite border Brain, spinal cord roof plate Spinal cord Spinal cord Brain Hindlimb Forelimb Thoracic somites Difference Xenopus Pax2, zebrash Pax2a Xenopus Xcad2, zebrash cdx1b Xenopus Xcad1, zebrash cdx1b Zebrash Tbx24 Zebrash msxB,C,E Xenopus Ath3 Xenopus Xash3 Zebrash gbx1 Newt Tbx5b Newt Tbx4b Snakedifferent order

x, c x, c c c, z x, c x, c, z x, c, z

See Table 1 for references. a m, mouse; c, chicken; x, Xenopus; z, zebrash. b Khan et al. (2002) report differences in Tbx expression in newt limbs. There are other differences reported for limbs of other urodele amphibians. These include Fgf-8 expression in the mesenchyme of axolotl limb buds, rather than the apical ectodermal ridge (Han et al., 2001), and differences in Shh expression (Stopper & Wagner, 2005).

Developmental Dynamics

Regulatory Genes
Genes were chosen, primarily based on expression patterns. A more stringent requirement for inclusion would be demonstrations of function in the development of a particular tissue or organ. In many cases, function was shown in one model animal by the most convenient method, i.e., mouse knockouts, zebrash mutants, or Xenopus overexpression, and expression was reported in other animals. For the gene collection used here (Table 1), gene expression was a sufcient criterion for inclusion. The two main classes of regulatory molecules are transcription factors and components of signaling pathways, usually the ligands. It was easier to include the transcription factors compared with ligands, although that may be partially an artifact of naming conventions. Both types of molecules are composed of families, whose members are often numbered. For example, there are nine Pax genes and more than 20 broblast growth factor (FGF) genes (Itoh, 2007). There are more transcription factor families compared with ligand families, of which there are mainly Wnt, FGF, Hh, Delta-Notch, and transforming growth factor-beta (TGFb). The TGFb superfamily is further split into bone morphogenetic proteins (BMPs), nodals, growth differentiation factors (GDFs), and others. Another important ligand is retinoic acid, which is

generated by several different enzymes. For many tissues and organs, activities of several signaling families are involved at different times and in different places. Sorting out whether a particular ligand or a different ortholog is used in different animals for the same activity is a major task, even in well-known cases. For example, Shh is expressed in the notochord, the oor plate, and the zone of polarizing activity of the limb, and there is considerable functional analysis. Complicating its placement in a Molecular Haeckel is the fact that the Xenopus Shh ortholog falls into the Ihh family phylogenetically (Varjosalo and Taipale, 2008). Because of the usage of multiple ligands in many tissues, less emphasis was placed on ligands than on transcription factors. Not only are there more transcription factor families, the conserved expression patterns of differently numbered family members are often well-documented. One example is the expression of the Hox genes in the spinal cord, the somites, and the limb. A second example is the tissue and organ specic expression of Pax and Tbx genes. There are interesting exceptions, however. For example, Pax8 is important for mouse thyroid development, but the related Pax2 and Pax2a are used in Xenopus and zebrash respectively (Table 2). Different Msx and Gbx members are

expressed in zebrash brain development compared with the tetrapods (Table 2). Other cases of differences of ortholog usage between zebrash and tetrapods are Tbx in somite segmentation and Cdx in intestine (Table 2). For the most part, this type of ortholog difference has been ignored. A future criterion for inclusion would be whether the differently numbered orthologs have the same activity; that is, can the gene from one animal substitute for a family member in a different species?

THE MOLECULAR HAECKELS

Our drawings are shown in Figures 17. Traditional colors have been used with blue for epidermis, green for central nervous system, light green for neural crest, red for mesoderm, and yellow/orange for endoderm. Each tissue or organ is constructed using the acronyms of the important gene expressions in the development of the tissue or organ. For some tissues, there are many gene expressions known, so the whole tissue texture is composed of acronyms. This is the case for the central nervous system, neural crest, somites, limb buds, heart, liver, and pancreas. For other tissues, only a few key expressions have been identied. These tissues are represented by a pattern with interspersed acronyms.

