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Ten years later, chick hairy2 expression unveils a molecular clock operating during limb development. This review revisits vertebrate embryo segmentation with ...
Molecular Clocks Underlying Vertebrate Embryo Segmentation: A 10-Year-Old hairy-Go-Round

REVIEW

Birth Defects Research (Part C) 81:65–83 (2007)

Raquel P. Andrade*, Isabel Palmeirim, and Fernanda Bajanca Segmentation of the vertebrate embryo body is a fundamental developmental process that occurs with strict temporal precision. Temporal control of this process is achieved through molecular segmentation clocks, evidenced by oscillations of gene expression in the unsegmented presomitic mesoderm (PSM, precursor tissue of the axial skeleton) and in the distal limb mesenchyme (limb chondrogenic precursor cells). The first segmentation clock gene, hairy1, was identified in the chick embryo PSM in 1997. Ten years later, chick hairy2 expression unveils a molecular clock operating during limb development. This review revisits vertebrate embryo segmentation with special emphasis on the current knowledge on somitogenesis and limb molecular clocks. A compilation of human congenital disorders that may arise from deregulated embryo clock mechanisms is presented here, in an attempt to reconcile different sources of information regarding vertebrate embryo development. Challenging open questions concerning the somitogenesis clock are presented and discussed, such as When?, Where?, How?, and What for? Hopefully the next decade will be equally rich in answers. Birth Defects Research (Part C) 81:65–83, 2007. VC 2007 Wiley-Liss, Inc. Key words: somitogenesis; clock; embryo segmentation; temporal control; human malformations; congenital disorders; presomitic mesoderm; limb; axial skeleton

INTRODUCTION ‘‘We are going to have a baby!’’ is one of the greatest joys one can experience throughout lifetime. The birth of a human baby, however, is nothing short of a miracle. In fact, several studies have shown that about 30% of natural conceptions with successful embryo implantation result in pregnancy loss (Wilcox et al., 1988; Wang et al., 2003). Of these, 83% occur before 4 weeks of gestation (Wilcox et al., 1999), the time during which the most fundamental processes of embryo development take place. Moreover, an equally high percent-

age (30%) of conceptions do not result in embryo implantation, and a total of 70% do not reach birth (reviewed in Macklon et al., 2002). Among an array of possible causes for conception loss is the deregulation of temporal control during embryonic development. In fact, alongside the three dimensions in which an embryo undergoes growth and differentiation, one can consider time as being a fourth dimension, which must experience equally tight regulatory mechanisms to assure that each tissue/ structure/organ is formed in the right place, at the right time. At the

cellular level, this implies that each cell must receive/produce the appropriate signal(s) at the right time and place. Considering that the time of embryo gestation is constant and specific for each species, many have questioned: How is embryonic temporal control achieved?

How is embryonic temporal control achieved?

VERTEBRATE EMBRYO BODY SEGMENTATION One of the best systems to study the molecular mechanisms of embryonic temporal regulation is somitogenesis. Somitogenesis is the process through which the vertebrate embryo body is segmented along its anterior-posterior (A-P) axis into repeated units, named somites (Fig. 1). Although somites are merely transient embryonic structures, they are of unequivocal importance for the layout of the vertebrate body plan. Somites are the earliest manifestation of the segmental pattern of the adult vertebrate body, clearly evidenced in the vertebrae, intervertebral discs, and ribs, and also

Raquel P. Andrade, Isabel Palmeirim, and Fernanda Bajanca are from the Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal. ˆncia e Tecnologia, Portugal; Grant numbers: SFRH/BPD/9432/2002; SFRH/BPD/17368/2004. Grant sponsor: Fundac ¸˜ ao para a Cie *Correspondence to: Raquel P. Andrade, Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057 Braga, Portugal. E-mail: [email protected] Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdrc.20094

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the embryo’s A-P axis, symmetrically positioned flanking the axial structures-neural tube and notochord. As time proceeds from gestation day 20, when the first pair of somites arises in the future cervical region of the human embryo, until the end of the fifth week, when a final number of 42 to 44 somite pairs are present (GilbertBarness and Debich—Spicer, 2004), somite pairs are periodically formed in rostral-caudal sequence through epithelialization of the anterior portion of the unsegmented mesenchymal presomitic mesoderm (PSM) while, concomitantly, the embryo grows caudally due to gastrulation (Fig. 2, caudal end).

Segmentation and Somite Formation

Figure 1. Chick embryo displaying one of the characteristic phases of cyclic hairy1 gene expression. The PSM, somites, and A-P orientation are depicted.

imposed upon ligaments, muscles, nerves, and blood vessels. Excellent reviews on somite formation and differentiation have been produced in the past years (Gossler and Hrabe de Angelis, 1998; Pourquie ´, 2001; Saga and Takeda, 2001; Kalcheim and Ben Yair, 2005), so we will merely introduce the most fundamental aspects of these processes in order to familiarize the reader with the main molecular players involved. Somites are epithelial balls of cells sequentially generated along

Extracellular matrix molecules assume great importance for somite epithelialization to occur (Gossler and Hrabe de Angelis, 1998; reviewed in Chong and Jiang, 2005). In fact, one of the most severe somitogenesis phenotypes occurs in Fibronectin (Fn1)null mouse embryos, where no distinguishable somites are formed (Georges-Labouesse et al., 1996). Rifes and collaborators (submitted) have observed that the PSM must be surrounded by an intact fibronectin matrix in order for morphological somites to form. These authors also showed that a major role of the ectoderm during somitogenesis is to provide the fibronectin protein required to build this matrix. Two other important genes involved in somite epithelialization are paraxis (Tcf15) and the protocadherin-encoding gene Papc (Burgess et al., 1996; Rhee et al., 2003; Linker et al., 2005). Mutants for Paraxis, however, can still form somite boundaries while no epithelialization takes place, indicating that these two processes are somewhat independent (Burgess et al., 1996). Other molecules known to be required for morphological segmentation of the PSM include the Eph/Ephrin and cadherin families of proteins (reviewed in Kalcheim and Ben Yair, 2005).

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Somites are subdivided into rostral and caudal halves (Fig. 2, mature somites), clearly distinct in terms of gene expression patterns and of fundamental importance for the patterned development of the axial skeleton and peripheral nervous system (reviewed in Saga and Takeda, 2001). Although formation of morphological somites only occurs at the anterior tip of the PSM, molecular segmentation is already observed along the anterior third of the PSM tissue (Palmeirim et al., 1998), where somite A-P polarity is specified by Mesp2 and Delta/Notch signaling (Saga et al., 1997; Takahashi et al., 2000). Dll1 and notch1 are expressed in a striped pattern in the rostral third of the PSM, delimiting the posterior borders of the next somites to be formed (Palmeirim et al., 1998). On the other hand, Mesp2 expression in the anterior PSM delimits the next segments anteriorly. Here, Mesp2 represses Dll1 and induces Lfng expression, which in turn inhibits Notch activity (Morimoto et al., 2005). In this manner, Mesp2 ensures gene expression segregation between the prospective rostral (Mesp2 and Lfng) and caudal (Dll1 and Notch1) somite compartments. The maintenance of rostral and caudal identities in newly formed somites is tightly dependent on tbx18 and uncx4.1 gene expression, respectively (Bussen et al., 2004; Mansouri et al., 2000). In vitro cultured PSM explants devoid of ectoderm do not form somites, although a clear segmental pattern of gene expression is present, evidence suggesting that segment specification in the PSM is independent of epithelial somite formation (Palmeirim et al., 1998). In accordance with this is the observation that although somite formation is perturbed in Dll1, Psen1, Mesp2, and Paraxis mutant embryos, the paraxial mesoderm still retains a metameric arrangement and the resulting axial skeleton, although malformed, is clearly segmented (Burgess et al., 1996; Hrabe de Angelis et al., 1997; Saga et al., 1997; Wong et al., 1997).

