Paraxis: A New Basic Helix-Loop-Helix Proteinby Joanne Archer, Trisha Armishaw and Cindi Corbett |
Before neurulation, a mid-dorsal strip of mesoderm separates from the rest of the mesodermal sheet to form the notochord (in the chick, this is done by the Hensen's node). Paraxial mesoderm is located immediately adjacent to the notochord and becomes segmented in a rostral to caudal progression at day 8.0 of development. The segmentation is accompanied by the formation of segmental clefts at the rostral and caudal sides of the prospective somites. The somites now form from the condensation of paraxial mesoderm.
Initially, the somites appear as epithelial spheres. But shortly after their development, the somites become compartmentalized into the dorsal-lateral dermamyotome and the ventral-medial sclerotome. The cells of the sclerotome will surround the neural tube to give rise to the vertebrae, the invertebral discs and the ribs. Once the sclerotome cells are in place they begin to differentiate into cartilage, which is eventually replaced by bone. The dermamyotome also subdivides into the upper layer of dermatome, which will become the dermis of dorsal skin, and the myotome, which expands ventrally and gives rise to the axial musculature.
Relatively little is known about the genetic pathways that control the formation and patterning of mesoderm in mammals nor the pathways that direct the formation of somites with their subsequent subdivision into dermatome, myotome and sclerotome.
Substantial progress has been made in identifying genes that control muscle cell determination. The myogenic basic helix-loop-helix (bHLH) factors (MyoD, myogenin, myf5, and MRF4) belong to a family of rapidly growing bHLH proteins that regulate cell proliferation and differentiation. bHLH proteins contain a basic region comprised of positively charged amino acids that mediate binding to DNA and a HLH domain that facilitates dimerization. The bHLH proteins are regulated by dimerization. When the bHLH proteins dimerize to the bHLH proteins of the E protein class, the resulting heterodimers activate gene expression by binding to an E box consensus sequence in target DNA. Basic helix-loop-helix protein function is also attenuated when they bind to the HLH proteins of the Id protein family. This down-regulation occurs because the Id protiens lack the basic region that facilitates binding to the DNA.
Thus, these heterodimeric complexes are incapable of binding to the DNA. In most cases, these bHLH proteins belong to smaller subfamilies whose members show overlapping but distinct expression along different points in the pathways that lead to cell differentiation.
Recently Burgess, Cserjesi, Ligan and Olson cloned a gene encoding a bHLH protein named Scleraxis. Scleraxis is expressed in the sclerotome in the precursors of the axial skeleton, long bones and in the condensing cartilage of the nose and face as well as in the connective tissue. Because other bHLH proteins belong to small families whose members share high homology within the bHLH regions, Burgess et al. searched for related bHLH proteins by low stringency screening of a day 13 mouse embryo cDNA library. A portion of the Scleraxis cDNA containing the bHLH region was used as a probe.
Burgess et al. found three cDNA clones that hybridized to the Scleraxis cDNA probe under low, but not high, stringency conditions. The sequencing of these 3 cDNA clones revealed that they were overlapping and encoded a bHLH protein that shared extensive homology with the bHLH regions of scleraxis. Burgess et al. named this new bHLH protein Paraxis because of its expression in the paraxial mesoderm. Paraxis shares an 89% amino acid identity with Scleraxis in the bHLH region, where Scleraxis and Paraxis only differ at 6 residues. Of all the bHLH proteins cloned to date, Paraxis showed the highest homology to Scleraxis although it does share some homology with other bHLH proteins.
Paraxis has been found to contain the conserved amino acids that define the bHLH family of transcription factors. It contains conserved amino acids in the basic region that have been shown in other bHLH proteins to mediate DNA binding. But, also note that in the basic region, the central portion that influences DNA sequence recognition is unlike that of any other bHLH. This is to be expected as binding of Paraxis to a different portion of the DNA would influence the transcription of different genes.
Paraxis is expressed in the paraxial mesoderm where its expression progresses rostro-caudally immediately preceding somite formation. Paraxis is also expressed in the newly-formed epithelial somites. As somites mature, Paraxis expression is down-regulated in the myotome. These expression patterns of Paraxis were determined during mouse embryogenesis by in situ hybridization experiments beginning at day 7.5 of development. For these experiments, sense and antisense [35S]UTP-labeled probes were synthesized from a 1.2kb Paraxis cDNA insert cloned into pBSKII (a plasmid vector) using T3 and T7 RNA polymerase, respectively. The probes for Paraxis were derived from the regions of the Paraxis mRNAs that are not conserved between genes. Hence, there was no cross-hybridization of the Paraxis probe to Scleraxis transcripts.
Results from the in situ hybridization experiments showed that the Paraxis transcripts can first be detected at a low level on day 7.5 of development in mesodermal cells in the posterior portion of the embryo that are fated to become the paraxial mesoderm. At day 8.5 of development, the Paraxis transcripts are present at high levels throughout the uncompartmentalized epithelial somites. Paraxis expression is also observed in the newly compartmentalized dermatome, myotome and sclerotome.