MOLECULAR HAECKEL 1915

Fig. 1. Ectoderm. The skin has been removed from the embryo to reveal the brain and spinal cord (green), the midbrainhindbrain boundary (turquoise) and the cranial and trunk neural crest (light green).

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Fig. 2. Mesoderm. The skin has been removed to reveal the mesoderm. The anterior somites have formed, while somitogenesis continues posteriorly. The somites, intermediate mesoderm, heart, and limb buds are composed of gene acronyms, while the lateral plate mesoderm and the mesenchyme 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 these organs are well-represented by gene acronyms. There are some common gene expressions within the endoderm, as well as some specific ones for stomach, intestine and cloaca.

For some tissues, not only is gene expression conserved but also the spatial location within the tissue of the cells expressing that gene is conserved. Hox genes are expressed with anteriorposterior polarity in the neural tube and

the somites and with proximaldistal polarity in the limb buds. Other examples are the dorsalventral pattern of gene expressions in the neural tube, the regional expressions within the somite, the posterior expression of Shh in the

limb bud, and the expressions of the limb bud ectoderm. In these cases, the placement of acronyms reected the conserved spatial locations. When viewed as reduced drawings (Figs. 14), the acronyms are not

1916 ELINSON AND KEZMOH

legible. The acronyms can be seen in the full-sized drawings, and examples of the eye, heart, liver, and pancreas are presented (Figs. 57). The complete full-sized drawings are in the Supplementary Material (Supp. Figs. S14, which are available online).

FUTURE MOLECULAR HAECKELS

Constructing the Molecular Haeckels provides a glimpse into the difculties that Haeckel must have encountered in drawing his pictures. Which details should be emphasized and which differences should be ignored? Genes were included if they were expressed during development of the same tissue or organ in a range of animals. A more stringent requirement for inclusion would be a demonstration of an important developmental function of the gene in both a teleost and an amniote. A second example is the decision to included different orthologs within a family of transcription factors, mentioned earlier (Table 2). The molecular database is much richer than the morphological one used by Haeckel. We have identied more than 160 conserved associations of regulatory genes involved in the early development of specic tissues. Many of the genes are expressed in conserved locations within a tissue, adding to the number of molecular characters than can be used. Nonetheless, there may be a bias in selecting the genes. Many of the included genes were discovered because they have a strong effect on

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Fig. 4.

Fig. 5.

Fig. 6.

Fig. 4. Cross-section at the forelimb level. Dorsoventral and other spatially restricted gene expressions are depicted for the spinal cord, somite, and both the ectoderm and mesoderm of the limb bud. Fig. 5. Eye. The eye region is presented at full-size to show specific eye expressions as well as surrounding brain expressions. The full-size ectoderm picture, from which this figure 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. 7. Liver and pancreas. The full-size endoderm picture, from which this figure was taken, is Supp. Fig. S3.

Fig. 7.

MOLECULAR HAECKEL 1917

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development in one species. Once that was known, its ortholog was cloned and analyzed in other species. This search process is biased toward commonality, the very feature highlighted by the Molecular Haeckels. Future data, generated by microarrays, should give unbiased pictures of genes expressed in particular developmental events in different animals. The problem then becomes identifying gene expression differences that have signicant functional differences between animals. There are several ways to advance the Molecular Haeckels. One way is to design them using the enlarging data base of gene regulatory networks (GRN). Rather than individual gene expressions, tissues would be constructed with conserved pathways and molecular interactions. Giudice and Onorato (2003) provided a prototype for this type of image. A second way to advance is to include timing and the order of gene expression. That would require computer animations of Molecular Haeckels. Haeckels pictures have not been forgotten after a century, because they contain a core truth of commonality among vertebrate embryos. These commonalities are more apparent than ever.

ACKNOWLEDGMENT
The production of the drawings was supported by a NSF grant to R.P.E.

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