Developmental basis for hind-limb loss in dolphins and origin of the cetacean bodyplan.

Among mammals, modern cetaceans (whales, dolphins, and porpoises) are unusual in the absence of hind limbs. However, cetacean embryos do initiate hind-limb bud development. In dolphins, the bud arrests and degenerates around the fifth gestational week. Initial limb outgrowth in amniotes is maintained by two signaling centers, the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA). Our data indicate that the cetacean hind-limb bud forms an AER and that this structure expresses Fgf8 initially, but that neither the AER nor Fgf8 expression is maintained. Moreover, Sonic hedgehog (Shh), which mediates the signaling activity of the ZPA, is absent from the dolphin hind-limb bud. We find that failure to establish a ZPA is associated with the absence of Hand2, an upstream regulator of Shh. Interpreting our results in the context of both the cetacean fossil record and the known functions of Shh suggests that reduction of Shh expression may have occurred approximately 41 million years ago and led to the loss of distal limb elements. The total loss of Shh expression may account for the further loss of hind-limb elements that occurred near the origin of the modern suborders of cetaceans approximately 34 million years ago. Integration of paleontological and developmental data suggests that hind-limb size was reduced by gradually operating microevolutionary changes. Long after locomotor function was totally lost, modulation of developmental control genes eliminated most of the hind-limb skeleton. Hence, macroevolutionary changes in gene expression did not drive the initial reduction in hind-limb size.

Branching, segmentation and the metapterygial axis: pattern versus process in the vertebrate limb.

Explanations of the patterns of vertebrate fin and limb evolution are improving as specific hypotheses based on molecular and developmental data are proposed and tested. Comparative analyses of gene expression patterns and functions in developing limbs, and morphological patterns in embryonic, adult and fossil limbs point to digit specification as a key developmental innovation associated with the origin of tetrapods. Digit development during the fin-to-limb transition involved sustained proximodistal outgrowth and a new phase of Hox gene expression in the distal fin bud. These patterning changes in the distal limb have been explained by the linked concepts of the metapterygial axis and the digital arch. These have been proposed to account for the generation of limb pattern by sequential branching and segmentation of precartilagenous elements along the proximodistal axis of the limb. While these ideas have been very fruitful, they have become increasingly difficult to reconcile with experimental and comparative studies of fin and limb development. Here we argue that limb development does not involve a branching mechanism, and reassess the concept of a metapterygial axis in limb development and evolution.

Developmental mechanisms of vertebrate limb evolution.

Over the past few years, our understanding of the evolution of limbs has been improved by important new discoveries in the fossil record. Additionally, rapid progress has been made in identifying the molecular basis of vertebrate limb development. It is now possible to integrate these two areas of research in order to identify the molecular developmental mechanisms underlying the evolution of paired appendages in vertebrates. After the origin of paired appendages, several vertebrate lineages reduced or eliminated fins and limbs and returned to the limbless condition. Examples include eels, caecilians, snakes, slow worms and several marine mammals. Analyses of fossil and extant vertebrates show that evolution of limblessness frequently occurred together with elongation of the trunk and loss of clear morphological boundaries in the vertebral column. This may be suggestive of a common developmental mechanism linking these two processes. We have addressed this question by analysing python embryonic development at tissue, cellular and molecular levels, and we have identified a developmental mechanism which may account for evolution of limb loss in these animals.

Distribution of polarizing activity and potential for limb formation in mouse and chick embryos and possible relationships to polydactyly.

A central feature of the tetrapod body plan is that two pairs of limbs develop at specific positions along the head-to-tail axis. However, the potential to form limbs in chick embryos is more widespread. This could have implications for understanding the basis of limb abnormalities. Here we extend the analysis to mouse embryos and examine systematically the potential of tissues in different regions outside the limbs to contribute to limb structures. We show that the ability of ectoderm to form an apical ridge in response to FGF4 in both mouse and chick embryos exists throughout the flank as does ability of mesenchyme to provide a polarizing region signal. In addition, neck tissue has weak polarizing activity. We show, in chick embryos, that polarizing activity of tissues correlates with the ability either to express Shh or to induce Shh expression. We also show that cells from chick tail can give rise to limb structures. Taken together these observations suggest that naturally occurring polydactyly could involve recruitment of cells from regions adjacent to the limb buds. We show that cells from neck, flank and tail can migrate into limb buds in response to FGF4, which mimics extension of the apical ectodermal ridge. Furthermore, when we apply simultaneously a polarizing signal and a limb induction signal to early chick flank, this leads to limb duplications.

