Vertebrate limb regeneration and the origin of limb stem cells

The existence of multipotent cells in the adult tissues and organs of those vertebrates that are capable of regeneration has been accepted for decades. Although studies of vertebrate limb regeneration have yet to identify many of the specific molecules involved in regeneration, numerous tissue grafting experiments and studies of cell lineage have contributed significantly to an understanding of the origin, activation, proliferation and cell-cell interactions of these progenitor cells. This has allowed the development of ideas about the regulation of pattern formation to restore the structure and function of lost tissues and organs. An understanding of the molecular mechanisms controlling these processes has lagged behind the dramatic advances achieved with other model organisms. However, given the intense, new research interest in stem cells over the past few years, there is good reason to be encouraged that insights about the biology of mammalian stem cells will accelerate progress in understanding the biology of regeneration in organisms that can regenerate. Advances in regeneration research will then feed back in terms of devising new strategies for therapies to induce regeneration in organisms such as humans that have traditionally been viewed as incapable of regeneration.     

Vertebrate limb development and possible clues to diversity in limb form

Chick embryos are good models for vertebrate development. The principles that underlie chick wing development have been discovered and there is increasing knowledge about the molecules involved. The importance of identifying molecules is that this provides a direct link to understanding the genetic basis of diversity in form. Chick wing development will be compared with limb development in other vertebrates. Possible mechanisms that could lead to variations in form, including limb reductions and limblessness, differences between fore- and hindlimbs, limb proportions, and interdigital webbing can be suggested.     

Pattern regulation in the embryonic chick limb: Supernumerary limb formation with anterior (non-ZPA) limb bud tissue

The formation of supernumerary limbs and limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. A “wedge” (ectoderm and mesoderm) of anterior or mid donor right wing bud (stage 21) was inserted in a slit made in a host right limb bud (stage 21) at the same position as its position of origin or to a more posterior position. The AER of the donor tissue and host wing bud were aligned with each other. Donor tissue was grafted with its dorsalventral polarity the same as the host's limb bud or reversed to that of the host's. Depending on the position of origin of the donor limb bud tissue and the position to which it was transplanted in a host, supernumerary wings or wing structures formed. Furthermore, depending on the orientation of the graft in the host, supernumerary limbs with either left or right asymmetry developed. The results of experiments performed here are considered in light of two current models which have been used to describe supernumerary limb formation: one based on local, short-range, cell-cell interactions and the other based on long-range positional signaling via a diffusible morphogen.     

Early hominin limb proportions

Recent analyses and new fossil discoveries suggest that the evolution of hominin limb length proportions is complex, with evolutionary reversals and a decoupling of proportions within and between limbs. This study takes into account intraspecific variation to test whether or not the limb proportions of four early hominin associated skeletons (AL 288-1, OH 62, BOU-VP-12/1, and KNM-WT 15000) can be considered to be significantly different from one another. Exact randomization methods were used to compare the differences between pairs of fossil skeletons to the differences observed between all possible pairs of individuals within large samples of Gorilla gorilla, Pan troglodytes, Pongo pygmaeus, and Homo sapiens. Although the difference in humerofemoral proportions between OH 62 and AL 288-1 does not exceed variation in the extant samples, it is rare. When humerofemoral midshaft circumferences are compared, the difference between OH 62 and AL 288-1 is fairly common in extant species. This, in combination with error associated with the limb lengths estimates, suggests that it may be premature to consider H. (or Australopithecus) habilis as having more apelike limb proportions than those in A. afarensis. The humerofemoral index of BOU-VP-12/1 differs significantly from both OH 62 and AL 288-1, but not from KNM-WT 15000. Published length estimates, if correct, suggest that the relative forearm length of BOU-VP-12/1 is unique among hominins, exceeding those of the African apes and resembling the proportions in Pongo.Evidence that A. afarensis exhibited a less apelike upper:lower limb design than A. africanus (and possibly H. habilis) suggests that, if A. afarensis is broadly ancestral to A. africanus, the latter did not simply inherit primitive morphology associated with arboreality, but is derived in this regard. The fact that the limb proportions of OH 62 (and possibly KNM-ER 3735) are no more human like than those of AL 288-1 underscores the primitive body design of H. habilis.     

