Hypothermia modulates circadian clock gene expression in lizard peripheral tissues

The molecular mechanisms whereby the circadian clock responds to temperature changes are poorly understood. The ruin lizard Podarcis sicula has historically proven to be a valuable vertebrate model for exploring the influence of temperature on circadian physiology. It is an ectotherm that naturally experiences an impressive range of temperatures during the course of the year. However, no tools have been available to dissect the molecular basis of the clock in this organism. Here, we report the cloning of three lizard clock gene homologs (Period2, Cryptochrome1, and Clock) that have a close phylogenetic relationship with avian clock genes. These genes are expressed in many tissues and show a rhythmic expression profile at 29 degrees C in light-dark and constant darkness lighting conditions, with phases comparable to their mammalian and avian counterparts. Interestingly, we show that at low temperatures (6 degrees C), cycling clock gene expression is attenuated in peripheral clocks with a characteristic increase in basal expression levels. We speculate that this represents a conserved vertebrate clock gene response to low temperatures. Furthermore, these results bring new insight into the issue of whether circadian clock function is compatible with hypothermia.     

The chick embryo: a leading model in somitogenesis studies

The vertebrate body is built on a metameric organization which consists of a repetition of functionally equivalent units, each comprising a vertebra, its associated muscles, peripheral nerves and blood vessels. This periodic pattern is established during embryogenesis by the somitogenesis process. Somites are generated in a rhythmic fashion from the presomitic mesoderm and they subsequently differentiate to give rise to the vertebrae and skeletal muscles of the body. Somitogenesis has been very actively studied in the chick embryo since the 19th century and many of the landmark experiments that led to our current understanding of the vertebrate segmentation process have been performed in this organism. Somite formation involves an oscillator, the segmentation clock whose periodic signal is converted into the periodic array of somite boundaries by a spacing mechanism relying on a traveling threshold of FGF signaling regressing in concert with body axis extension.     

Molecular clocks underlying vertebrate embryo segmentation: A 10-year-old hairy-go-round

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. © 2007 Wiley‐Liss, Inc.     

A brief history of circadian time

Recent progress in clock research has revealed major molecular components in the mechanisms responsible for circadian time keeping in mammals. The first vertebrate clock mutation (tau) was discovered in the Syrian hamster more than a decade ago and, using the power of comparative genomics, this gene has now been cloned. We now know that tau is the mammalian homologue of a Drosophila circadian clock component (double-time) that plays an important role in regulating clock protein turnover.     

Photoporation of biomolecules into single cells in living vertebrate embryos induced by a femtosecond laser amplifier

Introduction of biomolecules into cells in living animals is one of the most important techniques in molecular and developmental biology research, and has potentially broad biomedical implications. Here we report that biomolecules can be introduced into single cells in living vertebrate embryos by photoporation using a femtosecond laser amplifier with a high pulse energy and a low repetition rate. First, we confirmed the efficiency of this photoporation technique by introducing dextran, morpholino oligonucleotides, or DNA plasmids into targeted single cells of zebrafish, chick, shark, and mouse embryos. Second, we demonstrated that femtosecond laser irradiation efficiently delivered DNA plasmids into single neurons of chick embryos. Finally, we successfully manipulated the fate of single neurons in zebrafish embryos by delivering mRNA. Our observations suggest that photoporation using a femtosecond laser with a high pulse energy and low repetition rate offers a novel way to manipulate the function(s) of individual cells in a wide range of vertebrate embryos by introduction of selected biomolecules.     

Molecular basis of left-right asymmetry

In vertebrates visceral asymmetry is conserved along the left-right axis within the body. Only a small percentage of randomization (situs ambiguus), or complete reversal (situs inversus) of normal internal organ position and structural asymmetry is found in humans. A breakdown in left-right asymmetry is occasionally associated with severe malformations of the organs, clearly indicating that the regulated asymmetric patterning could have an evolutionary advantage over allowing random placement of visceral organs. Genetic, molecular and cell transplantation experiments in humans, mice, zebrafish, chick and Xenopus have advanced our understanding of how initiation and establishment of left-right asymmetry occurs in the vertebrate embryo. In particular, the chick embryo has served as an extraordinary animal model to manipulate genes, cells and tissues. This chick model system has enabled us to reveal the genetic pathways that occur during left-right development. Indeed, genes with asymmetric expression domains have been identified and well characterized using the chick as a model system. The present review summarizes the molecular and experimental studies employed to gain a better understanding of left-right asymmetry pattern formation from the first split of symmetry in embryos, to the exhibition of asymmetric morphologies in organs.     

