Download Chordate Evolution and Autonomous Specification of Cell Fate: The

AMER. ZOOL., 37:237-249 (1997)
Chordate Evolution and Autonomous Specification of Cell Fate: The
Ascidian Embryo Model1
J. R. WHITTAKER
Department of Biology, University of New Brunswick, Fredericton,
New Brunswick E3B 6E1, Canada
TWO parallel themes emerge in the history of the investigation of the
ascidian tunicate [Urochordata] embryo: the realization that the larval stage is
probably a surviving example of the earliest chordate body plan from which vertebrates arose, and secondly the unusual degree of autonomous specification of cell
fate involved in the development of ascidian larval parts. Such developmental autonomy in larval structures results in patterns of development referred to as "mosaic." This paper follows the progress of these two themes from their beginnings
in the second half of the nineteenth century to their status at the present time.
Romer's concept of vertebrates as a "dual animal" (somatic and visceral) stands
out as a landmark perception in support of the theory of vertebrate origin by
paedomorphosis through a merger of the pelagic larval and benthic adult stages
of a tunicate-like animal. The present contribution attempts to unite the two themes
by postulating that autonomous specification further enhanced the modular nature
of the developing tunicate embryo and permitted natural selection to act differentially on the largely independent organ systems of larvae and paedomorphs, in
what amounts to a mosaic selection pattern. This, in turn, favored the very rapid
emergence and radiation of the chordates during the Cambrian explosion.
SYNOPSIS.
INTRODUCTION
During the past century, there have been
two major parallel lines of inquiry related
to the larval development of ascidian tunicates (phylum Chordata, subphylum Urochordata, class Ascidiacea). One of these
has been the discovery of the tunicate larva
and steady progress in uncovering particulars of ascidian larval structure. Many of
these details prove to be features of chordate affinity which suggest in turn that the
present day ascidian larva preserves the
primitive chordate body plan from which
vertebrates arose in the course of evolution
(Garstang, 1928; Berrill, 1955). The other
line of very long-time inquiry, recently
summarized by Satoh (1994), is a progression of findings about the developmental
regulation of the ascidian larval form.
These observations and experiments indicate a relatively autonomous specification
in the embryonic development of the strict-
ly larval features, which later in evolution
become the major chordate features of vertebrates.
This essay reviews certain highlights in
these two streams of investigation and discovery and suggests how the findings may
intersect. Their convergence lies in the possibility that the autonomous specification
mechanisms found strongly expressed in
early larval development may be one of the
two major reasons, including paedomorphosis, for the rapid evolution of higher
chordates, and hence of vertebrates, from a
urochordate larva-like common ancestor.
A CHORDATE-LIKE LARVA
Larvae of the ascidian tunicates were
known for some time before their internal
anatomy was studied histologically by Alexander Kowalevsky (1866). One of the
first descriptions and illustrations of an ascidian larva appeared in Henri Milne-Edward's (1842) monograph on colonial as1
From the Symposium Forces in Developmental Bi- cidians. In it he described and depicted
ology Research: Then and Now presented at the Annual Meeting of the Society for Comparative and In- some of the embryonic, larval and metategrative Biology, 26-30 December 1995, at Washing- morphic stages of Aplidium (Amaroucium)
proliferum which he and J. V. Audouin had
ton, D.C.
237
238
J. R. WHITTAKER
first recorded in 1828. Between 1816 and
1850, at least four other investigators independently published figures depicting the
ascidian larva: J. C. Savigny, Michael Sars,
John Dalyell, and Louis Agassiz. Charles
Darwin had studied the larvae of a colonial
ascidian in 1833 during his voyage on the
H.M.S. Beagle (Darwin, 1871).
The findings of Kowalevsky (1866,
1871) concerning the internal structures of
the ascidian larva and their embryonic development came as a surprise to the zoological community. At the time there was
no controversy or even great puzzlement
over tunicate affinities. Most authorities, including Milne-Edwards and the venerable
Karl von Baer, believed them to be allied
most closely to the Mollusca, as suggested
by the classifications of Cuvier and Lamarck. Perhaps for this reason no one of
note, except for Ernst Haeckel (1868), paid
any immediate attention to the 1866 publication which claimed ascidian larval similarities to vertebrates.
Kowalevsky's initial and subsequent
claim was to have found the mode of development of the ascidian larva {Phallusia
mammillata and Ciona intestinalis) to be
closely similar to that occurring in lower
vertebrates and amphioxus. The tadpoleshaped larvae of both species had a long
motile tail with lateral muscle bands, and
an internal rod-shaped structure which was
notochord in its appearance, function and
mode of development. Not until 1871 did
Kowalevsky correctly describe the larval
nervous system and its formation, as a dorsal tubular design formed by a vertebratelike neurulation process, and depict the subnotochordal endodermal strand. Meanwhile
Kupffer (1869, 1870), using Ciona intestinalis, had also described the formation of the
anterior brain vesicle and posterior hollow
nerve cord and confirmed others of Kowalevsky's observations. The presence in the
ascidian larva of an obvious notochord and
a dorsal tubular nervous system, as well as
its general resemblance to the vertebrate
body plan was compelling evidence that ascidians were primitive relatives of the vertebrates.
After Kupffer's confirmation of Kowalevsky's findings, they became so widely
accepted that by 1871 Charles Darwin was
able to write in The Descent of Man, and
Selection in Relation to Sex, " . . . we have
at last gained a clue to the source from
whence the Vertebrata have been derived.
We should thus be justified in believing that
at an extremely remote period a group of
animals existed, resembling in many respects the larvae of our present Ascidians,
which diverged into two great branches—
the one retrograding in development and
producing the present class of Ascidians,
the other rising to the crown and summit of
the animal kingdom by giving birth to the
Vertebrata."