FGF and genes encoding transcription factors in early limb specification.

SnR, twist and Fgf10 are expressed in presumptive limb territories of early chick embryos. When FGF-2/FGF-8 beads are implanted in chick flank, an ectopic limb develops and SnR is irreversibly activated as early as 1 h. Ectopic Fgf10 and twist expression are activated much later at 17 and 20 h, respectively. FGF-10 can also induce SnR, but much later, and in this case activation occurs simultaneously with that of twist and Fgf10 via the Fgf8- expressing ridge. Tbx-4 and Tbx-5 are expressed in leg and wing forming regions, respectively, in a similar pattern to SnR and twist. FGF-2 leads to ectopic expression of Tbx-4 and Tbx-5 as rapidly as ectopic expression of SnR, but the patterns of ectopic transcripts suggest that induction of SnR and Tbx gene expression occur via different pathways.

Morphological analysis of the mammalian postcranium: a developmental perspective.

The past two decades have greatly improved our knowledge of vertebrate skeletal morphogenesis. It is now clear that bony morphology lacks individual descriptive specification and instead results from an interplay between positional information assigned during early limb bud deployment and its "execution" by highly conserved cellular response programs of derived connective tissue cells (e.g., chondroblasts and osteoblasts). Selection must therefore act on positional information and its apportionment, rather than on more individuated aspects of presumptive adult morphology. We suggest a trait classification system that can help integrate these findings in both functional and phylogenetic examinations of fossil mammals and provide examples from the human fossil record.

Developmental basis of limblessness and axial patterning in snakes.

The evolution of snakes involved major changes in vertebrate body plan organization, but the developmental basis of those changes is unknown. The python axial skeleton consists of hundreds of similar vertebrae, forelimbs are absent and hindlimbs are severely reduced. Combined limb loss and trunk elongation is found in many vertebrate taxa, suggesting that these changes may be linked by a common developmental mechanism. Here we show that Hox gene expression domains are expanded along the body axis in python embryos, and that this can account for both the absence of forelimbs and the expansion of thoracic identity in the axial skeleton. Hindlimb buds are initiated, but apical-ridge and polarizing-region signalling pathways that are normally required for limb development are not activated. Leg bud outgrowth and signalling by Sonic hedgehog in pythons can be rescued by application of fibroblast growth factor or by recombination with chick apical ridge. The failure to activate these signalling pathways during normal python development may also stem from changes in Hox gene expression that occurred early in snake evolution.

Molecular control of vertebrate limb development, evolution and congenital malformations.

The vertebrate limb is a powerful model system for studying the cellular and molecular interactions that determine morphological pattern during embryonic development. Recent advances in our understanding of these interactions have shed new light on the molecular mechanisms of vertebrate limb development, evolution and congenital malformations. The transfer of information has, until recently, been largely one way, with developmental studies informing our understanding of the fossil record and clinical limb anomalies; however, evolutionary and clinical studies are now beginning to shed light onto one another and onto basic developmental processes. In this review, we discuss recent advances in these fields and how they are interacting to improve our understanding of vertebrate limb biology.

Hox9 genes and vertebrate limb specification.

Development of paired appendages at appropriate levels along the primary body axis is a hallmark of the body plan of jawed vertebrates. Hox genes are good candidates for encoding position in lateral plate mesoderm along the body axis and thus for determining where limbs are formed. Local application of fibroblast growth factors (FGFs) to the anterior prospective flank of a chick embryo induces development of an ectopic wing, and FGF applied to posterior flank induces an ectopic leg. If particular combinations of Hox gene expression determine where wings and legs develop, then formation of additional limbs from flank should involve changes in Hox gene expression that reflect the type of limb induced. Here we show that the same population of flank cells can be induced to form either a wing or a leg, and that induction of these ectopic limbs is accompanied by specific changes in expression of three Hox genes in lateral plate mesoderm. This then reproduces, in the flank, expression patterns found at normal limb levels. Hox gene expression is reprogrammed in lateral plate mesoderm, but is unaffected in paraxial mesoderm. Independent regulation of Hox gene expression in lateral plate mesoderm may have been a key step in the evolution of paired appendages.