Evidence for medial/lateral specification and positional information within the presomitic mesoderm.

In the vertebrate embryo, segmentation is built on repetitive structures, named somites, which are formed progressively from the most rostral part of presomitic mesoderm, every 90 minutes in the avian embryo. The discovery of the cyclic expression of several genes, occurring every 90 minutes in each presomitic cell, has shown that there is a molecular clock linked to somitogenesis. We demonstrate that a dynamic expression pattern of the cycling genes is already evident at the level of the prospective presomitic territory. The analysis of this expression pattern, correlated with a quail/chick fate-map, identifies a 'wave' of expression travelling along the future medial/lateral presomitic axis. Further analysis also reveals the existence of a medial/lateral asynchrony of expression at the level of presomitic mesoderm. This work suggests that the molecular clock is providing cellular positional information not only along the anterior/posterior but also along the medial/lateral presomitic axis. Finally, by using an in vitro culture system, we show that the information for morphological somite formation and molecular segmentation is segregated within the medial/lateral presomitic axis. Medial presomitic cells are able to form somites and express segmentation markers in the absence of lateral presomitic cells. By contrast, and surprisingly, lateral presomitic cells that are deprived of their medial counterparts are not able to organise themselves into somites and lose the expression of genes known to be important for vertebrate segmentation, such as Delta-1, Notch-1, paraxis, hairy1, hairy2 and lunatic fringe.     

Developmental origins and evolution of jaws: new interpretation of "maxillary" and "mandibular"

Cartilage of the vertebrate jaw is derived from cranial neural crest cells that migrate to the first pharyngeal arch and form a dorsal "maxillary" and a ventral "mandibular" condensation. It has been assumed that the former gives rise to palatoquadrate and the latter to Meckel's (mandibular) cartilage. In anamniotes, these condensations were thought to form the framework for the bones of the adult jaw and, in amniotes, appear to prefigure the maxillary and mandibular facial prominences. Here, we directly test the contributions of these neural crest condensations in axolotl and chick embryos, as representatives of anamniote and amniote vertebrate groups, using molecular and morphological markers in combination with vital dye labeling of late-migrating cranial neural crest cells. Surprisingly, we find that both palatoquadrate and Meckel's cartilage derive solely from the ventral "mandibular" condensation. In contrast, the dorsal "maxillary" condensation contributes to trabecular cartilage of the neurocranium and forms part of the frontonasal process but does not contribute to jaw joints as previously assumed. These studies reveal the morphogenetic processes by which cranial neural crest cells within the first arch build the primordia for jaw cartilages and anterior cranium.     

Watch-ing out for chick limb development

Time control is a crucial issue during embryonic development.Nevertheless, little is known about how embryonic cells measuretime. Until recently, the only molecular clock known to operateduring vertebrate embryonic development was the somitogenesisclock, exclusively functioning in coordinating the precise timingof each new pair of somites formed from the presomitic mesoderm.We have recently evidenced that a similar molecular clock alsounderlies the timing at which autopod chondrogenic precursorsare laid down to form a skeletal limb element. In addition,we herein suggest that the molecular clock is not the only parallelismthat can be established between somitogenesis and limb-bud development.In an evolutionary perspective, we support the previously proposedidea that the molecular mechanisms involved in the segmentationof the body axis may have been partially reused in the mesodermof the lateral plate, thereby allowing the emergence of pairedappendages.     

The generation and diversification of spinal motor neurons: signals and responses

Motor neurons are probably the best characterised neuronal class in the vertebrate central nervous system and have become a paradigm for understanding the mechanisms that control the development of vertebrate neurons. For many investigators working on this problem the chick embryo is the model system of choice and from these studies a picture of the steps involved in motor neuron generation has begun to emerge. These findings suggest that motor neuron generation is shaped by extracellular signals that regulate intrinsic, cell-autonomous determinants at sequential steps during development. The chick embryo has played a prominent role in identifying the sources of these signals, defining their molecular identities and determining the cell intrinsic programs they regulate.     

p68, a DEAD-box RNA helicase, is expressed in chordate embryo neural and mesodermal tissues

