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Classic theories of vertebrate head segmentation clearly exemplify the idealistic nature of comparative embryology prior to the 20th century. Comparative embryology aimed at recognizing the basic, primary structure that is shared by all vertebrates, either as an archetype or an ancestral developmental pattern. Modern evolutionary developmental (Evo-Devo) studies are also based on comparison, and therefore have a tendency to reduce complex embryonic anatomy into overly simplified patterns. Here again, a basic segmental plan for the head has been sought among chordates. We convened a symposium that brought together leading researchers dealing with this problem, in a number of different evolutionary and developmental contexts. Here we give an overview of the outcome and the status of the field in this modern era of Evo-Devo. We emphasize the fact that the head segmentation problem is not fully resolved, and we discuss new directions in the search for hints for a way out of this maze.

Embryogenesis involves two distinct processes. On the one hand, cells must specialize, acquiring fates appropriate to their positions (differentiation); on the other hand, they must physically construct the embryo through coordinated mechanical activity (morphogenesis). In early vertebrate development, fibroblast growth factor (FGF) regulates multiple embryonic events, including germ layer differentiation and morphogenesis; the cellular components that direct FGF signaling to evoke these different responses remain largely unknown. We show here that the copper transporter 1 (Ctr1) protein is a critical router of FGF signals during early embryogenesis. Ctr1 both promotes the differentiation and inhibits the morphogenesis of mesoderm and neurectoderm in embryos of the frog Xenopus laevis, thereby coordinating normal development. Signal sorting by Ctr1 involves the activation of the Ras-MAP kinase cascade and appears to be independent of its role in copper transport. Mouse embryonic stem (ES) cells deficient for Ctrl ([Ctr1.sup.-1-]) retain characteristics of pluripotency under conditions that favor differentiation in wild-type ES cells, indicating a conserved role for Ctr1 during amphibian and mammalian cell fate determination. Our studies support a model in which vertebrate Ctr1 functions as a key regulator of the differentiation capacity of both stem and progenitor cell populations. Xenopus | FGF | pluripotent | ERK | Laloo

Across vertebrates, there are three principal skeletal tissues: bone, persistent cartilage, and replacement cartilage. Although each tissue has a different evolutionary history and functional morphology, they also share many features. For example, they function as structural supports, they are comprised of cells embedded in collagen-rich extracellular matrix, and they derive from a common embryonic stem cell, the osteochondroprogenitor. Occasionally, homologous skeletal elements can change tissue type through phylogeny. Together, these observations raise the possibility that skeletal tissue identity is determined by a shared set of genes. Here, we show that misexpression of either Sox9 or Runx2 can substitute bone with replacement cartilage or can convert persistent cartilage into replacement cartilage and vice versa. Our data also suggest that these transcription factors function in a molecular hierarchy in which chondrogenic factors dominate. We propose a binary molecular code that determines whether skeletal tissues form as bone, persistent cartilage, or replacement cartilage. Finally, these data provide insights into the roles that master regulatory genes play during evolutionary change of the vertebrate skeleton. Keywords: Sox9; Runx2; Persistent cartilage; Replacement cartilage; Bone; Transcription factors; Differentiation; Secondary cartilage; Dominance; Skeletal evolution

Developmental biology strives to decipher how a single cell gives rise to a complex, multicellular organism. Most animals share specific families of genes that regulate major aspects of body pattern. At the same time, biologists recognize that the ontogenic path varies from species to species. Thus, dissecting the molecular genetic mechanisms underlying development in different species should elucidate common principles of development but may also shed some light on the evolution of diverse animal forms. Our labs share an interest in vertebrate limb development and while most of this research in this field has been limited to the chick and mouse, we would like to broaden the scope of this work to include bats. Why study bats? One of the most productive adaptations of the vertebrate limb has been for flight. Bats are the most successful mammals, contributing close to one-quarter of all mammalian species. Unlike bird wings, the lifting surface of bat wings is composed of a membrane that extends between the forelimb digits as well as between the limbs and body wall. In addition, bat limbs demonstrate several other unique characteristics including elongated zeugopod and autopod skeletal elements. We have begun cloning and characterizing the expression of a number of genes from Carollia, with particular interest in those known to regulate limb growth, patterning, or cell death in order to better understand the unique aspects of bat limb development.

In vertebrate development, the left-right axis is specified by a regulatory cascade of genes with left-right asymmetric expression patterns in the lateral plate mesoderm (LPM). The mechanisms by which organ primordia interpret these expression patterns in order to acquire asymmetric shapes or assume handed positions in the body cavity remain unknown. Here we define the developmental differences between the left and right sides of the Xenopus embryo that shape the asymmetries of the digestive organs. We show that the asymmetric morphogenesis of the digestive system does not result from fate differences between the left and right LPM. Likewise, we do not detect differential proliferation in the left and right sides of the gut during asymmetric morphogenesis. Rather, we find that key curvatures occur in defined regions of the GI tract as a result of differences in the rate of elongation of the left and right sides of the primitive gut tube. Importantly, in both normal and heterotaxic embryos, the asymmetric expression patterns of the homeobox gene, Pitx2, correlate closely with the location of the less extended, concave face of these curvatures. Finally, we show that ectopic expression of Pitx2 mRNA causes heterotaxic positioning of the digestive organs by inducing inappropriate concavities in the developing GI tract. These results suggest that the primitive gut interprets Pitx2 expression as a signal to create a concavity or slow the rate of tissue elongation during digestive organ morphogenesis. This work provides the first link between asymmetric gene expression and the morphogenetic mechanisms that orient and shape asymmetric organs.