Ascidians are, however, a dimorphic
form in which a non-feeding pelagic chordate-like larva or tadpole bears essentially
no resemblance to the sessile filter-feeding
adult ascidian. Since deuterostomes in general have these bentho-pelagic life cycles
(Jagersten, 1972), there is good reason to
believe that the ascidian larva evolved from
a previous larva rather than some earlier
adult form (Crowther and Whittaker, 1992).
The tailed larva and its nervous system has
presumably evolved at a very early geologic time and become adapted to serve the
adult needs of dispersion and site selection
(Berrill, 1955). Nonetheless, the adult ascidian shares at least two important characters with chordates, namely the gill openings in their branchial basket which develop
from prostigmata that are the likely homologues of vertebrate gill slits (Garstang,
1928), and an endostyle (ciliated groove) as
part of the filter-feeding apparatus. This endostyle occurs also in the ventral pharyngeal region of Branchiostoma (=Amphioxus) and the ammocoete larva of lampreys
(cyclostomes). When the ammocoete metamorphoses into the adult lamprey, some of
the endostyle cells are seen clearly to become part of the vertebrate thyroid gland
(Garstang, 1928).
A characteristic of the class Ascidiacea is
a thick outer mantle or tunic surrounding
the adult and which distinguishes the group
from other invertebrate taxa; this tunic contains a unique cellulose-like substance, tunicin. The sedentary, benthic and usually attached adult is essentially a "visceral" animal engaged in efficient filter feeding, food
ASCIDIAN EMBRYO MODEL
processing and reproduction. In many general ways the internal organs of adult tunicates serving these functions appear homologous to those of lamellibranch molluscs. Because of these organ similarities,
von Baer (1873) and some others never became reconciled to tunicates as chordates,
preferring instead to discount completely
the significance of ascidian larval anatomy.
Chordate phytogenies based on 18S ribosomal RNA gene sequence comparisons
(Turbeville et al, 1994; Wada and Satoh,
1994) and cladistic analysis of morphological and developmental features (Maisey,
1986; Schaeffer, 1987) affirm the central
position which urochordates occupy in
most theories of chordate evolution. The
cephalochordates and vertebrates are regarded as sister groups, and the urochordates (ascidians, larvaceans, salps) are the
sister group of the cephalochordate/vertebrate line. Whether the wholly pelagic larvaceans (Appendicularia) actually preceded
the ascidians in origin, as suggested by
Wada and Satoh (1994) and others, remains
an unresolved question. There are good
morphological and life cycle reasons in
support of larvacean derivation from a neotenized ascidian larva (Nielsen, 1995). In
addition, an 18S rDNA database is probably
insufficient in sequence complexity to resolve cladogenetic events separated by less
than about 40 million years (Myr) (Philippe
et al, 1994). The several tunicate groups
presumably originated within a time interval of less than 20 Myr (last section below).
CHORDATE CHARACTERS OF THE ASCIDIANS
The list of possible chordate traits in the
urochordate subphylum, especially those
evident in the ascidian larva, has become
progressively longer in the century and a
half since discovery of the larva. Comprehensive lists and discussions of these chordate characters have appeared at various
times, most recently by Katz (1983), Maisey (1986), Schaeffer (1987), Cripps (1990),
and Nielsen, (1995).
The ascidian larva has a bilaterally symmetrical body plan with definite head and
tail ends that is notably similar to that seen
in lower vertebrates. Except for lacking
segmentation and an obvious coelomic
239
body cavity, it looks much like the ammocoete larva of lampreys, and the larval
Branchiostoma. Ascidian larvae are tadpole-shaped with an anterior cephalic region and a long tail-like extension, containing contractile lateral muscle bands, and a
central incompressible axial skeletal rod,
the notochord, running the length of posterior body. Figure 1 shows two schematic
diagrams of their structural organization.
There is a tubular nervous system, with
a hollow brain in the dorsal cephalic region
connected to a hollow neural tube which
runs dorsally along the length of the tail and
above the notochord. In the central and ventral cephalic region one finds the endodermal mass of pharyngeal rudiment and associated digestive system of the ascidiozooid. The pharyngeal mass shows early
traces of an endostyle; in larvae of some
colonial species one finds "gill slit"-like
prostigmatae arising there even before
metamorphosis. An endodermal strand of
cells connected to the main mass of the cephalic endoderm occurs subnotochordally
along the length of the tail. Most authorities
have generally regarded the whole larval
tail as a postanal tail equivalent to that of
vertebrates. In actuality, the distal fifth of
the tail is composed of cells with structural
characteristics and embryological origins
that suggest it to be the "postanal" tail homologue (Crowther and Whittaker, 1992,
1994).
Aside from the notochord, the nervous
system is perhaps the most impressive chordate character. A bipartite brain is divided
into an anterior prosencephalon and a posterior deuterencephalon (Katz, 1983). The
brain and posterior neural tube develop in
what appears to be a completely vertebrate
manner. There is formation of a neurulalike stage and closure of a neural plate to
form a tubular structure by inrolling of the
plate of neuroectodermal cells. Even the
mode of primary neural induction is topographically similar to that of amphibians. In
each case cells in the roof of the archenteron induce formation of neural plate tissue
in competent ectoderm although it is different cells in each taxon: notochord and
endoderm in ascidians, mesoderm in amphibians.
240
J. R. WHTTTAKER
CAUDAL NEURAL TUBE
BRAIN
visceral ganglion
sensory vesicle
ocellus (pineal eye)
statocyte
cavity
neurohypophysis
B
endodermal
strand
endostyle
ENDODERM
FIG. 1. Schematic diagrams of organ systems within the hatched larva of Ciona inteslinalis. A. Structures seen
without the enveloping cephalic endodermal tissues. B. Endodermal tissues in relation to the notochord.