The p68 DEAD-box RNA helicases have been identified in diverse organisms, including yeast, invertebrates, and mammals. DEAD-box RNA helicases are thought to unwind duplexed RNAs, and the p68 family may participate in initiating nucleolar assembly. Recent evidence also suggests that they are developmentally regulated in chordate embryos. bobcat, a newly described member of this gene family, has been found in eggs and developing embryos of the ascidian urochordate, Molgula oculata. Antisense RNA experiments have implicated this gene in establishing basic chordate features, including the notochord and neural tube in ascidians (Swalla et al. 1999). We have isolated p68 homologs from chick and Xenopus in order to investigate their possible role in vertebrate development. We show that embryonic expression of p68 in chick, frog, and ascidian embryos is high in the developing brain and spinal cord as well as in the sensory vesicles. In frog embryos, p68 expression also marks the streams of migrating cranial neural crest cells throughout neural tube development and in tailbud stages, but neural crest expression is faint in chick embryos. Ascidian embryos also show mesodermal p68 expression during gastrulation and neurulation, and we document some p68 mesodermal expression in both chick and frog. Thus, as shown in these studies, p68 is expressed in early neural development and in various mesodermal tissues in a variety of chordate embryos, including chick, frog, and ascidian. Further functional experiments will be necessary to understand the role(s) p68 may play in vertebrate development.     

Chick Hairy1 protein interacts with Sap18, a component of the Sin3/HDAC transcriptional repressor complex

Background  

The vertebrate adult axial skeleton, trunk and limb skeletal muscles and dermis of the back all arise from early embryonic structures called somites. Somites are symmetrically positioned flanking the embryo axial structures (neural tube and notochord) and are periodically formed in a anterior-posterior direction from the presomitic mesoderm. The time required to form a somite pair is constant and species-specific. This extraordinary periodicity is proposed to depend on an underlying somitogenesis molecular clock, firstly evidenced by the cyclic expression of the chick hairy1 gene in the unsegmented presomitic mesoderm with a 90 min periodicity, corresponding to the time required to form a somite pair in the chick embryo. The number of hairy1 oscillations at any given moment is proposed to provide the cell with both temporal and positional information along the embryo's anterior-posterior axis. Nevertheless, how this is accomplished and what biological processes are involved is still unknown. Aiming at understanding the molecular events triggered by the somitogenesis clock Hairy1 protein, we have employed the yeast two-hybrid system to identify Hairy1 interaction partners.     

It's time to swim! Zebrafish and the circadian clock

The zebrafish represents a fascinating model for studying key aspects of the vertebrate circadian timing system. Easy access to early embryonic development has made this species ideal for investigating how the clock is first established during embryogenesis. In particular, the molecular basis for the functional development of the zebrafish pineal gland has received much attention. In addition to this dedicated clock and photoreceptor organ, and unlike the situation in mammals, the clocks in zebrafish peripheral tissues and even cell lines are entrainable by direct exposure to light thus providing unique insight into the function and evolution of the light input pathway. Finally, the small size, low maintenance costs and high fecundity of this fish together with the availability of genetic tools make this an attractive model for forward genetic analysis of the circadian clock. Here, we review the work that has established the zebrafish as a valuable clock model organism and highlight the key questions that will shape the future direction of research.     

Notch around the clock.

The establishment of a segmental pattern within the vertebrate body plan is achieved during embryogenesis by the somitogenesis process. Two molecular systems have been implicated in this phenomenon: a molecular clock linked to vertebrate segmentation and the Notch signalling pathway. Rhythmic expression of the Lunatic Fringe gene in the presomitic mesoderm has now provided a link between these two systems.     

Developmental control of chondrogenesis and osteogenesis

During vertebrate embryogenesis, bones of the vertebral column, pelvis, and upper and lower limbs, are formed on an initial cartilaginous model. This process, called endochondral ossification, is characterized by a precise series of events such as aggregation and differentiation of mesenchymal cells, and proliferation, hypertrophy and death of chondrocytes. Bone formation initiates in the collar surrounding the hypertrophic cartilage core that is eventually invaded by blood vessels and replaced by bone tissue and bone marrow. Over the last years we have extensively investigated cellular and molecular events leading to cartilage and bone formation. This has been partially accomplished by using a cell culture model developed in our laboratory. In several cases observations have been confirmed or directly made in the developing embryonic bone of normal and genetically modified chick and mouse embryos. In this article we will review our work in this field.