Oscillations of the snail genes in the presomitic mesoderm coordinate segmental patterning and morphogenesis in vertebrate somitogenesis

The segmented body plan of vertebrate embryos arises through segmentation of the paraxial mesoderm to form somites. The tight temporal and spatial control underlying this process of somitogenesis is regulated by the segmentation clock and the FGF signaling wavefront. Here, we report the cyclic mRNA expression of Snail 1 and Snail 2 in the mouse and chick presomitic mesoderm (PSM), respectively. Whereas Snail genes' oscillations are independent of NOTCH signaling, we show that they require WNT and FGF signaling. Overexpressing Snail 2 in the chick embryo prevents cyclic Lfng and Meso 1 expression in the PSM and disrupts somite formation. Moreover, cells mis-expressing Snail 2 fail to express Paraxis, remain mesenchymal, and are thereby inhibited from undergoing the epithelialization event that culminates in the formation of the epithelial somite. Thus, Snail genes define a class of cyclic genes that coordinate segmentation and PSM morphogenesis.     

Determination of somite cells: independence of cell differentiation and morphogenesis

Somites are mesodermal structures which appear transiently in vertebrates in the course of their development. Cells situated ventromedially in a somite differentiate into the sclerotome, which gives rise to cartilage, while the other part of the somite differentiates into dermomyotome which gives rise to muscle and dermis. The sclerotome is further divided into a rostral half, where neural crest cells settle and motor nerves grow, and a caudal half. To find out when these axes are determined and how they rule later development, especially the morphogenesis of cartilage derived from the somites, we transplanted the newly formed three caudal somites of 2.5-day-old quail embryos into chick embryos of about the same age, with reversal of some axes. The results were summarized as follows. (1) When transplantation reversed only the dorsoventral axis, one day after the operation the two caudal somites gave rise to normal dermomyotomes and sclerotomes, while the most rostral somite gave rise to a sclerotome abnormally situated just beneath ectoderm. These results suggest that the dorsoventral axis was not determined when the somites were formed, but began to be determined about three hours after their formation. (2) When the transplantation reversed only the rostrocaudal axis, two days after the operation the rudiments of dorsal root ganglia were formed at the caudal (originally rostral) halves of the transplanted sclerotomes. The rostrocaudal axis of the somites had therefore been determined when the somites were formed. (3) When the transplantation reversed both the dorsoventral and the rostrocaudal axes, two days after the operation, sclerotomes derived from the prospective dermomyotomal region of the somites were shown to keep their original rostrocaudal axis, judging from the position of the rudiments of ganglia. Combined with results 1 and 2, this suggested that the fate of the sclerotomal cells along the rostrocaudal axis was determined previously and independently of the determination of somite cell differentiation into dermomyotome and sclerotome. (4) In the 9.5-day-old chimeric embryos with rostrocaudally reversed somites, the morphology of vertebrae and ribs derived from the explanted somites were reversed along the rostrocaudal axis. The morphology of cartilage derived from the somites was shown to be determined intrinsically in the somites by the time these were formed from the segmental plate. The rostrocaudal pattern of the vertebral column is therefore controlled by factors intrinsic to the somitic mesoderm, and not by interactions between this mesoderm and the notochord and/or neural tube, arising after segmentation.     

Differential Axial Requirements for Lunatic Fringe and Hes7 Transcription during Mouse Somitogenesis

Vertebrate segmentation is regulated by the “segmentation clock”, which drives cyclic expression of several genes in the caudal presomitic mesoderm (PSM). One such gene is Lunatic fringe (Lfng), which encodes a modifier of Notch signalling, and which is also expressed in a stripe at the cranial end of the PSM, adjacent to the newly forming somite border. We have investigated the functional requirements for these modes of Lfng expression during somitogenesis by generating mice in which Lfng is expressed in the cranial stripe but strongly reduced in the caudal PSM, and find that requirements for Lfng activity alter during axial growth. Formation of cervical, thoracic and lumbar somites/vertebrae, but not sacral and adjacent tail somites/vertebrae, depends on caudal, cyclic Lfng expression. Indeed, the sacral region segments normally in the complete absence of Lfng and shows a reduced requirement for another oscillating gene, Hes7, indicating that the architecture of the clock alters as segmentation progresses. We present evidence that Lfng controls dorsal-ventral axis specification in the tail, and also suggest that Lfng controls the expression or activity of a long-range signal that regulates axial extension.     