In certain minor details the nervous system of the ascidian larva is strikingly similar to other chordate nervous systems. Ascidian larvae have tubulated bulb cell organs protruding into the brain cavity that
appear to be the homologues of coronet
cells in the saccus vasculosus of fishes
(Svane, 1982); ascidians, as well as Branchiostoma and vertebrates, have Reissner's
fibre in the canal of the posterior neural
tube (Olsson, 1972). Also within the ascidian brain wall is a dorsal eye structure (the
ocellus) projecting into the brain cavity;
this has been homologized by Eakin (1973)
and others with the pineal eye or pineal
body of vertebrates.
The endodermal strand, a frequently
overlooked larval structure, corresponds in
position to the intestine seen in Branchiostoma and cyclostomes and may be what has
given rise to the vertebrate intestine. It
serves no apparent "intestinal" purpose in
the ascidian larva or subsequent adult.
However, the strand does retain expression
of gut alkaline phosphatase activity during
its developmental stages (Fig. 2), and has
the same cell lineage origins as other endodermal cells which ultimately form the
digestive system of the ascidiozooid (Whittaker, 1990). Ultrastructural investigation of
the strand by Crowther and Whittaker (in
preparation) confirms it to be a single row
ASCIDIAN EMBRYO MODEL
FIG. 2. Dechorionated 11-h Ciona intestinalis embryo cleavage-arrested at 11-h with cytochalasin B and
reacted at 28 hr (following fixation) for alkaline phosphatase activity with the BCIP reagent. Details given
in Whittaker (1990). Bar = 25M-m.
of about 25 cells remaining intact throughout the whole larval period. Figure 3 illustrates the location and structure of such
cells.
Two important barriers to regarding the
ascidian larva as a surviving relict of the
earliest chordate form have been the apparent lack of coelom (that is, a body cavity
lined by mesoderm) and a segmentation/
metamerism involving various parts of the
body, particularly the muscle (Willmer,
1990). Berrill (1936) has claimed the pericardium to be a remnant of the coelom, and
according to Ivanova-Kasas (1988), ascidians show traces of the same three coelomic
compartments as found in other Deuterostomia.
Crowther and Whittaker (1994) used immunocytochemical staining with anti-tubulin antibodies to demonstrate regularly
spaced cilia pairs in two rows immediately
opposite to each other mid-dorsally and
mid-ventrally along the larval tail surface
of Ciona intestinalis. These immotile cilia,
which are embedded in the matrix of the
extracellular larval test of the flattened tail
fin, originate from pairs of cell bodies in
the mid-dorsal and mid-ventral peripheral
nerves running beneath the tail epidermis.
241
Such serially repeated and equidistantly
spaced cilia pairs (approximately ten dorsal-ventral sets) possibly indicate a primitive underlying segmentation pattern preceding chordate metamerism. The presence
of functional Hox genes in ascidians (Katsuyama et ai, 1995) is also consistent with
this suggestion.
Holland and Garcia-Fernandez (1996)
have addressed some important evolutionary questions in reviewing their own work
and that of others on Hox gene diversity in
primitive chordates and lower vertebrates.
In insects and vertebrates, the Hox genes
are involved in controlling the specification
of segment identity and the linear organization of body plan. Branchiostoma clearly
has only a single and archetypal Hox gene
cluster; according to recent findings of several cluster members in ascidian tunicates,
so apparently do they. Duplications or multiple clusters of Hox genes first occur in
hagfishes and lampreys; the genomes of
jawed vertebrates (teleost fishes, mouse, human) each possess four Hox gene clusters.
Hence, vertebrate evolution per se correlates with cluster duplication. Conversely,
the presence of only a single Hox gene cluster in amphioxus and ascidians is sufficient
to refute the fallacious suggestion first
made by Dohrn (1875) and later by others
{e.g., Jefferies, 1986) that lower chordates
are not primitive but originate by the serial
degeneration of vertebrates.
THE SOMATIC AND VISCERAL VERTEBRATE
ANIMAL
Romer (1972) described in reasoned detail how vertebrates combine the general
characters and features of what appear to be
two almost independent kinds of animal, a
sessile visceral feeding and reproductive
animal and a motile or somatic one. Others
before him have understood this dichotomy,
notably Garstang (1928) and Grave (1935),
but the clarity of Romer's synthesis is remarkable. He reasons that the obvious
source of this inherent duality is likely to
be a combination of the ascidian larva and
ascidian adult into one functionally integrated life cycle stage. This has been accomplished through acceleration and retar-
242
J. R. WHITTAKER
FIG. 3. Endodermal strand in the embryonic and larval tail of Ciona intestinalis. A. Light micrograph of a thin
(0.5jjim) sagittal section of a whole 11-h embryo. Stained with osmium tetroxide and cut in epoxy resin. Arrow
shows position of the endodermal strand. Bar = 25p.m. B. Transmission electron micrograph of a sagittal section
along the tail of a hatched larva showing an endodermal strand cell (arrow) below the notochord. Bar = 5|xm.
dation of certain processes at the developmental level.
According to Romer, a typical vertebrate
consists of an "external" or somatic animal
with quite functionally integrated organ
systems, including a nervous system with
sense organs as well as skeletal and muscular systems. This somatic covering interacts with the environment. A second "internal" or visceral animal consists largely
of a digestive system with its pharyngeal
and other appendages and a reproductive
system, each of which operates independently of much direct control by the somatic animal. When one looks in more detail at the following elements in the two
"animals," somatic and visceral musculature, somatic and visceral skeletal systems,
and the somatic and visceral nerve components, one notes that the differences in
the anatomical composition and embryological origins of the elements between the
two situations are so marked as to suggest
a basic dichotomy of vertebrate make-up.