The somitic mesoderm produces the axial skeleton and the striated muscle of both body and limbs of vertebrates. The limb skeleton and connective tissue develops from cells of the lateral plate mesoderm. To function properly, the elements of the appendicular and axial systems must be coordinated during development; for instance the limbs need to be in register with regional changes in the vertebral column. This patterning is realized during development as cells from the somites migrate into the lateral plate and differentiate into specific muscles. There is a boundary that separates the dorsal compartment (somitic cells only) from the ventral compartment (somitic and LP cells) that we call the somitic frontier. Previous experiments in avian embryos have shown that somitic mesoderm forming structures in the dorsal compartment of the body (axial skeleton, paravertebral muscles, dorsal dermis) is intrinsically patterned, i.e. it maintains its axial, morphological identity despite transplantation to a new anterior-posterior (AP) axial address. Furthermore, Hox gene expression is also maintained in transplanted tissue, strengthening the correlation between Hox gene activity and AP patterning of axial tissues. The somitic cells that cross the somitic frontier and form limb and abdominal muscles, however, appear to come under the influence of the lateral plate. It is uncertain whether the lateral plate mesoderm has an intrinsic axial identity with regard to both morphology and Hox expression. Using the quail-chick chimera system we are performing heterotopic transplants of lateral plate mesoderm to determine the nature of patterning information in the lateral plate, and the role of Hox gene expression in this tissue.

The transient segmentation of the vertebrate hindbrain into rhombomeres serves as a scaffold for motor and sensory connections between the hindbrain and peripheral tissues. The latter are patterned by neural crest, and the rhombomeric pattern is thus projected on the periphery also. The manner in which this phylogenetically ancient pattern is integrated into the late embryonic and mature CNS and craniofacial tissues of mammals is essentially unknown. Here we have used site-specific recombination to map the fates of rhombomere 4 and its derived neural crest in normal and hoxb-1 mutant mice. We replaced the hoxb-1 coding sequence with the coding sequence for Cre recombinase, creating an allele that was null for hoxb-1 function and that instead expressed the recombinase in a pattern that closely miraicked that of hoxb-1. Using this allele and the Cre-conditional R26R reporter strain, we were able to heritably mark the entire r-4 neuroepithelial cohort and all of the r4-derived neural crest. In addition to the previously recognized migration of VIIth nerve motor neurons from r4, we find that several other neuronal populations exit r4 rostrally and caudally, and that the rostral migration of at least one group, like the motor neurons of VII, is also dependent on hoxb-1 function. As in the chick, r4 derived neural crest formed sharp boundaries with r1/2 crest. Surprisingly, the malleus of the inner ear was observed to be a composite of first and second arch cartilages, a finding relevant to the evolution of the mammalian middle ear and the interpretation of several mutations of mice with middle ear phenotypes.

Suites of stereotyped cellular behaviors mediate the morphogenesis of vertebrate embryos. To determine the role that cytoskeleton-matrix interactions play in modulating cell movements, we have cloned zebrafish focal adhesion kinase (FAK). FAK RNA is expressed in the notochord. Subsequently, FAK protein becomes concentrated at regions of protrusive activity in the center of the notochord. This localization of FAK protein suggests that FAK may play a role in the increase of motility of notochord cells. FAK protein concentrates at the notochord-somite boundary when the cells intercalate to become a single layer. This new localization indicates a possible additional role for FAK, that of boundary maintainance. In the paraxial mesoderm, FAK RNA is expressed in the posterior half of each somite. FAK protein is predominantly localized at each somite boundary. We propose that focal contacts along the extracellular matrix-rich somite boundaries aid in the stabilization of the somite boundaries. Analysis of FAK expression in floating head, spadetail, and no tail embryos indicates that the notochord and somite expression domains of FAK are genetically separable. In conclusion, our results indicate a dual role for FAK in modulating cell movements during development: (1) FAK may regulate the increase in protrusive activity of intercalating cells, and (2) FAK may play a role in stabilizing tissue boundaries.

A few vertebrate animals, including teleost fish, have an exceptional ability to regenerate various structures, such as all types of fins, spinal cord and optic nerve. Fin regeneration in teleosts can be divided into stages of 1) wound healing; 2) disorganization of connective tissue; 2) proliferation and migration of mesenchymal cells that result in 3) recruitment of the blastema (mass of potentially multipotent mesenchyme cells); 4) differentiation and outgrowth of the regenerate. We have performed an Early Pressure (EP) mutagenesis screen, where we have analyzed 227 families for defects in caudal fin regeneration. As many of the genes involved in fin regeneration are likely to be important for ontogenic development, we screened for temperature-sensitive (TS) as well as non-TS mutations. We have recovered 10 independent mutations, of which 7 appear to be transmitted in a Mendelian fashion. Gross morphological analysis revealed that the mutations affect various stages of regeneration. We are currently analyzing expression of different markers in the mutant and wild type fins during regeneration as well as the effects of these mutations on embryonic development, requirements of these genes in embryonic development. Two mutations were mapped using EP technique, and efforts are under way to generate crosses for fine genetic and positional cloning of the mutant genes. Our results indicate that regeneration processes in zebrafish can be studied using classical genetic approaches.

One way or another, all species possess the ability to regenerate damaged tissues. The degree of regeneration, however, varies considerably among tissues within a body and among species, with urodeles being the most spectacular. Such differences in regenerative capacity are indicative of specific mechanisms that control the different types of regeneration. In this review the different types of regeneration in vertebrates and their basic characteristics are presented. The major cellular events, such as dedifferentiation and transdifferentiation, which allow complex organ and body part regeneration, are discussed and common molecular mechanisms are pinpointed.