Synchronised cycling gene oscillations in presomitic mesoderm cells require cell-cell contact

Segmentation of the vertebrate body axis is initiated early in development with the sequential formation of somites. Somitogenesis is temporally regulated by a molecular oscillator, the segmentation clock, which acts within presomitic mesoderm (PSM) cells to drive periodic expression of the cyclic genes. We have investigated the kinetics of the progression of cycling gene expression along the PSM. Here we show that c-hairy1 and c-hairy2 mRNA expression traverses the PSM in an entirely progressive manner and that both these genes and c-Lfng maintain a similar anterior limit of expression during each cycle. However, some differences are seen regarding both the onset of a new oscillation of these genes and the duration of their expression in the caudal PSM. We also investigated whether oscillating cyclic gene expression in the PSM is entirely cell autonomous. We find that while small PSM explants are still able to maintain their oscillation schedule, once they are dissociated, PSM cells are no longer able to maintain synchronous oscillations. The results imply that cell communication or a community effect is essential for the normal pattern of cyclic gene expression in these cells.     

Signals from trunk paraxial mesoderm induce pronephros formation in chick intermediate mesoderm

We used Pax-2 mRNA expression and Lim 1/2 antibody staining as markers for the conversion of chick intermediate mesoderm (IM) to pronephric tissue and Lmx-1 mRNA expression as a marker for mesonephros. Pronephric markers were strongly expressed caudal to the fifth somite by stage 9. To determine whether the pronephros was induced by adjacent tissues and, if so, to identify the inducing tissues and the timing of induction, we microsurgically dissected one side of chick embryos developing in culture and then incubated them for up to 3 days. The undisturbed contralateral side served as a control. Most embryos cut parallel to the rostrocaudal axis between the trunk paraxial mesoderm and IM before stage 8 developed a pronephros on the control side only. Embryos manipulated after stage 9 developed pronephric structures on both sides, but the caudal pronephric extension was attenuated on the cut side. These results suggest that a medial signal is required for pronephric development and show that the signal is propagated in a rostral to caudal sequence. In manipulated embryos cultured for 3 days in ovo, the mesonephros as well as the pronephros failed to develop on the experimental side. In contrast, embryos cut between the notochord and the trunk paraxial mesoderm formed pronephric structures on both sides, regardless of the stage at which the operation was performed, indicating that the signal arises from the paraxial mesoderm (PM) and not from axial mesoderm. This cut also served as a control for cuts between the PM and the IM and showed that signaling itself was blocked in the former experiments, not the migration of pronephric or mesonephric precursor cells from the primitive streak. Additional control experiments ruled out the need for signals from lateral plate mesoderm, ectoderm, or endoderm. To determine whether the trunk paraxial mesoderm caudal to the fifth somite maintains its inductive capacity in the absence of contact with more rostral tissue, embryos were transected. Those transected below the prospective level of the fifth somite expressed Pax-2 in both the rostral and the caudal isolates, whereas embryos transected rostral to this level expressed Pax-2 in the caudal isolate only. Thus, a rostral signal is not required to establish the normal pattern of Pax-2 expression and pronephros formation. To determine whether paraxial mesoderm is sufficient for pronephros induction, stage 7 or earlier chick lateral plate mesoderm was cocultured with caudal stage 8 or 9 quail somites in collagen gels. Pax-2 was expressed in chick tissues in 21 of 25 embryos. Isochronic transplantation of stage 4 or 5 quail node into caudal chick primitive streak resulted in the generation of ectopic somites. These somites induced ectopic pronephroi in lateral plate mesoderm, and the IM that received signals from both native and ectopic somites formed enlarged pronephroi with increased Pax-2 expression. We conclude that signals from a localized region of the trunk paraxial mesoderm are both required and sufficient for the induction of the pronephros from the chick IM. Studies to identify the molecular nature of the induction are in progress.     