"There seems to be a somewhat imperfect
welding, functionally and structurally, of
two . . . distinct beings" (Romer, 1972).
Textbooks and many monographs almost
universally favor an explanation of vertebrate evolution that invokes neoteny or paedomorphosis of a primitive urochordatelike larva in which most of the larval characters are retained and upon which the development of the more visceral
ascidian-like adult features become superimposed. This theory is attributed to Garstang (1928) and Berrill (1955). Presumably then, an amphioxus-like cephalochordate becomes one of the first stable evolutionary products of this transformation. The
neoteny theory is so popular that one critic
has remarked: "The suspicion that vertebrates had a tunicate-tadpole-like ancestor
is well founded but neoteny of such a tadpole in the origin of vertebrates has the scientific status of a creation myth" (Jefferies,
1986, p. 350).
One of the major reasons for the popularity of the neoteny theory of vertebrate
origin is that the animal kingdom abounds
in examples of form changes which can be
attributed reasonably to a neoteny or paedomorphosis (McKinney and McNamara,
1991). Within primitive chordates (am-
ASCIDIAN EMBRYO MODEL
phioxids and lampreys), neoteny is not a
rare event (Bone, 1957; Zanandrea, 1957)
and there are also many recorded examples
of ascidian larvae failing to metamorphose,
or of their metamorphosing without resorbing their tails (e.g., Berrill, 1955).
243
En
AUTONOMOUS SPECIFICATION
Two decades after Kowalevsky's investigations were laying the groundwork for
our understanding of the nature of the ascidian larva, a young trench investigator,
Laurent Chabry, began the first investigation into the so-called physiology of ascidian development. Chabry had noted in collecting ascidian embryos from the plankton
that when one of the two early blastomeres
within the chorionic membrane had become
damaged, leading to cytolysis of that cell, a
partial and incomplete embryo developed;
he realized that such embryos might possess a key to understanding the mechanisms
of development (Chabry, 1885). In pursuing the problem further for his Doctor of
Science thesis at the Sorbonne, Chabry invented the first micromanipulator for puncturing the egg chorion with a tiny lancet to
destroy selected blastomeres. He then used
it to discover the effects of destroying blastomeres in two- and four-cell stages of Ascidiella aspersa embryos.
Chabry (1887) first demonstrated that the
earliest blastomeres, from 2- and 4-cell
stages, were already specified in part for
producing the particular tissues making up
a region of the ascidian larva. His most famous diagrams depicting the result of destroying one of the first two blastomeres are
reproduced here in Figure 4A, B. Only halfgastrulae and half-larvae resulted from such
operations, a result duplicated later by
Conklin (1905) who punctured (also within
the chorion) the two cells resulting from
one of the first two blastomeres (Fig. 4C,
D). Cohen and Berrill (1936) achieved similar results after first dechorionating the embryos and actually removing the dead or inactive cells. Subsequently, Reverberi and
Minganti (1946) began a series of blastomere isolation and recombination experiments which showed even more clearly that
cell lineages in ascidians are effectively fate
maps for larval parts.
(0
FIG. 4. Results of the blastomere destruction experiments of Laurent Chabry (1887, figs. 108, 111) and
Edwin G. Conklin (1905, figs. 31, 32). (A) A halfgastrula subsequently developing after Chabry's destruction of one blastomere at the 2-cell stage. (B) A
half-larva formed from such a half-gastrula. (C, D)
Similar findings by Conklin, where two daughter-cells
were inactivated at the 4-cell stage. Ec, ectoderm; En,
endoderm; No, notochord; Np, neural plate.
Ascidian lineages themselves result from
an invariant cleavage pattern which always
segregates the same visibly distinct cytoplasmic regions of the zygote into lineages
having restricted tissue and organ fates.
This led to the conception that many aspects of ascidian larval development are
conditioned by spatially localized and subsequently segregated egg cytoplasmic "organ-forming" substances, of presumably
maternal origin. Davidson (1990) has characterized this as an autonomous specification process; he and others explain such
spatially distributed tissue determinants as
being maternally preformed gene regulatory factors or gene products of some kind.
The experimental basis of this general conception has been reviewed extensively by
Satoh (1994).
Conditional specification is dependent on
cell-cell interactions in which some kind of
inductive signal passes between cells that
causes them to change or modify their fate.
Terminal differentiation of the nervous system and the brain melanocytes of ascidian
embryos are regulated by such conditional
specification (Reverberi and Minganti,
1946). It is interesting, however, that the
competence of only certain ectodermal tis-
244
J. R. WHITTAKER
sues of ascidians to respond to inductive
signals may itself be controlled by autonomous specification (Satoh, 1994). Under
experimental conditions a modest degree of
neural expression may proceed even in the
absence of inductive interactions (Whittaker, in preparation).
For purposes of illustrating developmental autonomy, that of larval endodermal tissues will be discussed briefly. During the
late gastrulation of ascidian development,
the mass of endodermal tissues sharing
common lineages (founder cells) begins to
produce a strong histochemical localization
of alkaline phosphatase enzyme (Whittaker,
1977). The distribution of this enzyme
staining is illustrated in Fig. 2, where staining can also be seen in the endodermal
strand. Particular blastomeres of the 8- and
16-cell stages, when isolated and cultured,
result in partial embryos that produce alkaline phosphatase but only if they are blastomeres from an endodermal lineage (Whittaker, 1990). At the 8-cell stage the endodermal lineages are already restricted to the
vegetal quartet of cells and only cells from
these make alkaline phosphatase. Cytoplasmic transfer experiments, involving the fusion of different lineage blastomeres with
cytoplasmic fragments from various
regions, give results which indicate that cytoplasm from an endodermal lineage causes
non-lineage blastomeres to develop partial
embryos with strong alkaline phosphatase
activity (Nishida, 1993).