Limbs: a model for pattern formation within the vertebrate body plan.

Giant strides have been made in identifying the molecular basis of limb development. The four main phases are initiation of the limb bud, specification of limb pattern, differentiation of tissues and shaping of the limb, and growth of the miniature limb to the adult size. We will focus on the exciting advances that have been made in initiation and specification of limb pattern. The limb is a model system and the same sets of molecules are used at different times and places in vertebrate embryos. There is also remarkable conservation of the molecular mechanisms of limb development in insects and vertebrates.

Multiple functions of fibroblast growth factors in vertebrate development.

The biological consequences of ectopic FGF-4 expression were examined in chimaeric mouse embryos prepared with ES cells constitutively expressing FGF-4. The embryos exhibit abnormalities of the limbs and the anterior central nervous system. The limb phenotype consisted of the induction of early phases of limb development along the lateral ridge between the definitive fore and hind limb buds, where normally no limb outgrowth takes place. This phenotype was examined further by the implantation of beads soaked in FGF into the flank of developing chick embryos. This resulted in the induction of complete supernumerary limbs containing skeletal structures. These results indicated that FGFs have the ability to induce the outgrowth of limbs in an ectopic site and implicate FGFs in the initiation of normal limb development.

Fibroblast growth factors induce additional limb development from the flank of chick embryos.

Fibroblast growth factors (FGFs) act as signals in the developing limb and can maintain proliferation of limb bud mesenchyme cells. Remarkably, beads soaked in FGF-1, FGF-2, or FGF-4 and placed in the presumptive flank of chick embryos induce formation of ectopic limb buds, which can develop into complete limbs. The entire flank can produce additional limbs, but generally wings are formed anteriorly and legs posteriorly. FGF application activates Sonic hedgehog in cells with polarizing potential to make a discrete polarizing region. Hoxd-13 is also expressed in the ectopic bud, and an apical ectodermal ridge forms. A limb bud is thus established that can generate the appropriate signals to develop into a complete limb. The additional limbs have reversed polarity. This can be explained by the distribution of cells in the flank with potential polarizing activity. The results suggest that local production of an FGF may initiate limb development.

Z-contrast imaging of supported Pt and Pd clusters.

High-angle annular dark-field or Z-contrast microscopy was used to demonstrate that well dispersed metal supported catalysts consist of nanometer sized clusters. Depending upon the impregnated metal, different cluster sizes were observed. Grouping of Pd clusters could also be confirmed by analytical electron microscopy.

RADCOM: a computerized translation device for use during fluoroscopic examination of non-English-speaking patients.

We developed and have implemented RADCOM (RADiology COMmunicator), a computerized speech-based language translator for use during fluoroscopic examination of non-English-speaking patients. It is controlled completely by voice commands issued into a headset microphone. The system output is digital audio via a small speaker, in the native language of the patient. RADCOM currently supports more than 40 commands in more than a dozen languages. The language data base is easily expandable. We have performed more than 20 fluoroscopic examinations with the RADCOM system. The non-English-speaking patients respond well, following the selected instructions appropriately.

Frequency of certain urgent eye problems in an emergency room in Massachusetts.

The frequency and cost of emergency room care for certain ocular problems was surveyed. Superficial eye problems accounted for 2.67 percent of patients reporting to the emergency room, while 0.23 percent reported for penetrating injuries. Of the conditions surveyed, conjunctivitis was the most commonly presenting diagnosis. Abrasions and superficial foreign bodies were the next most common presentations. Together these three diagnoses accounted for nearly 75 percent of all superficial eye problems seen in the emergency room. The cost of each emergency room visit averaged $132. This study has implications for health care managers and for legislatures contemplating expanding the scope of optometric practice.