Interaction between Notch signalling and Lunatic fringe during somite boundary formation in the mouse.

BACKGROUND: The process of somitogenesis can be divided into three major events: the prepatterning of the mesoderm; the formation of boundaries between the prospective somites; and the cellular differentiation of the somites. Expression and functional studies have demonstrated the involvement of the murine Notch pathway in somitogenesis, although its precise role in this process is not yet well understood. We examined the effect of mutations in the Notch pathway elements Delta like 1 (Dll1), Notch1 and RBPJkappa on genes expressed in the presomitic mesoderm (PSM) and have defined the spatial relationships of Notch pathway gene expression in this region. RESULTS: We have shown that expression of Notch pathway genes in the PSM overlaps in the region where the boundary between the posterior and anterior halves of two consecutive somites will form. The Dll1, Notch1 and RBPJkappa mutations disrupt the expression of Lunatic fringe (L-fng), Jagged1, Mesp1, Mesp2 and Hes5 in the PSM. Furthermore, expression of EphA4, mCer 1 and uncx4.1, markers for the anterior-posterior subdivisions of the somites, is down-regulated to different extents in Notch pathway mutants, indicating a global alteration of pattern in the PSM. CONCLUSIONS: We propose a model for the mechanism of somite border formation in which the activity of Notch in the PSM is restricted by L-fng to a boundary-forming territory in the posterior half of the prospective somite. In this region, Notch function activates a set of genes that are involved in boundary formation and anterior-posterior somite identity.     

Compartmentalised expression of <Emphasis Type="Italic">Delta-like 1</Emphasis> in epithelial somites is required for the formation of intervertebral joints

Background  

Expression of the mouse Delta-like 1 (Dll1) gene in the presomitic mesoderm and in the caudal halves of somites of the developing embryo is required for the formation of epithelial somites and for the maintenance of caudal somite identity, respectively. The rostro-caudal polarity of somites is initiated early on within the presomitic mesoderm in nascent somites. Here we have investigated the requirement of restricted Dll1 expression in caudal somite compartments for the maintenance of rostro-caudal somite polarity and the morphogenesis of the axial skeleton. We did this by overexpressing a functional copy of the Dll1 gene throughout the paraxial mesoderm, in particular in anterior somite compartments, during somitogenesis in transgenic mice.     

The single amphioxus Mox gene: insights into the functional evolution of Mox genes,somites, and the asymmetry of amphioxus somitogenesis

Mox genes are members of the "extended" Hox-cluster group of Antennapedia-like homeobox genes. Homologues have been cloned from both invertebrate and vertebrate species, and are expressed in mesodermal tissues. In vertebrates, Mox1 and Mox2 are distinctly expressed during the formation of somites and differentiation of their derivatives. Somites are a distinguishing feature uniquely shared by cephalochordates and vertebrates. Here, we report the cloning and expression of the single amphioxus Mox gene. AmphiMox is expressed in the presomitic mesoderm (PSM) during early amphioxus somitogenesis and in nascent somites from the tail bud during the late phase. Once a somite is completely formed, AmphiMox is rapidly downregulated. We discuss the presence and extent of the PSM in both phases of amphioxus somitogenesis. We also propose a scenario for the functional evolution of Mox genes within chordates, in which Mox was co-opted for somite formation before the cephalochordate-vertebrate split. Novel expression sites found in vertebrates after somite formation postdated Mox duplication in the vertebrate stem lineage, and may be linked to the increase in complexity of vertebrate somites and their derivatives, e.g., the vertebrae. Furthermore, AmphiMox expression adds new data into a long-standing debate on the extent of the asymmetry of amphioxus somitogenesis.     