Similar kinds of experiments have now
been done with muscle, ectoderm, and notochordal lineages; their results also point
to an early autonomous specification (described by Satoh, 1994), with the odd exception of notochord. Specification of notochord cells appears to depend on blastomere interactions that occur before the
64-cell stage (Nakatani and Nishida, 1994).
This notochordal difference highlights an
important point about the determination of
cell fate. Eventually in embryogenesis, all
specification becomes autonomous in the
sense that no further information, either intrinsic or acquired by cell interactions, is
necessary to determine the ultimate organ
fate of cells. At some point in most kinds
of embryos there is a regionalization or
modularity with respect to fates. Tunicates
are unusual only with regard to how exceptionally early such specifications occur.
SELECTION AND THE LARVAL/ADULT
ACTION-SYSTEMS
In addition to his speculation about the
paedomorphic origin of vertebrates from ascidian-like larvae, Garstang (1922) made an
enduring contribution to our thinking about
larval evolution in general. He was among
the first to see that natural selection could
act just as powerfully on the developmental
or larval stages of an organism as upon the
adult end-product, especially when larva
and adult inhabit different zones of life.
Grave (1935) independently recognized a
similar dichotomy of parts in ascidians by
describing the independent changes in their
larval and adult action-systems during
metamorphosis.
The various tissues and organs of the ascidian larva, and probably of other tunicate
larvae as well, seem to have an extraordinary degree of developmental autonomy. In
this sense, their development is uncoupled,
and selection might be expected to act differentially and with considerable freedom
on variants with mutations affecting single
organs or systems. In fact, the whole of larval ontogeny, from the neurula stage onwards, can be dissociated from tissue
changes leading to ascidiozooids. This is
shown by two different kinds of observation: experimental preparations of half-larvae that result in normal ascidiozooids, and
the occurrence of so-called anural species
of ascidian in which major expressions of
the larval stage structures have been reduced or eliminated presumably by environmentally mediated selection.
Half-embryos, and subsequently half-larvae, were produced by puncturing the egg
chorion of Styela plicata embryos with a
tungsten needle and destroying one of the
blastomeres at the 2-cell stage (Nakauchi
and Takeshita, 1983). The lateral half-larvae resulting from this operation were able
to hatch, metamorphose, and ultimately develop into complete functional but smaller
ascidiozooids. Not only does this illustrate
that the adult action system has a largely
conditional specification, but further indi-
ASCIDIAN EMBRYO MODEL
cates its relative independence of interaction with larval tissues.
Some species of ascidian which find
themselves living on sand and mud flats
where wide dispersal and specific site selection are perhaps no longer likely to be
an advantage, are discovered to be developing without a larval stage. That is, the
larvae develop only about as far as the neural plate and neurula stages; most gene expressions for larval tissue-specific proteins
will be reduced or absent (Whittaker, 1979;
Jeffery and Swalla, 1990). Such structural
larval features as differentiated brain tissues, including melanocytes, muscle myofilaments and notochordal cell extensions do
not ordinarily occur. These tailless (anural)
larvae are not in any sense primordial or
more primitive. Larval loss occurs in the
quite advanced families, Molgulidae and
Styelidae; elegant 18S rDNA comparison
methods show that anural species apparently originate independently and sometimes
from sympatric tailed (urodele) species
within the same geographic area (Hadfield
et al., 1995). Anural development is polyphyletic in origin.
Perhaps the single most interesting experiment done with an anural species (Molgula occulta) was to fertilize their eggs with
sperm from a closely related urodele species (Molgula oculata). Some of the hybrid
embryos resulting from this cross developed a brain melanocyte and a short tail
rudiment containing extended notochord
cells (Swalla and Jeffery, 1990). The most
important deduction from the results of
such an interspecific hybridization experiment is that anural development may result
from loss-of-function mutants. Perhaps
some of the genes involved in suppression
of the larval features are possible "master
control genes" similar to the eyeless gene
of Drosophila which appears to control eye
formation (Haider et al, 1995). Genes encoding potential regulatory factors have already been identified with altered expression patterns in M. oculata and M. occulta
(Swalla et al., 1993).
The results of experiments with expression of lacZ fusion constructs containing
the 5' upstream promotor regions of a larval muscle actin gene from each species
245
suggest that loss of muscle cell differentiation is also accompanied by changes in the
structure of muscle actin genes themselves
rather than just in trans-acting regulatory
factor genes involved in their expression
(Kusakabe et al., 1996). An accumulation
of mutations leading to malfunction in the
structural genes for actin and other muscle
proteins may, however, have been preceded
by prior inactivation of regulatory genes
which initiate notochord differentiation and
tail organization in general.
There is evidence in some cases that individual larval organs and even features
within the organs are differentially suppressed during anural transformations. In
Molgula arenata (Whittaker, 1979) and
Molgula occulta (Swalla and Jeffery,
1990), there is still a residuum of histochemically detectable muscle acetylcholinesterase even when there is obviously no
structural muscle differentiation; in certain
other anural species enzyme activity appears to be absent. In the anural molgulid
species Bostrichobranchus digonas, expression of tyrosinase enzyme and melanin pigment occurs in 1-2 melanocytes that would
ordinarily form parts of brain sensory structures in urodele species. So far, no other
anural species have been reported as having
brain melanocytes. In B. digonas there are
no expressions of such muscle-specific
markers as acetylcholinesterase, a-actin,
and myosin heavy chain (Swalla and Jeffery, 1992).
Berrill (1945, 1955) and others have
compared larval structures in many ascidian
species and have assumed adaptationist explanations of the differences observed.