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Figure 2. Schematic representation of the gradient of differentiation of the PSM and somites along the embryo A-P (rostral-caudal) axis. The central scheme represents a whole-mount embryo and different steps of paraxial mesoderm development are depicted in the surrounding boxes, starting from top right and proceeding in a counter-clockwise direction, corresponding to a posterior to anterior developmental gradient in the embryo. Caudal end: cell ingression (arrows) at the node (asterisk) contributes to continuous growth of the PSM (brown) in the tail, while pairs of somites (orange) form at the anterior PSM. Epithelial somite: the mesenchymal PSM cells become epithelial as they aggregate to form a somite. Sclerotome formation: the ventral somite cells deepithelize to form the sclerotome (yellow), while the dorsal, dermomyotomal cells (green) remain epithelial. Myotome formation: cells from the dermomyotome (green) give rise to the myotome underneath (red). Mature somites: cells from the sclerotome (yellow) develop into the tendon-progenitor syndetome (blue), located between adjacent somites. Tissues giving rise to future structures of the adult body are indicated.

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Despite all that is known regarding gene expression profiles during somite formation, Kulesa and Fraser (2002) have shown that somite boundary establishment is not simply the result of gene expression patterns. Using in vivo time-lapse analysis in the chick embryo, these authors showed that a huge amount of cellular rearrangement takes place during somite detachment from the PSM and that cells can move across the presumptive somite border, violating gene expression limits proposed to demarcate somitic boundaries.

Somite Compartmentalization and Differentiation The somites positioned along the embryo A-P axis present a gradient of differentiation, since the most rostral ones are formed prior to the somites located more caudally. In this manner, one can observe an array of developmental steps of somite differentiation along the A-P axis of one single embryo (Fig. 2). Each newlyformed somite is composed of an epithelial layer surrounding a central core of mesenchymal cells (Fig. 2, epithelial somite) and is enclosed by extracellular matrix that connects it to adjacent structures, such as the ectoderm, endoderm, other somites, notochord, and neural tube. As the somite matures, its ventral portion undergoes an epithelial to mesenchymal transition, resulting in a dorsal-ventral (D-V) compartmentalization of the somite (Fig. 2, sclerotome formation). At the molecular level this process is dependent upon a strict balance between Wnt signaling from the surface ectoderm and dorsal neural tube, and sonic hedgehog (Shh) and Noggin signaling, which originate mainly from the notochord. Additionally, signaling events along the somite mediallateral (M-L) axis are going on between bone morphogenetic proteins (BMPs) from the lateral plate mesoderm and the notochordderived molecules (reviewed in Christ et al., 2000; Yusuf and Brand-Saberi, 2006).

Somite D-V compartmentalization clearly distinguishes the ventrally located mesenchymal sclerotome from the epithelial dermamyotome, positioned dorsally (Fig. 2, sclerotome formation). The sclerotome will form the cartilage and bone anlagen of the vertebrae and ribs (reviewed in Christ et al., 2000). The dermomyotome contains the precursor cells of the body skeletal muscles and also contributes to the dermis of the back. The precursors of the trunk skeletal muscles delaminate from the dermomyotome and organize underneath it forming the myotome (Fig. 2, myotome formation). Precursors of limb, diaphragm, and tongue skeletal muscles delaminate from the lateral dermomyotome and migrate toward their target sites, where they later differentiate. Finally, cells from the dorsal cranial and caudal edges of the sclerotome develop into the tendon-progenitor syndetome, located between adjacent somites (Fig. 2, mature somites). This compartment thus arises at the interface between somitic muscle and cartilage, precisely the cell lineages that tendons will later connect (Brent et al., 2003). The sclerotome is further subdivided into rostral and caudal regions by a cleft (von Ebner’s fissure) and these two compartments present different combinations of gene expression (reviewed in Kuan et al., 2004). Later in development, the anterior compartment of one somite will fuse with the posterior region of the adjacent somite and give rise to vertebrae, in addition to intervertebral discs, and ribs (Fig. 2, mature somites). This process is referred to as somitic resegmentation. If we consider the vertebrae as the adult body’s segmental units, the sclerotome compartments can in fact be viewed as parasegments (Meinhardt, 1982), such as those that are formed during Drosophila embryo segmentation (reviewed in Deutsch, 2004). A consequence of somite resegmentation is that the boundaries of somites and vertebral bodies are shifted in space by a half-somite. Since myotomes are in phase with the somites,

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muscles will end up over-bridging the skeletal elements (Fig. 2, mature somites), which is essential for the ability of the vertebrate to move the axial skeleton (reviewed in Christ et al., 1998). The metameric arrangement of the adult vertebrate body, however, is not restricted to the structures directly derived from somites. Migrating neural crest cells and outgrowing motor and sensory axons can only invade the rostral somite half due to the different properties of the sclerotome compartments, and this imposes a segmental patterning on the peripheral nervous system (reviewed in Gossler and Hrabe de Angelis, 1998; Kuan et al., 2004). Although all somites along the embryo A-P axis look morphologically alike, the most rostral ones give rise to nonsegmented structures—the basal occipital and the sphenoid bone on the basis of the skull (Huang et al., 2000). Accordingly, the rostral-most somites present distinct molecular characteristics from the more caudallypositioned ones (Rodrigues et al., 2006).

THE SOMITOGENESIS CLOCK: COMING OF AGE Temporal control is of fundamental importance during somitogenesis. This is clearly evidenced by the fact that the time required to form a new somite pair, as well as the total number of somites formed, is constant and species-specific. For example, in the chick embryo a somite pair is formed every 90 min, to a total of 52 somite pairs. Moreover, the notion of the time in which each somite must be formed is an intrinsic property of the PSM tissue. This was shown by in vivo microsurgical experiments in the chick embryo where the rostralmost part of the PSM was grafted into a more caudal area of host PSM (Packard, 1978). In these conditions, the grafted tissue segmented as if it remained in the donor embryonic environment, suggesting that this tissue was already committed to segment in that exact temporal sequence.

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This property was also evidenced by experiments that involved excising the PSM portion that was about to be segmented and grafting it in a 1808-rotated position. The somites in this tissue arose in a posterior-to-anterior orientation, according to the time in which they would segment in the donor embryo, as opposed to the A-P sequence of events in the host tissue (Palmeirim et al., 1998; Dubrulle et al., 2001). Several theoretical models were proposed to account for the periodic segmentation of the vertebrate embryo body (reviewed in Schnell et al., 2000), but the clock and wavefront model proposed by Cooke and Zeeman (1976) has found widest acceptance and applicability. This model proposes that a biochemical oscillator (clock) is operating synchronously in PSM cells while a gradient of maturation (wavefront) sweeps the embryo along the A-P axis. The confrontation between the wavefront signal and PSM cells in a biochemically permissive state for somite formation would determine the making of a new somite.

