Interplay between the molecular signals that control vertebrate limb development

Vertebrate limbs display three obvious axes of asymmetry. These three axes are referred to as proximal-distal (Pr-D; shoulder to digit tips), anterior-posterior (A-P; thumb to little finger), and dorsal-ventral (D-V; back of hand to palm). At a molecular level, it is now possible to define the signals that control patterning of each of the three axes of the developing limb. These signals do not work in isolation though but rather their activity must be integrated such that the various limb elements are coordinately formed with relation to these three axes. This review will provide an overview of the intricate medley amongst the molecular signals that serve to establish and coordinate patterning information along the three primary axes of the limb.     

Muscle development during vertebrate limb outgrowth

Limb development has become one of the model systems for studying vertebrate development. One crucial aspect in limb development is the origin, differentiation and patterning of muscle. Much progress has been made in recent years towards understanding this process. One of the general observations is that the genes involved in limb muscle development appear to be very similar to those involved in muscle development in other regions of the embryo. In this review, we summarize some of the genes and mechanisms that regulate limb muscle development and discuss various avenues along which a deeper understanding can be gained of how muscle cells originate and differentiate in different tissues during vertebrate development.     

Molecular evolution of the vertebrate immune system

Adaptive immunity is unique to the vertebrates, and the molecules involved (including immunoglobulins, T cell receptors and the major histocompatibility complex molecules) seem to have diversified very rapidly early in vertebrate history. Reconstruction of gene phylogenies has yielded insights into the evolutionary origin of a number of molecular systems, including the complement system and the major histocompatibility complex (MHC). These analyses have indicated that the C5 component of complement arose by gene duplication prior to the divergence of C3 and C4, which suggests that the alternative complement pathway was the first to evolve. In the case of the MHC, phylogenetic analysis supports the hypothesis that MHC class II molecules evolved before class I molecules. The fact that the MHC-linked proteasome components that specifically produce peptides for presentation by class I MHC appear to have originated before the separation of jawed and jawless vertebrates suggests that the MHC itself may have been present at this time. Immmune system gene families have evolved by gene duplication, interlocus recombination and (in some cases) positive Darwinian selection favoring diversity at the amino acid level.     

Achieving bilateral symmetry during vertebrate limb development

While the various internal organs of vertebrates display many obvious left–right asymmetries in their location and/or morphology, external features exhibit a high degree of bilateral symmetry. How this external bilateral symmetry is established during development is largely unknown. In this review, we explore several mechanisms, in place during development, that regulate the final size of the limb. These mechanisms rely on the presence of positive signaling feedback loops during limb bud growth. Through the activity of these signaling loops and their eventual breakdown when the limb bud has reached a certain size, bilateral symmetry can be achieved.     

Retinoid receptors in vertebrate limb development.

Although the precise role of retinoids in limb development remains obscure, the finding that retinoic acid can produce major alterations in limb patterning suggests that this ligand might be involved in the process of limb morphogenesis. Here we describe the patterns of expression of retinoic acid receptors and cytosolic retinoid binding proteins during the course of limb morphogenesis. Examining the distribution of these molecules in the limb and correlating their presence with important processes in limb development could help elucidate their possible functions.     

Morphogen gradients in vertebrate limb development.

The developing limb is an excellent model for pattern formation in vertebrate embryos. Signalling by the polarizing region controls limb pattern across the antero-posterior axis of the chick limb. It was suggested first on theoretical grounds that signalling by the polarizing region could involve a morphogen gradient. Embryological manipulations provided evidence consistent with this model and, more recently, signalling molecules associated with the polarizing region have been identified and tested for their role as morphogens. It is still not clear whether any of the known molecules act directly as a morphogen. The extension of the morphogen model to patterning along the other axes of the limb has been proposed but this may not be applicable.     

Comparative genomics in vertebrate evolution and development

The vast quantities of publicly available DNA sequencing data and genome resources are enabling biologists to investigate age-old problems in biology that were not addressable previously. In this review, we discuss how comparative genomics is practiced and how the data can be used to make biological inferences with respect to vertebrate evolution and development. Examples are taken from the well-known HOX clusters, which are always a high-priority target for genomic analyses due to their inferred role in the evolution of metazoans. In addition, we briefly discuss the application of genomic approaches to problems in comparative endocrinology.     

Opposing RA and FGF signals control proximodistal vertebrate limb development through regulation of Meis genes

Vertebrate limbs develop in a temporal proximodistal sequence, with proximal regions specified and generated earlier than distal ones. Whereas considerable information is available on the mechanisms promoting limb growth, those involved in determining the proximodistal identity of limb parts remain largely unknown. We show here that retinoic acid (RA) is an upstream activator of the proximal determinant genes Meis1 and Meis2. RA promotes proximalization of limb cells and endogenous RA signaling is required to maintain the proximal Meis domain in the limb. RA synthesis and signaling range, which initially span the entire lateral plate mesoderm, become restricted to proximal limb domains by the apical ectodermal ridge (AER) activity following limb initiation. We identify fibroblast growth factor (FGF) as the main molecule responsible for this AER activity and propose a model integrating the role of FGF in limb cell proliferation, with a specific function in promoting distalization through inhibition of RA production and signaling.     