Changes seem to have occurred frequently
involving reductions, deletions or modifications of the two brain sensory structures,
the ocellus and statocyte. These alterations
occur without obvious correlated changes in
other organs and are deduced to be adaptations associated with shifts in sensory input needs related to altered habitat preference for a newly evolving species. In other
species there are variations in tail muscle
cell number which reflect increased larval
size. The numbers range from 36 muscle
cells in larvae of solitary ascidians to as
many as 1,134 in Ecteinascidia turbinata,
246
J. R. WHITTAKER
an exceptionally large larva of a colonial
species. There are no attendant changes in
the number (about 40) of the adjacent notochordal cells. Possibly increased muscle
cell numbers favor a greater swimming efficiency in the larger larvae, whereas
changing the number of the incompressible
notochordal cells would have little selective
advantage. Larval organ systems clearly appear to be modified independently of each
other.
As Gould (1992) reminds us, one of the
first (1812) arguments against the possibility of evolution was rooted in Baron Cuvier's correlation of parts and their absolute
dissociability. Animal parts were presumed
to be so closely integrated that even minor
changes would occasion a virtually prohibitive compensatory reorganization in order
to maintain function.
In minor key, Gould and Lewontin
(1979) have raised the same specter that
single-feature adaptations of body parts,
such as those postulated above in ascidians,
do not occur easily or rapidly in evolution
because of the highly integrated nature of
developmental processes. Raff (1996),
however, has revealed the somewhat illusory nature of this perception by emphasizing the compartmental and modular behavior of embryonic regions in many kinds of
organisms. Such modularity can be interpreted as permitting natural selection to operate quite independently on individual
body parts, essentially in a pattern of "mosaic evolution."
THE CAMBRIAN EXPLOSION AND CHORDATE
RADIATION
One of the surprises of the last decade
has been confirmation of the apparent speed
at which the major animal phyla seem to
have evolved during the Cambrian period,
510—545 Myr ago. Over an interval of
about 15-20 Myr, virtually all the metazoan
phyla, including the chordates, burst into
being (Briggs et al., 1994). An amphioxuslike cephalochordate creature, Pikaea gracilens, has been recovered from the Burgess
Shale formation of 520 Myr ago (Briggs et
al., 1994). The larvacean specialist Lohmann (1922) identified a larvacean tunicate
fossil, Oesia disjuncta, from the Burgess
Shale, and another larvacean species was
described from the Early Cambrian (Zhang,
1987), although both designations remain
controversial. A fossil colonial ascidian,
Palaeobotryllus, has been claimed from the
Late Cambrian (Miiller, 1977).
These fossil species, and others more
problematical in their interpretation, indicate nonetheless that chordates have radiated explosively during Cambrian times.
Identification of the conodont-bearing animal from fossils of the much later Ordovician and Carboniferous periods, and the discovery that their tooth-like conodont elements were functional teeth with a microstructure similar to the dermal bone and
mesodentine of vertebrates, indicates these
animals to be primitive vertebrate-like chordates (Janvier, 1995). Conodont fossils first
occur in Upper Cambrian strata, as do fragments of an agnathan fish, Anatolepis
(Smith et al., 1996).
On the basis of the fossil record as presently known, tunicates seemingly arose at
some time in the Lower Cambrian. Paradoxically, it is the character of early autonomous specification in their development
that may have favored their success both as
a group and as a malleable precursor to
more complex pre-vertebrate forms such as
amphioxids. Given both their long history
and their present day species numbers, estimated at approximately 3000 (Satoh,
1994), urochordates appear to be a welladapted group. Although most of the species are ascidians living subtidally in varied
niches, tunicates have radiated successfully
into both pelagic and abyssal environments.
In contrast, living cephalochordates as
represented by two main genera (Branchiostoma and Asymmetron=Epigonichthys) are undoubtedly collateral survivors
of a once connecting group to the vertebrates, yet they are much less successful in
their diversity. These modern forms are
niche specialists limited to virtually a single
kind of environment (coarse, current-swept
sand) with only about 25 species worldwide. Not surprisingly, amphioxids are
more conditionally specified (regulative) in
their early stages and much less autonomously specified in their early embryogenesis than ascidians (Reverberi, 1971).
ASCIDIAN EMBRYO MODEL
A thesis presented here is that the innate
property of extreme autonomous specification in the early ascidian embryo is a basis
for dissociation of the various development
programs in larval organs, and their separation from those in the ascidiozooid. Because of this dissociability and a consequent rather strict modularity, natural selection might be expected to act more rapidly
on the individual organ systems. Presumably independent organ changes would favor the success of ascidian species in making rapid adjustments to their preferred ecological niches, but equally favor rapid evolutionary change among the "dual animal"
paedomorphs postulated by Garstang
(1928), Berrill (1955) and Romer (1972).
Consideration of the continuous small
improvements of design required to produce a structure as elaborate and precise as
a vertebrate eye, beginning with a patch of
light-sensitive cells, gives what is thought
to be an over-estimate of about 1800 steps
taking place in about 400,000 generations
(Nilsson and Pelger, 1994). The generation
time for small and medium-sized aquatic
animals such as ascidians is usually one
year. Even without a developmental regulatory mechanism that strongly favored selection on a very modular basis, important
structural changes in organs could occur
rapidly. During the Cambrian period rapid
changes in the diverse structural features of
chordates did unquestionably occur.
ACKNOWLEDGMENTS
Robert J. Crowther collaborated in much
of the original investigative work discussed
herein and contributed helpful discussions
of the ideas. The work was supported in
part by a grant from NSERC Canada, and
by funds from the University of New
Brunswick.
REFERENCES
Baer, K. E. von. 1873. Entwickelt sich die Larve der
einfachen Ascidien in der ersten Zeit nach dem
Typus der Wirbelthiere? Mem. Acad. Imp. Sciences St. Petersbourg 19 (Ser. 7): 1-35.