A Molecular Clock Underlying Somitogenesis The biochemical clock oscillating in PSM cells proposed by the somitogenesis clock and wavefront theoretical model (Cooke and Zeeman, 1976) remained without experimental support for 20 years. In 1997, however, Palmeirim et al. (1997) reported the periodic expression of hairy1, the avian homolog of the Drosophila segmentation gene hairy, in chick embryo PSM (Fig. 1) (Pascoal and Palmeirim, 2007). Chick hairy1 gene was shown to be cyclically expressed in PSM cells, with the same periodicity as somite formation (90 min). The authors observed that although hairy1 expression resembles a wave sweeping the PSM in a posterior-to-anterior direction, the coordinated pulses of gene expression are not due to cell movement or to the propagation of a signal along that axis. Contrarily, cyclic hairy1

mRNA expression is an intrinsic property of PSM cells. These are slightly out-of-phase relatively to each other along the PSM A-P axis, thus forming a kinematic wave (Palmeirim et al., 1997; Masamizu et al., 2006). The discovery of a molecular clock controlling vertebrate embryo somite formation was considered by the publishers of the journal Nature to be a 20th century milestone in developmental biology (Skipper, 2004).

Chick hairy1 gene was shown to be cyclically expressed in PSM cells, with the same periodicity as somite formation (90 min)

Soon after, the Notch modulator gene lfng was also found to be cyclically expressed in chick (McGrew et al., 1998; Aulehla and Johnson, 1999) and mouse PSMs (Forsberg et al., 1998). Mutations affecting Notch signaling are known to result in somite formation defects (reviewed in Gridley, 2006) and the report of cyclic lfng expression established what is nowadays an undisputed link between the somitogenesis clock and the Notch signaling pathway. Jouve et al. (2000) further confirmed Notch signaling as an important component of the segmentation clock, since mouse Hes1 expression ceased to cycle in Notch receptor Delta1 mutant embryo PSMs. By the onset of the 21st century, many developmental biology research groups had focused their attention on this surprising property of PSM gene expression and the subsequent years were enriched with the report of many other genes whose cyclic expression in the embryo PSM correlated with somite formation (Fig. 3). These genes were

identified in multiple animal models used in developmental biology studies and many of them belong to the Notch signaling pathway (Fig. 3) (Rida et al., 2004; Andrade et al., 2005; Freitas et al., 2005; Bray, 2006), with great relevance to the Hairy/ Enhancer-of-Split (Hes) family of genes (reviewed in Kageyama et al., 2007b). Notch signaling has been shown to play an essential role in the synchronization of PSM cells with respect to their oscillations in gene expression (Jiang et al., 2000; Lewis, 2003; Horikawa et al., 2006). The requirement for synchronized molecular oscillations between adjacent PSM cells had in fact been postulated as a requirement for periodic somite formation in the clock and wavefront model (Cooke and Zeeman, 1976). With the significant technological advances made since the proposal of the model, real-time imaging of cyclic gene expression in both single cells and whole mount embryos clearly revealed that Notch-dependent cell–cell communication is essential for synchronization of the somitogenesis clock genes (Horikawa et al., 2006; Masamizu et al., 2006). These and other experiments demonstrated that clock gene cyclic expression is stable in both period and amplitude in the PSM, while in dissociated cells gene oscillations fluctuate and rapidly fall out of synchrony (Hirata et al., 2002; Maroto et al., 2005; Masamizu et al., 2006; Horikawa et al., 2006). Thus, Notch-mediated intercellular communication not only ensures synchronization, but also stabilization and robustness of the somitogenesis clock (Horikawa et al., 2006; Masamizu et al., 2006). Accordingly, mutants in components of the Notch signaling pathway present gradually increasing segmentation defects, while their first five to eight somites are preserved. This has been explained by the fact that desynchronization among neighboring cells is also a gradual process, and segmentation proceeds until a severe lack of coor-

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Figure 3. Timetable of major events in the elucidation of the molecular clocks and gradients underlying embryo segment formation. Genes with cyclic expression patterns in the PSM (unless otherwise stated) of different animal models are indicated with distinct colors (1Palmeirim et al., 1997; 2McGrew et al., 1998; 3Forsberg et al., 1998; 4Jouve et al., 2000; 5Leimeister et al., 2000; 6 Holley et al., 2000; 7Jiang et al., 2000; 8Dubrulle et al., 2001; 9Bessho et al., 2001; 10Dunwoodie et al., 2002; 11Oates and Ho, 2002; 12Hirata et al., 2002; 13Aulehla et al., 2003; 14Li et al., 2003; 15Diez del Corral et al., 2003; 16Ishikawa et al., 2004; 17 Elmasri et al., 2004; 18Maruhashi et al., 2005; 19Gajewski et al., 2006; 20Dale et al., 2006; 21Suriben et al., 2006; 22Deque ´ant et al., 2006; 23William et al., 2007; 24Pascoal et al., 2007b; 25Shankaran et al., 2007).

dination causes somite formation to fail altogether (Jiang et al., 2000; Lewis, 2003). Synchronized clock gene oscillations are most evident in the caudal part of the PSM (Horikawa et al., 2006), after which clock gene expression resembles a wave traveling from the posterior toward the anterior tip of the PSM, where it is arrested by Mesp2 activity (Morimoto et al., 2005; reviewed in Saga, 2007), forming a stripe of mRNA that demarcates the caudal border of the next somite to be formed (Palmeirim

et al., 1997; reviewed in Aulehla and Pourquie ´, 2006). The propagation of the clock signal along the PSM A-P axis is periodic and strictly unidirectional. Understanding how the latter is ensured is not straightforward, since both Notch receptor and its ligand (Delta) are coexpressed in the PSM cells. Zhu and Dhar (2006) addressed this issue and simulated Notch signaling using a multicellular model. They found that the unidirectional propagation of the clock along the mouse PSM A-P axis is independent of fibroblast growth factor

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(FGF) signaling present in the PSM and may be guaranteed by transient blocks of receptor activity produced by a Notch-Lfng-Hes7 triangulation (Zhu and Dhar, 2006; reviewed in Kageyama et al., 2007a). Notch activation induces both Lfng (Cole et al., 2002; Morales et al., 2002) and Hes7, which display cyclic expression in PSM cells due to negative feedback loop regulation upon their own promoters (Bessho et al., 2003; Dale et al., 2003; Serth et al., 2003; Chen et al., 2005). In turn, Lfng represses

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Notch signaling (Dale et al., 2003), possibly involving Dll3 (Ladi et al., 2005; Zhu and Dhar, 2006), and its transcription is repressed by Hes7 (Bessho et al., 2003). Transcriptional and translational delays in this process are the basis of this robust system (Lewis, 2003), which prevents reciprocating signaling among cells, which would ultimately result in segmentation defects. Accordingly, such defects are observed when this system is perturbed experimentally, for example, by either up- or down-regulation of Lfng expression (Dale et al., 2003; Serth et al., 2003). Although the involvement of the Notch signaling pathway in the somitogenesis clock was established earlier, chronologically speaking, today there is unequivocal evidence that the Wnt signaling pathway plays an equally pivotal role in this molecular mechanism (reviewed in Aulehla and Pourquie ´, 2006). Genes belonging to the Wnt pathway have also been described to present cyclic gene expression in embryonic PSM (Fig. 3) and cross-talk between Notch and Wnt pathways were unveiled. Namely, Wnt antagonist Nkd1 presents cyclic gene expression in phase with Lfng in mouse PSM. While Nkd1 induction depends on Wnt3a, its oscillations are regulated by Notch-induced Hes7 (Ishikawa et al., 2004). On the other hand, Notch ligand Dll1 is cyclically expressed in the mouse embryonic PSM (Maruhashi et al., 2005) and was found to be induced by a cooperative action of the mesenchymal marker gene Tbx6 and the Wnt signaling pathway components lymphoid enhancer factor/T-cell factor (LEF/ TCF) (White et al., 2003; Hofmann et al., 2004; White and Chapman, 2005). Lfng and Hes7 oscillations are also dependent on Wnt signaling (Aulehla et al., 2003; Nakaya et al., 2005; Satoh et al., 2006), revealing a reciprocal interaction of Notch and Wnt signaling pathways in the somitogenesis clock. The FGF signaling pathway has also been implicated in the somitogenesis clock mechanism. Kawa-