Oromandibular limb hypogenesis syndromes

The oromandibular limb hypogenesis syndrome is a group of anomalies affecting the mandible, tongue, and maxilla with or without reductive limb anomalies. Their genetic origin is uncertain, and no drug-induced teratogen has been clearly identified. Although many similarities exist on both an embryologic and clinical level, distinction between these entities is appropriate. A new classification system with these principles in mind is presented. Two cases are presented of glossopalatine ankylosis with hypodactyly representing the thirteenth and fourteenth cited in the world literature. One patient presented with a fatal pulmonary hypoplasia not previously reported in association with this syndrome. Three of the 14 cases with reductive limb anomalies reported have had fatal outcomes.     

Lipotyphla limb myology comparison

Fore- and hindlimb muscles were dissected in four species of Lipotyphla: the western European hedgehog Erinaceus europaeus (Erinaceidae, Erinaceinae); the moonrat Echinosorex gymnura (Erinaceidae, Hylomyinae or Galericinae); the tailless tenrec Tenrec ecaudatus (Tenrecidae, Tenrecinae); and the common European white-toothed shrew Crocidura russula (Soricidae, Soricinae). This work completely reviews the limb musculature of these walking mammals. Twelve myological characters were evaluated in order to disclose phylogenetic relationships. The cladogram obtained supported previous ones based on cranial and dental characters. This study shows that myological characters are valuable in phylogenetic analyses.     

Hyaluronan in limb morphogenesis

Hyaluronan (HA) is a large glycosaminoglycan that is not only a structural component of extracellular matrices, but also interacts with cell surface receptors to promote cell proliferation, migration, and intracellular signaling. HA is a major component of the extracellular matrix of the distal subapical mesenchymal cells of the developing limb bud that are undergoing proliferation, directed migration, and patterning in response to the apical ectodermal ridge (AER), and has the functional potential to be involved in these processes. Here we show that the HA synthase Has2 is abundantly expressed by the distal subridge mesodermal cells of the chick limb bud and also by the AER itself. Has2 expression and HA production are downregulated in the proximal central core of the limb bud during the formation of the precartilage condensations of the skeletal elements, suggesting that downregulation of HA may be necessary for the close juxtaposition of cells and the resulting cell-cell interactions that trigger cartilage differentiation during condensation. Overexpression of Has2 in the mesoderm of the chick limb bud in vivo results in the formation of shortened and severely malformed limbs that lack one or more skeletal elements. Skeletal elements that do form in limbs overexpressing Has2 are reduced in length, exhibit abnormal morphology, and are positioned inappropriately. We also demonstrate that sustained HA production in micromass cultures of limb mesenchymal cells inhibits formation of precartilage condensations and subsequent chondrogenesis, indicating that downregulation of HA is indeed necessary for formation of the precartilage condensations that trigger cartilage differentiation. Taken together these results suggest involvement of HA in various aspects of limb morphogenesis.     

Regeneration inducers in limb regeneration

Limb regeneration ability, which can be observed in amphibians, has been investigated as a representative phenomenon of organ regeneration. Recently, an alternative experimental system called the accessory limb model was developed to investigate early regulation of amphibian limb regeneration. The accessory limb model contributed to identification of limb regeneration inducers in urodele amphibians. Furthermore, the accessory limb model may be applied to other species to explore universality of regeneration mechanisms. This review aims to connect the insights recently gained to emboss universality of regeneration mechanisms among species. The defined molecules (BMP7 (or2) + FGF2 + FGF8) can transform skin wound healing to organ (limb) regeneration responses. The same molecules can initiate regeneration responses in some species.     

Wnt signalling during limb development

Wnts control a number of processes during limb development--from initiating outgrowth and controlling patterning, to regulating cell differentiation in a number of tissues. Interactions of Wnt signalling pathway components with those of other signalling pathways have revealed new mechanisms of modulating Wnt signalling, which may explain how different responses to Wnt signalling are elicited in different cells. Given the number of Wnts that are expressed in the limb and their ability to induce differential responses, the challenge will be to dissect precisely how Wnt signalling is regulated and how it controls limb development at a cellular level, together with the other signalling pathways, to produce the functional limb capable of coordinated precise movements.     

The expression pattern of tomoregulin-1 in urodele limb regeneration and mouse limb development

Tomoregulin-1 (TMEFF1) was first identified as a gene implicated in pituitary secretion in Xenopus laevis. The predicted structure of TMEFF1 is that of a transmembrane protein with a highly conserved cytoplasmic tail, two follistatin domains and one modified EGF domain in its extracellular region. We report the cloning of the newt orthologue, and show that the expression of TMEFF1 is upregulated in the blastema during limb regeneration, and is also expressed in mouse embryonic limb development.