Molecular evolution of proteins involved in vertebrate phototransduction

Vision is one of the most important senses for vertebrates. As a result, vertebrates have evolved a highly organized system of retinal photoreceptors. Light triggers an enzymatic cascade, called the phototransduction cascade, that leads to the hyperpolarization of photoreceptors. It is expected that a systematic comparison of phototransduction cascades of various vertebrates can provide insights into the diversity of vertebrate photoreceptors and into the evolution of vertebrate vision. However, only a few attempts have been made to compare each phototransduction protein participating in this cascade. Here, we determine phylogenetic trees of the vertebrate phototransduction proteins and compare them. It is demonstrated that vertebrate opsin sequences fall into five fundamental subfamilies. It is speculated that this is crucial for the diversity of the spectral sensitivity observed in vertebrate photoreceptors and provides the vertebrates with the molecular tools to discriminate the color of incident light. Other phototransduction proteins can be classified into only a few subfamilies. Cones generally share isoforms of phototransduction proteins that are different from those found in rods. The difference in sensitivity to light between rods and cones is likely due to the difference in the molecular properties of these isoforms. The phototransduction proteins seem to have co-evolved as a system. Switching the expression of these isoforms may characterize individual vertebrate photoreceptors.     

Modularity in vertebrate brain development and evolution.

Embryonic modularity and functional modularity are two principles of brain organization. Embryonic modules are histogenetic fields that are specified by position-dependent expression of patterning genes. Within each embryonic module, secondary and higher-level pattern formation takes places during development, finally giving rise to brain nuclei and cortical layers. Defined subsets of these structures become connected by fiber tracts to form the information-processing neural circuits, which represent the functional modules of the brain. We review evidence that a group of cell adhesion molecules, the cadherins, provides an adhesive code for both types of modularity, based on a preferentially homotypic binding mechanism. Embryonic modularity is transformed into functional modularity, in part by translating early-generated positional information into an array of adhesive cues, which regulate the binding of functional neural structures distributed across the embryonic modules. Brain modularity may provide a basis for adaptability in evolution.     

The role of Hox genes during vertebrate limb development

The potential role of Hox genes during vertebrate limb development was brought into focus by gene expression analyses in mice (P Dolle, JC Izpisua-Belmonte, H Falkenstein, A Renucci, D Duboule, Nature 1989, 342:767-772), at a time when limb growth and patterning were thought to depend upon two distinct and rather independent systems of coordinates; one for the anterior-to-posterior axis and the other for the proximal-to-distal axis (see D Duboule, P Dolle, EMBO J 1989, 8:1497-1505). Over the past years, the function and regulation of these genes have been addressed using both gain-of-function and loss-of-function approaches in chick and mice. The use of multiple mutations either in cis-configuration in trans-configuration or in cis/trans configurations, has confirmed that Hox genes are essential for proper limb development, where they participate in both the growth and organization of the structures. Even though their molecular mechanisms of action remain somewhat elusive, the results of these extensive genetic analyses confirm that, during the development of the limbs, the various axes cannot be considered in isolation from each other and that a more holistic view of limb development should prevail over a simple cartesian, chess grid-like approach of these complex structures. With this in mind, the functional input of Hox genes during limb growth and development can now be re-assessed.     

Molecular evolution of shark and other vertebrate DNases I.

We purified pancreatic deoxyribonuclease I (DNase I) from the shark Heterodontus japonicus using three-step column chromatography. Although its enzymatic properties resembled those of other vertebrate DNases I, shark DNase I was unique in being a basic protein. Full-length cDNAs encoding the DNases I of two shark species, H. japonicus and Triakis scyllia, were constructed from their total pancreatic RNAs using RACE. Nucleotide sequence analyses revealed two structural alterations unique to shark enzymes: substitution of two Cys residues at positions 101 and 104 (which are well conserved in all other vertebrate DNases I) and insertion of an additional Thr or Asn residue into an essential Ca(2+)-binding site. Site-directed mutagenesis of shark DNase I indicated that both of these alterations reduced the stability of the enzyme. When the signal sequence region of human DNase I (which has a high alpha-helical structure content) was replaced with its amphibian, fish and shark counterparts (which have low alpha-helical structure contents), the activity expressed by the chimeric mutant constructs in transfected mammalian cells was approximately half that of the wild-type enzyme. In contrast, substitution of the human signal sequence region into the amphibian, fish and shark enzymes produced higher activity compared with the wild-types. The vertebrate DNase I family may have acquired high stability and effective expression of the enzyme protein through structural alterations in both the mature protein and its signal sequence regions during molecular evolution.