mura et al. (2005) reported that the Hairy/Enhancer-of-Split molecule Her13.2 was pivotal in linking FGF signaling to Notch-related cyclic gene expression and, ultimately, proper somite segmentation in the zebrafish embryo. FGFinduced Her13.2 acts as a dimerization partner for Her1, and the formation of this heterodimer is required for transcriptional feedback repression on Her1 promoter, ensuring its cyclic expression, and for somitic border formation throughout the entire A-P axis, in both zebrafish and medaka embryos (Kawamura et al., 2005; Sieger et al., 2006). Another study described cyclic expression of snail1 and snail2 in mouse and chick embryos, respectively, which required FGF and Wnt pathways and was Notch-independent. Nevertheless, Snail2 misexpression ceased lfng oscillations and impaired epithelial somite formation (Dale et al., 2006). More recently, as a result of an exhaustive microarray dissection of mRNA profiles during a cycle of clock gene expression, an unexpectedly large number of genes were found to be cyclically expressed in the mouse PSM (Deque ´ant et al., 2006). It was possible to cluster many of these genes into three groups: the Notch, Wnt, and FGF signaling pathways. This study showed that the FGF and Notch clusters of cycling genes are activated in parallel, while genes belonging to the Wnt cluster present an opposite cycling phase to the Notch/FGF cluster (Deque ´ant et al., 2006). This had in fact already been shown for Axin2 (Aulehla et al., 2003). With the current knowledge of how some of these genes interact to regulate gene expression or protein activity, it is clear that the three pathways may function in parallel and be integrated into one molecular clock (Deque ´ant et al., 2006). Somitogenesis is a fundamental process in embryogenesis, and the involvement and reciprocal regulation of multiple pathways in the molecular clock underlying somite formation may reflect the existence of fail-safe mechanisms that

ensure accurate embryo body patterning (Huppert et al., 2005). The exponentially growing complexity of the segmentation clock system seems to impel us to start considering the significance of cyclic signaling pathway activities, and not so much of individual cyclic genes, in order to grasp this extraordinary biological mechanism.

A Wavefront of Differentiation Using in situ hybridization, Dubrulle et al. (2001) showed that fgf8 was expressed as a gradient diminishing toward the anterior tip of the PSM. This mRNA gradient does not result from graded transcriptional induction along the A-P axis, but arises from progressive decay of the fgf8 mRNA generated at the tail bud of the embryo (Dubrulle and Pourquie ´, 2004). Opposed to this is a gradient of the gene encoding the retinoic acid (RA)-synthesizing enzyme, raldh2, which presents highest expression in already formed somites, diminishing in the rostral PSM (Diez del Corral et al., 2003). These two opposing gradients counteract each other and define what has been termed the determination front (Fig. 4) (Dubrulle et al., 2001; reviewed in Diez del Corral and Storey, 2004). A-P inversion experiments of PSM tissue showed that PSM cells located caudal to this front are not yet determined to form a somite, while those positioned rostrally to the determination front are already committed to incorporate a segment. Moreover, when the determination front is experimentally displaced along the PSM axis by overexpressing or inhibiting FGF8, the resulting formed somites were smaller or bigger, respectively (Dubrulle et al., 2001). The FGF8 wavefront was shown to generate and act through a gradient of mitogen-activated protein kinase (MAPK)/extracellular signalregulated kinase 1 (ERK1) signaling throughout the PSM A-P axis in both zebrafish (Sawada et al., 2001) and chick (Delfini et al., 2005) embryos. A wavefront of Wnt signaling has also been described in the

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Figure 4. Schematic representation of the caudal portion of an embryo (PSM in blue, and the two most-recently formed somites), indicating the expression patterns of molecules involved in the spatial/ temporal control of somitogenesis. Swirls represent cyclic gene expression and triangles represent molecular gradients. pM, prospective medial PSM; pL, prospective lateral PSM; arrows indicate future cell position in the PSM.

embryo PSM (Aulehla et al., 2003). Wnt3a is expressed along a gradient similar to Fgf8 and acts upstream of the latter, i.e., Fgf8 is downregulated in Wnt3a mutants. Moreover, in the absence of Wnt3a, Notch-dependent gene oscillations were impaired (Aulehla and Herrmann, 2004). Cyclic expression of Notch-related genes is also dependent on FGF-induced her13.2 in zebrafish (Kawamura et al., 2005) and Wnt/FGF-activated snail2 expression in chick (Dale et al., 2006), demonstrating an integration of the clock and wavefront in somite segmentation control. Further evidence for the clock and wavefront interaction came from studies using smadinteracting protein 1 (Sip1) mutants (Maruhashi et al., 2005).

Sip1 deficiency caused a rostral displacement of the expression of Raldh2, Fgf8, Wnt3a, Dll3, and Tbx6 genes, and concomitant rostral expansion of the region of cyclic expression of the genes Lfng, Hes7, and Dll1, supporting a close link between clock and wavefront activities. Sip1 was proposed to modulate the mutually repressive Raldh2/Fgf8 interaction, thus regulating the positioning of somite boundaries by the determination front (Maruhashi et al., 2005). From the reports summarized in the previous section, with special emphasis on the microarray characterization of the molecular components of the mouse somitogenesis clock (Deque ´ant et al., 2006), we are now aware that Notch, Wnt, and FGF pathways are interdependent players of the same clocked mechanism. Moreover, although components of the Wnt signaling pathway presenting cyclic gene expression had already been described, this was not so until now for the FGF pathway. What this may imply is that although the wavefront signalWnt3a, Fgf8/ERK—is graded along the PSM axis, its downstream activities/outputs may have a cyclic nature, evidencing an intimate relationship between clock and wavefront during segment formation. The description of the molecular players that participate in the somitogenesis process has traditionally associated the Notch signaling pathway with the clock in the clock and wavefront model proposed by Cooke and Zeeman (1976), and the FGF and Wnt pathways with the wavefront of differentiation. The longstanding simplistic view of the segregation of the components of these pathways into two separate groupsclock genes and wavefront genesmay have clouded our judgement when interpreting experimental results for too long.

THE CLOCK IS OPERATING ALONG THE PSM M-L AXIS Temporal control of embryo development is required for correct growth and patterning along all

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embryonic axes: A-P, M-L, and D-V. A newly-formed somite is clearly a three dimensional structure consisting of an epithelial ball of cells surrounding a mesenchymal core. As reviewed in previous sections, maturing somites contain precursor cells of very different adult structures. These are differentially located in the somite, with a clear distinction of cell identities along the three somite axes (Olivera-Martinez et al., 2000). Before being incorporated into a somite, cells differently located in the rostral-most PSM tissue already manifest distinct properties, which are particularly evidenced in the process of somite border establishment. Ventrally-located PSM cells signal dorsal cells for somite fissure formation (Sato and Takahashi, 2005) and A-P somite detachment from the PSM progresses in an M-L direction (Kulesa and Fraser, 2002). Moreover, Freitas et al. (2001) demonstrated that it is the medial portion of the PSM that drives segmentation. The medial PSM (M-PSM) is capable of segmenting into somites when isolated from the lateral PSM (L-PSM) while, in the absence of M-PSM, the lateral portion cannot segment and loses the expression of the molecular segmentation markers (Freitas et al., 2001). It is then natural that these cells with such different cellular behaviors and fates should also have specific origins and/or undergo particular specifications along development. The medial and lateral portions of the PSM originate from different cell populations during chick embryo development (Selleck and Stern, 1991; Catala et al., 1996; Freitas et al., 2001; Iimura et al., 2007). In a six-somite stage chick embryo, cyclic expression of hairy1, hairy2, and lfng is already observed along the prospective PSM M-L axis (Fig. 4) and an asynchrony between M-PSM and L-PSM is evidenced by the appearance of oblique stripes of expression of these genes in the PSM (Fig. 1) (Freitas et al., 2001). This study evidences that the somitogenesis clock is operating along at least two PSM axes: A-P and M-L.