Berrill, N. J. 1936. Studies in tunicate development.
Part V.—The evolution and classification of ascidians. Phil. Trans. Roy. Soc. B 226:43-70.
Berrill, N. J. 1945. Size and organization in the development of ascidians. In W. E. Le Gross Clark
247
and P. B. Medawar (eds.), Essays on growth and
form presented to D'Arcy Wentworth Thompson,
pp. 231-263. Clarendon Press, Oxford.
Berrill, N. J. 1955. The Origin of Vertebrates. Clarendon Press, Oxford.
Bone, Q. 1957. The problem of the 'amphioxides'
larva. Nature 180:1462-1464.
Briggs, D. E. G., D. H. Erwin, and F J. Collier. 1994.
The Fossils of the Burgess Shale. Smithsonian Institution Press, Washington.
Chabry, L. 1885. Monstres nouveaux chez les ascidies. Compt. Rend. Soc. Biol. 37:42-44.
Chabry, L. 1887. Contribution a l'embryologie normale et teratologique des Ascidies simples. J.
Anat. Physiol. (Paris) 23:167-319.
Cohen, A. and N. J. Berrill. 1936. The development
of isolated blastomeres of the ascidian egg. J. Exp.
Zool. 74:91-117.
Conklin, E. G. 1905. Mosaic development in ascidian
eggs. J. Exp. Zool. 2:145-223.
Cripps, A. P. 1990. A new stem craniate from the
Ordovician of Morocco and the search for the sister group of the Craniata. Zool. J. Linn. Soc. 100:
27-71.
Crowther, R. J. and J. R. Whittaker. 1992. Structure
of the caudal neural tube in an ascidian larva: Vestiges of its possible evolutionary origin from a
ciliated band. J. Neurobiol. 23:280-292.
Crowther, R. J. and J. R. Whittaker. 1994. Serial repetition of cilia pairs along the tail surface of an
ascidian larva. J. Exp. Zool. 268:9—16.
Darwin, C. 1871. The Descent of Man, and Selection
in Relation to Sex. Vol. 1, pp. 205—206. John Murray, London.
Davidson, E. H. 1990. How embryos work: A comparative view of diverse modes of cell fate specification. Development 108:365-389.
Dohrn, A. 1875. Der Ursprung der Wirbelthiere und
das Princip des Funktionswechsels. Genealogische
Skizzen. Verlag von Wilhelm Engelmann, Leipzig.
Eakin, R. M. 1973. The Third Eye, pp. 18-23. University of California Press, Berkeley.
Garstang, W. 1922. The theory of recapitulation: A
critical re-statement of the biogenetic law. J. Linn.
Soc. (Zool.) 35:81-101.
Garstang, W. 1928. The morphology of the Tunicata,
and its bearings on the phylogeny of the Chordata.
Quart. J. Microsc. Sci. 72:51-187.
Grave, C. 1935. Metamorphosis of ascidian larvae.
Carnegie Inst. Wash. Papers from the Tortugas
Laboratory 29:209-292.
Gould, S. J. 1992. Ontogeny and phylogeny- revisited
and reunited. BioEssays 14:275-279.
Gould, S. J. and R. C. Lewontin. 1979. The scoundrels of San Marco and the Panglossian paradigm:
A critique of the adaptationist programme. Proc.
Roy. Soc. Lond. B 205:281-298.
Hadfield, K. A., B. J. Swalla, and W. R. Jeffery. 1995.
Multiple origins of anural development in ascidians inferred from rRNA sequences. J. Mol. Biol.
40:413-427.
Haeckel, E. 1868. Natiirliche Schopfungsgeschichte.
Berlin: Reimer.
248
J. R. WHITTAKER
Haider, G., P. Callaerts, and W. J. Gehring. 1995. Induction of ectopic eyes by targeted expression of
the eyeless gene in Drosophila. Science 267:
1788-1792.
Holland, P. W. H. and J. Garcia-Fernandez. 1996. Hox
genes and chordate evolution. Dev. Biol. 173:
382-395.
Ivanova-Kazas, O. M 1988. Coelom derivatives in
Tunicata in relation to the evolution of lower chordata [In Russian]. Zool. Zh. 67:5-16.
Jagersten, G. 1972. Evolution of the metazoan life cycle. A comprehensive theory. Academic Press,
London.
Janvier, P. 1995. Vertebrate origins: Conodonts join
the club. Nature 374:761-762.
Jefferies, R. P. S. 1986. The ancestry of the vertebrates. British Museum, London.
Jeffery, W. R. and B. J. Swalla. 1990. Anural development in ascidians: Evolutionary modification
and elimination of the tadpole larva. Sem. Dev.
Biol. 1:253-261.
Katsuyama, Y., S. Wada, S. Yasugi, and H. Saiga.
1995. Expression of the labial group Hox gene
HrHox-1 and its alteration induced by retinoic
acid in development of the ascidian Halocynthia
roretzi. Development 121:3197-3205.
Katz, M. J. 1983. Comparative anatomy of the tunicate tadpole, Ciona intestinalis. Biol. Bull. Woods
Hole 164:1-27.
Kowalevsky, A. 1866. Entwickelungsgeschichte der
einfachen Ascidien. M6m. Acad. Imp. Sci. St. Petersbourg 10 (Sen 7): 1-19.
Kowalevsky, A. 1871. Weitere Studien iiber die Entwicklung der einfachen Ascidien. Arch. Mikr.
Anat. 7:101-130.
Kupffer, C. 1869. Die Stammerverwandtschaft
zwischen Ascidien und Wirbelthieren. Arch. Mikr.
Anat. 5:459-463.
Kupffer, C. 1870. Die Stammerverwandtschaft
zwischen Ascidien und Wirbelthieren. Arch. Mikr.