Genetic analysis of molecular oscillators in mammalian somitogenesis: clues for studies of human vertebral disorders

The repeating pattern of the human vertebral column is shaped early in development, by a process called somitogenesis. In this embryonic process, pairs of mesodermal segments called somites are serially laid down along the developing neural tube. Somitogenesis is an iterative process, repeating at regular time intervals until the last somite is formed. This process lays down the vertebrate body axis from head to tail, making for a progression of developmental steps along the rostral-caudal axis. In this review, the roles of the Notch, Wnt, fibroblast growth factor, retinoic acid and other pathways are described during the following key steps in somitogenesis: formation of the presomitic mesoderm (PSM) and establishment of molecular gradients; prepatterning of the PSM by molecular oscillators; patterning of rostral-caudal polarity within the somite; formation of somite borders; and maturation and resegmentation of somites to form musculoskeletal tissues. Disruption of somitogenesis can lead to severe vertebral birth defects such as spondylocostal dysostosis (SCD). Genetic studies in the mouse have been instrumental in finding mutations in this disorder, and ongoing mouse studies should provide functional insights and additional candidate genes to help in efforts to identify genes causing human spinal birth defects.     

A comparative analysis of Meox1 and Meox2 in the developing somites and limbs of the chick embryo

We have examined the expression pattern of the avian Meox1 homeobox gene during early development and up to late limb bud stages. Its expression pattern indicates that it is involved in somite specification and differentiation. The domains of expression are similar but different to those of Meox2. Meox1 is expressed from stage 6 in the pre-somitic mesoderm and as development proceeds, in the tail bud, the dermomyotome of the rostral somites and in the dermomyotome and sclerotome of the caudal somites, the lateral rectus muscle, truncus arteriosus of the heart and the limb buds. Unlike Meox1, Meox2 is not expressed in the pre-somitic mesoderm, but is expressed first in somites formed from stage 11 onwards. In the developing limb, both genes are expressed in the dorsal and ventral limb mesoderm in adjacent domains with a small region of overlap. In the limb bud, Meox1 is co-expressed with Meox2 but neither Meox gene is co-expressed with MyoD. These expression patterns suggest that these two genes have overlapping and distinct functions in development.     

Notch Is a Critical Component of the Mouse Somitogenesis Oscillator and Is Essential for the Formation of the Somites

Segmentation of the vertebrate body axis is initiated through somitogenesis, whereby epithelial somites bud off in pairs periodically from the rostral end of the unsegmented presomitic mesoderm (PSM). The periodicity of somitogenesis is governed by a molecular oscillator that drives periodic waves of clock gene expression caudo-rostrally through the PSM with a periodicity that matches somite formation. To date the clock genes comprise components of the Notch, Wnt, and FGF pathways. The literature contains controversial reports as to the absolute role(s) of Notch signalling during the process of somite formation. Recent data in the zebrafish have suggested that the only role of Notch signalling is to synchronise clock gene oscillations across the PSM and that somite formation can continue in the absence of Notch activity. However, it is not clear in the mouse if an FGF/Wnt-based oscillator is sufficient to generate segmented structures, such as the somites, in the absence of all Notch activity. We have investigated the requirement for Notch signalling in the mouse somitogenesis clock by analysing embryos carrying a mutation in different components of the Notch pathway, such as Lunatic fringe (Lfng), Hes7, Rbpj, and presenilin1/presenilin2 (Psen1/Psen2), and by pharmacological blocking of the Notch pathway. In contrast to the fish studies, we show that mouse embryos lacking all Notch activity do not show oscillatory activity, as evidenced by the absence of waves of clock gene expression across the PSM, and they do not develop somites. We propose that, at least in the mouse embryo, Notch activity is absolutely essential for the formation of a segmented body axis.