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ENSURING LEFT—RIGHT SYMMETRY DURING SOMITOGENESIS Somites are sequentially formed along the vertebrate embryo A-P axis in a remarkably symmetric manner. Accordingly, somitogenesis clock gene expression is strictly symmetrical in the PSM tissue flanking both sides of the embryonic axial structures. This explains why all somite-derived tissues are symmetrical in the adult body. On the inside, however, the vertebrate body is greatly asymmetrical in both the positioning and structure of the internal organs (reviewed in Raya and Belmonte, 2006; Levin, 2006). Notch-induced Nodal expression in the left lateral plate mesoderm during early phases of development mediates the establishment of the asymmetry of internal organs (Raya et al., 2003), and depends on an asymmetric activation of Notch activity around the embryo node (Raya et al., 2004). While several molecular players involved in embryo left-right (L-R) determination have been described, somite symmetry along the A-P axis was classically considered to result from a ‘‘default’’ state (Brent, 2005). Work performed in recent years, however, has shown that embryo bilateral symmetry requires strict regulation along the L-R body axis (Nakaya et al., 2005; Kawakami et al., 2005; Sau ´de et al., 2005; Vermot et al., 2005; Vermot and Pourquie ´, 2005; Sirbu and Duester, 2006; Morales et al., 2007). The first molecular link between L-R asymmetry establishment and bilateral synchronization of the somitogenesis clock was the terra gene in chick and zebrafish (Sau ´de et al., 2005). Terra is expressed asymmetrically at the node of chick embryos. The absence of terra leads to a randomization of lateral plate mesoderm markers and, consequently, of heart position, asymmetric oscillations of clock gene expression in the PSM and unbiased formation of extra somites on one side of the PSM. This study suggests that Terra promotes L-R patterning very early in

development and, at the same time, ensures bilaterally symmetric somite formation through a possible collaboration with other symmetry-promoting signaling pathways, like RA (Kawakami et al., 2005; Sau ´de et al., 2005; Vermot et al., 2005; Vermot and Pourquie ´, 2005). In the absence of RA in the PSM, symmetrical cyclic gene expression is lost and asymmetric somites are formed (Kawakami et al., 2005; Vermot et al., 2005; Vermot and Pourquie ´, 2005). In addition, in the absence of the Notch pathway component, Su(H), asymmetric gene expression is also observed and this seems to be due to a misregulation in the PSM RA levels (Echeverri and Oates, 2007). Recently, it has

A recent study showed that a molecular clock is also operating during limb had development

been shown that proper RA levels are important to prevent asymmetric Snail1 expression in the mouse PSM (Morales et al., 2007). Wnt3a is also a component of the molecular L-R pathway (Nakaya et al., 2005). Wnt3a regulates the expression of the Notch pathway gene Dll1, which induces differential Nodal gene expression. Wnt3a mutants present laterality disorders and lose clock cyclic gene expression. This reveals an intimate early connection between L-R determination and somitogenesis, which is mediated by Wnt signaling (Nakaya et al., 2005).

A MOLECULAR CLOCK IS OPERATING DURING LIMB DEVELOPMENT The vertebrate forelimb is segmented along the proximal-distal

(P-D) axis into its various skeletal structures: the humerus, the radius and ulna, and the autopod elements metacarpals and phalanges. Limb P-D outgrowth is driven by a signaling center named the apical ectodermal ridge (AER). The AER is a specialized ectodermal structure formed along the distal tip of the limb bud that expresses FGFs, shown to be the signal required for limb structure formation along the P-D axis (reviewed in Martin, 1998; Tickle, 2004). Similarly to what occurs for somitogenesis, temporal control during limb element formation is of critical importance. How is time measured during limb formation? A recent study showed that a molecular clock is also operating during limb had development (Pascoal et al., 2007b). Pascoal et al. (2007b) showed that the somitogenesis clock gene hairy2 is expressed cyclically in the distal mesenchyme of chick embryo forelimbs, with a 6-hr periodicity. They also determined that a new autopod limb element requires 12 hr to be laid down, implying that two cycles of hairy2 gene expression occur during the specification of an autopod limb element. Consequently, these authors proposed that the segmentation clock is not an exclusive property of PSM tissue, but may be a more general way to count time during vertebrate development, providing positional information to different types of cells (Pascoal et al., 2007b). In fact, many different cell types have already been shown to be able to generate gene expression oscillations (Hirata et al., 2002; Kageyama et al., 2007b; William et al., 2007). Despite the presence of several segmentation clock molecules in a widespread range of developing tissues, a clocked system has been harder to detect possibly due to lack of synchronization among cells (Hirata et al., 2002; Pascoal et al., 2007b). Could the molecular mechanisms involved in segment formation along the embryo A-P body and P-D limb axes be related? Another somitogenesis clock gene,

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hairy1, has also been implicated in limb size regulation (Vasiliauskas et al., 2003). Moreover, an mRNA decay-generated gradient of FGF signaling activity through Mkp3 (Kawakami et al., 2003) has been described along the P-D axis of the chick embryo limb (Pascoal et al., 2007a). This is reminiscent of the FGF/ERK1 gradient along the PSM also produced by progressive fgf8 mRNA degradation (Dubrulle et al., 2001; Dubrulle and Pourquie ´, 2004). A good system to provide further insight into the limb segmentation clock and, ultimately, to the biological functions of molecular clocks in embryo development, is the realtime bioluminescent imaging system employed to visualize mouse embryo Hes1 expression (Masamizu et al., 2006). These are exciting times since the required technological advances to answer these and other fundamental questions are now available to the scientific community.

EMBRYO DEFECTS AND HUMAN DISORDERS: CLOCK-RELATED? What would be the expected outcome(s) of developmental timecontrol disorders in human embryos? The molecular clock underlying somitogenesis, hence axial skeleton formation, has been thoroughly studied in animal models. Mouse embryos mutated in some individual components of the segmentation clock present phenotypic alterations, which are specially evident at the level of vertebrae and ribs. On the other hand, several human syndromes have been described that are characterized by skeletal malformations. A very first approach to unveil genes belonging to a putative human embryonic segmentation clock is to compare human skeletal malformation phenotypes with those that result from mutations in somitogenesis clock genes in animal models.

Mutant Mouse Embryo Defects To understand the biological functions of the somitogenesis clock

genes, embryos of several mutant mice have been produced and analyzed during the last decade. Impairment of Notch pathwayrelated genes cycling in the PSM produces defects in somitogenesis. This contrasts with the absence of PSM segmentation defects in embryos mutated in FGF or Wnt pathway-related cycling genes, revealing that, among these pathways, Notch plays a central role in the process of somite formation.

What would be the expected outcome(s) of developmental time, control disorders in human embryos?