Anat. 6:115-172.
Kusakabe, X, B. J. Swalla, N. Satoh, and W. R. Jeffery.
1996. Mechanism of an evolutionary change in
muscle cell differentiation in ascidians with different modes of development. Dev. Biol. 174:
379-392.
Lohmann, H. 1922. Oesia disjuncta Walcott, eine Appendicularie aus dem Kambrium. Mitt. Zool.
Staat. Zool. Mus. Hamburg 38 (1920):69-76.
Maisey, J. G. 1986. Heads and tails: A chordate phylogeny. Cladistics 2:201-256.
McKinney, M. L. and K. J. McNamara. 1991. Heterochrony. The evolution of ontogeny. Plenum
Press, New York.
Milne-Edwards, H. 1842. Observations sur les Ascidies composees des cotes de la Manche. Mem.
Acad. Sci. Paris 18:217-326.
Miiller, K. J. 1977. Palaeobotryllus from the Upper
Cambrian of Nevada—a probable ascidian. Lethaia 10:107-118.
Nakatani, Y. and H. Nishida. 1994. Induction of notochord during ascidian embryogenesis. Dev.
Biol. 166:289-299.
Nakauchi, M. and T. Takeshita. 1983. Ascidian one-
half embryos can develop into functional adult ascidians. J. Exp. Zool. 227:155-158.
Nielsen, C. 1995. Animal Evolution. Interrelationships of the living phyla, pp. 407—421. Oxford
Univ. Press, Oxford.
Nilsson, D.-E. and S. Pelger. 1994. A pessimistic estimate of the time required for an eye to evolve.
Proc. R. Soc. Lond. B 256:53-58.
Nishida, H. 1993. Localized regions of egg cytoplasm
that promote expression of endoderm-specific alkaline phosphatase in embryos of the ascidian
Halocynthia roretzi. Development 118:1-7.
Olsson, R. 1972. Reissner's fiber in ascidian tadpole
larvae. Acta Zool. (Stockh.) 53:17-21.
Philippe, H., A. Chenuil, and A. Adoutte. 1994. Can
the Cambrian explosion be inferred through molecular phylogeny? Development 1994 Suppl.:15—
25.
Raff, R. A. 1996. The shape of life. Genes, development, and the evolution of animal form, pp. 321—
361. The University of Chicago Press, Chicago.
Reverberi, G. 1971. Amphioxus. In G. Reverberi
(ed.), Experimental embryology of marine and
fresh-water invertebrates, Chap. 14, pp. 551—572
Amsterdam: North-Holland.
Reverberi, G. and A. Minganti. 1946. Fenomeni di
evocazione nello sviluppo di Ascidie. Resultati
dell'indagine sperimentale sull'uovo di Ascidiella
aspersa e di Ascidia malaca allo stadio di otto
blastomeri. Pubbl. Staz. Zool. Napoli 20:199-252.
Romer, A. S. 1972. The vertebrate as a dual animalsomatic and visceral. In T. Dobzhansky, M. K.
Hecht and W. C. Steere (eds.), Evolutionary Biology 6:121—156. Appleton-Century-Crofts, New
York.
Satoh, N. 1994. Developmental biology of ascidians,
pp. 69-131. Cambridge University Press, Cambridge.
Schaeffer, B. 1987. Deuterostome monophyly and
phylogeny. Evol. Biol. 21:179-235.
Smith, M. P., I. J. Sansom, and J. E. Repetski. 1996.
Histology of the first fish. Nature 380:702-704.
Svane, I. 1982. Possible ascidian counterpart to the
vertebrate saccus vasculosus with reference to
Pyura tessellata (Forbes) and Boltenia echinata
(L.). Acta Zool. (Stockh.) 63:85-89.
Swalla, B. J. and W. R. Jeffery. 1990. Interspecific
hybridization between an anural and urodele ascidian: Differential expression of urodele features
suggests multiple mechanisms control anural development. Dev. Biol. 142:319-334.
Swalla, B. J. and W R. Jeffery. 1992. Vestigial brain
melanocyte development during embryogenesis of
an anural ascidian. Develop. Growth & Differ. 34:
17-25.
Swalla, B. J., K. W. Makabe, N. Satoh, and W. R.
Jeffery. 1993. Novel genes expressed differentially in ascidians with alternate modes of development. Development 119:307-318.
Turbeville, J. M., J. R. Schulz, and R. A. Raff. 1994.
Deuterostome phylogeny and the sister group of
the chordates: evidence from molecules and morphology. Mol. Biol. Evol. 11:648-655.
Wada, H. and N. Satoh. 1994. Details of the evolu-
ASCIDIAN EMBRYO MODEL
tionary history from invertebrates to vertebrates,
as deduced from the sequences of 18S rDNA.
Proc. Natl. Acad. Sci. U.S.A. 91:1801-1804.
Whittaker, J. R. 1977. Segregation during cleavage of
a factor determining endodermal alkaline phosphatase development in ascidian embryos. J. Exp.
Zool. 202:139-154.
Whittaker, J. R. 1979. Development of vestigial tail
muscle acetylcholinesterase in embryos of an anural ascidian species. Biol. Bull. Woods Hole 156:
393-407.
249
Whittaker, J. R. 1990. Determination of alkaline phosphatase expression in endodermal cell lineages of
an ascidian embryo. Biol. Bull. Woods Hole 178:
222-230.
Willmer, P. 1990. Invertebrate relationships. Patterns
in animal evolution. Cambridge University Press,
Cambridge.
Zanandrea, G. 1957. Neoteny in a lamprey. Nature
179:925-926.
Zhang, A. 1987. Fossil appendicularians in the Early
Cambrian. Scientia Sinica B 30:888-896.