Mutations affecting Notch pathway components of the mouse somitogenesis clock with cyclic expression pattern, Lfng, Hes7, Dll1, impair somitogenesis. Among the features most commonly present in these embryos are incomplete somite segmentation or unclear boundaries (fused somites), disrupted A-P somite polarity, abnormal somite size and/or shape, and failure in somite differentiation (Hrabe de Angelis et al., 1997; Zhang and Gridley, 1998; Bessho et al., 2001; Hirata et al., 2004). For the cases in which it is possible to follow development in these embryos, the breakdown of paraxial mesoderm development is translated into vertebrae and rib malformations, namely in decreased number, fusions, and other morphological abnormalities (Bessho et al., 2001; Cordes et al., 2004). Mutations in Notch1 and its ligand Dll3, which present a stable expression pattern in the PSM, also affect downstream signaling, thus presenting a similar range of defects (Gruneberg, 1961; Swiatek et al., 1994; Conlon et al., 1995; Kusumi et al., 1998). However, it is interesting to notice that

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Notch1 deficiencies are much less severe than mutations in other Notch-related genes, especially contrasting with Presenilin-null embryos that completely lack somites, which was explained by a proposed c-secretase-independent role for presenilins in somite segmentation (Wong et al., 1997; Huppert et al., 2005). Strikingly, mutations in other Notch-target genes reported to oscillate in the mouse embryo PSM, such as Hes1, Hes5, and Hey1, do not seem to produce somite defects (Ishibashi et al., 1995; Hatakeyama et al., 2004; Fischer et al., 2004), revealing a certain degree of system robustness. Cyclic gene expression in the mouse PSM was also reported for some members of the Wnt and FGF signaling pathways, but the majority of mutants in these genes do not present obvious segmentation defects. This is the case for the FGF downstream targets, Bcl2l11, Dusp6, and Sprouty2 (Bouillet et al., 1999; Taketomi et al., 2005; Li et al., 2007), and the Wnt pathway genes axin2, dkk1, c-myc, sp5, and Tnfrsf19 (Davis et al., 1993; Harrison et al., 2000; Mukhopadhyay et al., 2001; Shao et al., 2005; Yu et al., 2005). A possible exception is the FGF pathway component Shp2. In Shp2-null embryos growth is usually arrested before segmentation occurs, although poorly developed somites could be observed in some cases (Saxton et al., 1997).

Human Axial Skeleton Malformations Studies on the expression of somitogenesis clock genes during human embryonic development are missing for obvious reasons, so little is known about gene expression patterns in the PSM of human embryos. A first step in determining human genes with a potential cyclic expression pattern during development was published recently in a very interesting study that identified a broad range of genes presenting an oscillating pattern of expression in a human mesenchymal stem cell model

A 10-YEAR-OLD EMBRYO SEGMENTATION CLOCK 75

(William et al., 2007). Interestingly, these comprise AXIN1, DACT1, DKK1, FGF2, HES1, HEY1, and SNAI2 genes. A link between the potential to be part of a putative human segmentation clock and the real biological functions of these genes might be revealed by the study of human malformations. Several congenital malformations of the human axial skeleton were described that result from segmentation defects during embryonic development (see also Martinez-Frias and Urioste, 1994; reviewed in Pourquie ´ and Kusumi, 2001; Gridley, 2006). These include missing or fused vertebrae, absent, fused or bifurcated ribs, and other morphological abnormalities that resemble the mouse mutants on clock genes, described above. In some cases, deletion of a specific gene was identified as causative and, interestingly, the phenotype coincides with that of the mutated mouse homolog gene, raising the possibility that a segmentation clock mechanism might be conserved, at least partially, among mammals. The most striking and better characterized human axial skeleton congenital malformations are spondylocostal dysostosis and the Alagille syndrome. Advances on the human genome sequencing project, however, may provide new tools for the comprehension of other, less understood syndromes and associations. Spondylocostal dysostosis is the common name for a group of disorders characterized by segmentation defects affecting the whole vertebral column. The most frequent features of this syndrome are vertebral anomalies such as hemivertebrae, fused vertebrae, and truncal shortening, accompanied by deformity of the ribs, which are often misaligned, with points of fusion and often reduced in number. Notably, all described types of these disorders are caused by Notch pathway-related genes: DLL3 mutations were found to be responsible for the JarchoLevin syndrome or spondylocostal dysostosis type 1 (SCDO1; MIM

#277300) (Bulman et al., 2000); mutations in the MESP2 gene characterize spondylocostal dysostosis type 2 (SCDO2; MIM #608681) (Whittock et al., 2004); and, recently, mutations in the LFNG gene were identified in individuals diagnosed for spondylocostal dysostosis type 3 (SCDO3; MIM #609813) (Sparrow et al., 2006). The spondylocostal dysostosis vertebral phenotype is reminiscent of that shown by mutations on mouse homolog genes, Dll3, Mesp2, and Lfng (Gruneberg, 1961; Saga et al., 1997; Zhang and Gridley, 1998), which makes these excellent animal models for spondylocostal dysostosis syndrome studies in what concerns axial skeleton formation (Table 1). Another interesting Notch-related disorder is the Alagille syndrome, which is an autosomal dominant multisystem anomaly that shows, among other symptoms, abnormal vertebrae formation (‘‘butterfly’’ shaped) and a decrease in interpediculate distance in the lumbar spine. Alagille syndrome type1 (ALGS1; MIM #118450) is caused by mutations in the JAG1 gene (Li et al., 1997; Oda et al., 1997). Another form of the Alagille syndrome (ALGS2; MIM #610205) was recently identified in two individuals caused by mutations in the NOTCH2 gene (McDaniell et al., 2006). Interestingly, a phenotype similar to the Alagille syndrome, but without reference to abnormal skeletal morphology, was described in mice heterozygous for the homologue genes Jag1 and Notch2 (Hamada et al., 1999; Xue et al., 1999; McCright et al., 2001; Tsai et al., 2001). This could result either from species—specific differences or from an incomplete phenotype description. The Silverman-Handmaker type of dyssegmental dysplasia (DDSH; MIM #224410), a lethal autosomal recessive form of dwarfism with characteristic anisospondyly and micromelia, is caused by mutations in the gene encoding HSPG2 (Arikawa-Hirasawa et al., 2001a). The term dyssegmental dysplasia originally refers to the abnormal

size and shape of the vertebral bodies (anisospondyly) that are characteristics of this disorder, classically attributed to errors in segmentation (reviewed in Arikawa-Hirasawa et al., 2001b). Hspg2-deficiency originates a similar phenotype in the mouse, and analysis of embryonic development indicated defective cartilage organization and differentiation (Costell et al., 1999; Arikawa-Hirasawa et al., 1999). Interestingly, Hspg2 was reported to be expressed cyclically in the mouse PSM (Deque ´ant et al., 2006), which suggests the possibility that the disorganization of chondrocyte precursors detected up to date might be caused by earlier segmentation defects. It is important to remark that heparan sulfate proteoglycans (hspg), are known to promote FGF activity (Rapraeger, 1995; Vlodavsky et al., 1996). In addition, mutations in several FGFs and FGF-receptors have been implicated in skeletal development disorders, most of them leading to skeletal dysplasias characterized by craniosynostosis, limb abnormalities, and short stature (reviewed in Chen and Deng, 2005). An FGFR2 mutation was reported to cause the Apert syndrome (Acrocephalosyndactyly; MIM #101200) which, among other features, presents fused cervical vertebrae (Kreiborg et al., 1992). Despite several studies using the Fgfr2-null mice as an animal model (Chen et al., 2003; Wang et al., 2005), the analysis of vertebral column defects has been neglected. Many other congenital malformations of the axial skeleton have been described, although causative mutations remain obscure. These include Klippel-Feil syndrome (regionalized cervical vertebral fusion), Goldenhar syndrome (hemifacial microsomia with radial defects), and syndrome associations such as ‘‘Mullerian duct aplasia, unilateral renal agenesis, and cervicothoracic somite anomalies’’ (MURCS) and ‘‘vertebral-analcardiac-tracheo-esophageal-renallimb’’ (VACTERL), among many others (reviewed in Pourquie ´

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Alagille syndrome type1 (ALGS1; MIM #118450)

Spondylocostal dysostosis type3 (SCDO3; MIM #609813)

Spondylocostal dysostosis type2 (SCDO2; MIM #608681)

Spondylocostal dysostosis type1/Jarcho– Levin syndrome (SCDO1; MIM #277300)

Human disorder

Human: JAG1

Mouse homolog: Lfng

Human: LFNG

Mouse homolog: Mesp2

Cervical and lumbar spine anomalies; multiple vertebral ossification centers in the thoracic spine Reduced number of cervical vertebrae, disrupted metameric pattern and fusion of vertebrae; reduced number of ribs, bifurcation and fusion Abnormal shape and segmentation of vertebral bodies; ‘butterfly’ vertebrae, short interpediculate distance in the lumbar spine

Severe vertebral and rib deformities: highly disorganized thoracic vertebrae; absent coccygeal vertebrae; multiple ossification centers per vertebra; ribs sometimes fused or absent; disorganized vertebrae cartilage primordia Severe disruption of the thoracic and upper cervical spine with multiple hemivertebrae Vertebrae and rib fusions; amorphous vertebral bodies with irregular alignment of ossification centers

Mouse model: Dll3-null, Dll3pu(pudgy)

Human: MESP2

Hemivertebrae and block vertebrae along entire vertebral column, deformity in form and number of ribs

Axial skeleton

Human: DLL3

Associated mutations

Hand malformations: short ulnae, short scaphoids, and short distal phalanges; reported presence of supernumerary digital flexion creases; foreshortening of the fingers

Not reported

Long, slender fingers; camptodactyly of the left index finger

Not reported

Not reported

Not reported

Reported cases of digital dysplasia

Limbs

Zhang and Gridley (1998); Evrard et al. (1998)

Li et al. (1997); Oda et al. (1997); Rosenfield et al. (1980); Raymond et al. (1989); Kamath et al. (2002) Short stature; abnormalities in the eye, heart, nervous system, craniofacies, liver and kidney

Sparrow et al. (2006)

Saga et al. (1997)

Whittock et al. (2004)

Bulman et al. (2000); Turnpenny et al. (2003); PerezComas and Garcia-Castro (1974) Gruneberg (1961); Kusumi et al. (1998); Dunwoodie et al. (2002)

References

Fused dorsal root ganglia; reproductive system disorders

No other abnormalities reported Spina bifida; abnormal axon outgrowth and fused dorsal root ganglia No other abnormalities reported

Reported some cases of occipitofacial dysplasia; abnormalities urinary system; congenital heart disease Decreased body length; kinked neural tube; abnormal spinal ganglia and nerve morphology

Additional defects reported

Information Regarding Mouse Syndrome Models or Mutants in Homolog Genes*

TABLE 1. Compilation of Human Skeletal Disorders Associated with Specific Gene Mutations, Along with

76 ANDRADE

Not reported

Skeletal abnormalities

Abnormal apoptosis in somites Marked differences in size and shape of the vertebral bodies (anisospondyly) Abnormal size and shape of ribs; vertebral bodies are increased in size and have an abnormal shape

Mouse homolog: Notch2

Human: HSPG2

Fused cervical vertebrae (in 68% cases)

Not reported

Human: FGFR2

Mouse model: Fgfr2hypomorphic

Mouse homolog: Hspg2

Not reported

Mouse model: heterozygous Jag1null/ Notch2hypomorphic Human: NOTCH2

Soft tissue and bony syndactyly of fingers and toes, occasional rhizomelic shortening elbow ankylosis No obvious abnormalities in limb lengths and no syndactyly

Shortened and bent long bones of the limbs; equinovarus deformities of the feet Long bones are about half the size of wild type and have a bended shape

Not reported

Not reported

No defects in somitogenesis or axial skeleton were observed until embryonic death Not detected

Mouse homolog: Jag1

Limbs

Axial skeleton

Associated mutations

Craniofacial anomalies, craniosynostosis; respiratory, cardiovascular, nervous system anomalies

Death between E10.5E12.5; abnormal neuronal and vascular development Growth retardation; defects in heart, liver, eye, and kidney development. Hepatic, cardiac, and ophthalmologic manifestations; renal and vascular systems less frequently perturbed Death by E11.5; abnormal apoptosis in CNS Lethal form of dwarfism; craniofacial and eye abnormalities, pulmonary hypoplasia Lethality from E10.5 to perinatally depending of severity; craniofacial, heart, nervous system abnormalities Craniofacial, mental, internal organ anomalies; craniosynostosis

Additional defects reported

Note that most are rare disorders with very few case descriptions and some phenotypes were not reported for all the affected individuals.

*

Apert syndrome (acrocephalosyndactyly, ACS1; MIM #101200)

Dyssegmental dysplasia, SilvermanHandmaker type (DDSH; MIM #224410)

Alagille syndrome type2 (ALGS2; MIM #610205)

Human disorder

TABLE 1. (Continued)

Park et al. (1995); Wilkie et al. (1995); Apert (1906); Kreiborg et al. (1992) Wang et al. (2005); Chen et al. (2003)

Arikawa-Hirasawa et al. (1999); Costell et al. (1999)

Arikawa-Hirasawa et al. (2001a)

Hamada et al. (1999)

McDaniell et al. (2006)

McCright et al. (2001, 2002)

Tsai et al. (2001); Xue et al. (1999)

References

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and Kusumi, 2001; Gridley, 2006; Kusumi and Turnpenny, 2007). Future studies should put effort in combining the knowledge of human geneticists, physicians, and developmental biologists in an attempt to dissect the origins of these and other congenital disorders.

Association of Human Axial Skeleton Congenital Malformations with Limb Abnormalities: Clues to Conserved Clock Mechanisms? Given the recent description of a molecular clock mechanism underlying limb autopod formation reminiscent of the originally described somitogenesis clock, it seems important to point out that most frequently a multiplicity of phenotypes are generated when clockrelated genes are mutated (Table 1). Cases in which both axial skeleton and limb deformities occur could give some insight on the common versus divergent nature of the embryonic segmentation clocks operating in the PSM and in the limb bud. Some of the described cases of mutations in human DLL3, LFNG, JAG1, HSPG2, and FGFR2 genes present concomitant vertebral and limb malformations (Table 1). The latter include alterations in bone length (SCDO3, DDSH, ALGS1, and ACS1), polydactyly (SCDO1), supernumerary digital flexion creases (ALGS1), and syndactyly of fingers and toes (ACS1). With the exception of the Hspg2 mouse mutant, none of the other corresponding mutant mice recapitulate mutant human limb phenotypes. However, despite extensive characterization of axial skeleton malformations in Dll3, Lfng, and Jag1 mutant mice, description of their limb phenotypes has been neglected. This is clearly the case where knowledge on human conditions may give inputs into limb developmental studies, since we are now compelled to look deeper into these mutant mice phenotypes. An interesting contribution for understanding the relationship

between somitogenesis and limb segmentation clocks comes from the phenotype caused by the loss of Presenilins in the mouse embryo. While somites do not form at all in the absence of presenilin activity (Huppert et al., 2005), limb-conditional mutant embryos have clinched forelimb digits and digit truncation with some distal phalanx missing (loss of limb elements) (Pan et al., 2005). These defects are comparable to axial skeleton anomalies caused by mutations in somitogenesis clock genes. Presenilin knockout (KO) mutant limb phenotypes could reflect the same c-secretase-independent activity of Presenilin that has been reported in somitogenesis (Huppert et al., 2005).

Future studies should put effort in combining the knowledge of human geneticists, physicians, and developmental biologists in an attempt to dissect the origins of these and other congenital disorders.

One must be cautious, however, when trying to draw conclusions from the comparison of human malformations and mutant animal models. Such perils are evidenced by: 1) the reported absence of axial skeleton defects in Jag1 mouse mutants, as opposed to clear malformations in Alagille syndrome type 1 patients; and 2) normal limb development in the Fgfr2 hypomorphic mice model for the human Apert syndrome, which is characterized by syndactyly phenotypes (Table 1). These divergences may indicate that what

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is conserved among species are the pathways and general mechanisms associated with the segmentation clock, not so much the individual players with a specific function. Therefore, real input into the origins of human skeletal malformations may only arise when we have fully mastered the segmentation clock mechanisms.

A 10-YEAR-OLD CLOCK Ten years have passed since the discovery of the molecular clock underlying vertebrate embryo segmentation (Palmeirim et al., 1997). Although we are now closer to grasping how the clock works, we have not yet begun to uncover the next fundamental question: What does it do? The cells located at the anterior end of the PSM are older than those that have just been generated by gastrulation at the caudal-most region, so the temporal order in which the PSM cells are aligned along the PSM A-P axis is translated into positional information. The somitogenesis clock has been proposed to be translating temporal information into positional information in PSM cells (Andrade et al., 2005; Freitas et al., 2005). When a cell crosses the determination front it will have undergone a finite number of cyclic gene oscillations. More rostrally-positioned PSM cells will have undergone more cycles of gene expression than cells located in a more caudal region, at any given time. A possibility is that increasing gene oscillations would result in progressive changes in cell protein content/biological activity throughout the PSM A-P axis, dictating the cell’s responsiveness to the determination front when the time is right. An alternative way for cells to register their A-P position in the PSM over time is through the wavefront gradient. PSM cells may be endowed with intrinsic positional information by Fgf8 or Wnt3a expression during gastrulation that would gradually decrease in a concerted spatial-temporal manner (Aulehla and Pourquie ´, 2006). This could be done autonomously or by driv-

A 10-YEAR-OLD EMBRYO SEGMENTATION CLOCK 79

ing clock gene oscillations, which would in turn produce the putative changes in cell behavior. The somitogenesis clock has also been proposed to be linked to vertebral axial specification through Hox gene expression regulation (Dubrulle et al., 2001; Za ´ka ´ny et al., 2001; Cordes et al., 2004). Although different anterior limits of expression of the different Hox genes can be clearly seen in the developing somites along the embryo A-P axis, it is while vertebrae-precursor cells are still in the PSM that the Hox genes truly specify vertebral identity (Carapuc ¸o et al., 2005). Studies performed by Za ´ka ´ny et al. (2001) suggest that Hox gene transcription in the PSM can be regulated by Notch-dependent clock gene oscillations. Accordingly, homeotic transformations were obtained in mouse embryos without Delta1 activity or impaired cyclic Lfng expression (either by knockout or overexpression) (Cordes et al., 2004), linking the segmentation clock to Hox-mediated axial patterning of the vertebrate body. If this is so and how it is achieved awaits further experimental clarification. We will be on the verge of making new breakthroughs regarding our knowledge on vertebrate embryo segmentation as soon as we are able to discern the biological significance of cyclic gene expression—cyclic protein production— cyclic biochemical activity. For this to happen, we must progress from gene expression studies to examine the functional proteins and their activities. The microarray study by Deque ´ant et al. (2006) showed that the molecular clock will most probably involve dynamic assembly/disassembly of protein complexes. Hes protein members are known to act as dimers and can recruit corepressor complexes for transcriptional regulation (reviewed in Kageyama et al., 2007a). In the chick embryo, Hairy1—Hairy1, Hey2—Hey2, Hairy1—Hey1, and Hairy1—Hey2 protein interactions are known to occur (Leimeister et al., 2000), and it could be the

combinatorial effect of multiple interacting cyclically produced proteins that ultimately results in the correct regulation of downstream target genes. Not much work has been done, however, to characterize the protein interactions involved in the somitogenesis clock. Direct experimental identification of such interactions is tedious and can be overwhelming. Computational modeling can be of great assistance in this task, since it can predict protein—protein interactions with biological relevance for the system (Roy et al., 2006; Cinquin, 2007).

We will be on the verge of making new breakthroughs regarding our knowledge on vertebrate embryo segmentation as soon as we are able to discern the biological significance of cyclic gene expression—cyclic protein production—cyclic biochemical activity.

The simultaneous involvement of at least three signaling pathways—Notch, FGF, and Wnt—in the somite segmentation clock (Deque ´ant et al., 2006) indicates that there must be an upstream signal regulating the overall pace of the process. What is the nature of this ‘‘pacemaker’’? Is it also responsible for initiating the clock? Theoretically, a very early ‘‘trigger’’ mechanism could initiate the clock, a ‘‘pacemaker’’ would maintain controlled clock ticking, and regulatory loops of gene expression would ensure signal propagation in the right direction (discussed above). The search for

such a pacemaker, upstream of all Notch, Wnt, and FGF pathways is on its way and will be indispensable to reach a full understanding of the nature and biological relevance of the somitogenesis clock. Other important issues that remain to be addressed are: What is the molecular nature of the initial ‘‘trigger’’ of the embryo segmentation clock? When and where does it go off? Is it exclusive to the somitogenesis process or is it a starting point for all embryonic temporal control? Moreover, are there several ‘‘independent clocks’’ ruling specific morphogenetic events or is there a unique embryonic molecular clock regulated by a universal ‘‘pacemaker’’? The knowledge gathered until now only allows us to know that components of the Notch signaling pathway start cycling in the prospective paraxial mesoderm in very early phases of chick embryonic development, correlating with ingression of its precursors into the primitive streak (Jouve et al., 2002) and that a similar molecular clock mechanism is also operating in another embryonic segmented structure—the limb (Pascoal et al., 2007b). The challenge remains and a new decade arises . . . !

ACKNOWLEDGMENTS We thank So ´lveig Thorsteinsdo ´ttir, Leonor Sau ´de, Susana Pascoal, and Ana Berta Sousa for input on the manuscript. R.P.A., l.P., and F.B. are members of the EU/FP6—Network of Excellence-‘‘Cells into Organs’’ (www. cellsintoorgans.net). Supported ˆncia by the Fundac ¸˜ ao para a Cie e Tecnologia, Portugal (grants SFRH/BPD/9432/2002 and SFRH/ BPD/17368/2004, to R.P.A. and F.B., respectively).

REFERENCES Andrade RP, Pascoal S, Palmeirim I. 2005. Thinking clockwise. Brain Res Brain Res Rev 49:114–119. Apert ME. 1906. De l’acrocephalosyndactylie. Bull Mem Soc Med Hop Paris 23:1310–1330.

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