By 9.5 days of development of the embryo, expression of Paraxis can be seen in the newly-formed caudal somites. Although there is down-regulation of Paraxis expression in the myotome, expression is maintained in the dermatome and sclerotome after the myotome forms. A human homolog has recently been found as well: bHLH-EC2. This is down-regulated in the myotome as in the mouse, but there is no bHLH-EC2 expression in the sclerotome.
Burgess et al. also prepared a tangential section of a mouse embryo at day 9.5 of development, and it illustrates Paraxis expression in the differentiated somites and in the dorsal region of the forelimb. A question is raised as to how Paraxis can be found in the forelimb when its expression is down-regulated in the myotome. Skeletal muscle in the limbs is derived from myogenic precursors that migrate from the ventral-lateral edge of the dermamyotome. Thus, the Paraxis-expressing cells observed in these forelimbs and hindlimbs are probably derived from the dermamyotome, in which Paraxis is expressed in high levels.
Whole mount in situ hybridization of a 9.5 postcoital mouse embryo was also conducted. In the whole mount, Paraxis expression was localized to the more rostral region in which somites were about to form; expression preceeded somite formation by a distance equivalent to 2 somites. Thus, the Paraxis expression declines as the somites mature toward the rostral end, and expression of Paraxis in the paraxial mesoderm and newly-formed somites progressed as a wave caudally between days 8.0 and 12.5 of mouse embryo development.
Northern blot analysis was also used to determine expression of Paraxis in adult muscle tissues. Paraxis was only detected in skeletal muscle and heart muscle. But Paraxis expression was not significantly expressed in these tissues before birth, and it is still unknown exactly which cell types in these tissues are expressing Paraxis. More research is needed in this area.
The expression of Paraxis in the somites preceded that of Scleraxis, which is expressed in the sclerotome during early development (approximately 10.5 days of development). Within the sclerotome, Scleraxis is expressed initially in a pattern overlapping that of Paraxis, but as Paraxis expression declines, Scleraxis expression increases in the chondroblasts that will give rise to the prevertebrae and invertebral discs. Scleraxis expression is also observed in other chondrogenic progenitors throughout the embryo (such as in the precursors of the axial and appendicular skeleton and in the cranial mesenchyme of the frontal-nasal mass).
It can be concluded that Paraxis and Scleraxis represent a new subclass of bHLH proteins that are expressed in the paraxial mesoderm and its derivatives. The overlapping expression patterns of Paraxis and Scleraxis suggest that they both function in a regulatory pathway that leads to the formation/ differentiation of somatic cell lineages. Because the expression of Paraxis is uniform throughout the epithelial somite, it seems likely that Paraxis alone does not determine the subsequent specification of somite cells. Burgess et al. investigated whether the forced expression of Paraxis could induce the expression of muscle or sclerotomal markers in transfected fibroblasts, but they observed no expression of either the muscle or sclerotomal markers. Therefore, if Paraxis is required for the formation of somite derived lineages, it may require other factors or environmental cues for its activity. (These factors may by supplied by the neural tube/ notochord, as experiments have shown that the fates of somites are plastic and are dependent on signals from the neural tube and notochord to specify whether they become committed to myogenic, chondrogenic or dermal cell lineages.)
Ordahl and Le Douarin did transplantation experiments with chick and quail epithelial somites and changed the lateral and medial halves of the somites. The exchanged halves showed plasticity, and their subsequent fates depended on their location with respect to the notochord/neural tube. It remains to be determined whether Paraxis expression requires signals from the neural tube and notochord.
Interestingly, the expression patterns of Paraxis and Scleraxis are similar to the expression patterns of the Pax genes. The Pax genes alter expression in the paraxial mesoderm and its derivatives, but unlike Paraxis and Scleraxis, the Pax genes are also expressed in the neural tube. Therefore, it will be very interesting to determine if Paraxis and Scleraxis act in the same developmental pathways as the Pax genes. If so, these genes may give clues to the roles of Paraxis and Scleraxis in somite development.
In conclusion, the sequential and overlapping patterns of the expression of Paraxis and Scleraxis in the paraxial mesoderm and the somites indicate that they are involved in somite formation and establishment of the somite-derived cell lineages. Since Paraxis and Scleraxis are bHLH proteins, they are thought to act like other bHLH proteins such as the myogenic bHLH proteins ,which are known to specify muscle cell fates. Still more experimentation with Paraxis and Scleraxis is needed to provide insight into their potential roles in development of the somites and somite-derived cell lineages.
| Gametogenesis | |||||
| The Foundations of Developmental Biology | |
The Developmental Biology Journal Club | |||
Browder, L.W., Erickson, C.A. and Jeffery, W.R. Developmental Biology. Third edition. Saunders College Pub. Philadelphia.
Burgess, R., P. Cserjesi, K.L. Ligon and E.N. Olson. 1995. Paraxis: a basic helix-loop-helix protein expressed in paraxial mesoderm and developing somites. Develop. Biol. 168: 296-306.
Ordahl, C.P. and N.M. Le Douarin. 1992. Two myogenic lineages within the developing somite. Development 114: 339-353.
Joanne Archer, Trisha Armishaw and Cindi Corbett. 1997. Paraxis: A New Basic Helix-Loop-Helix Protein. In L.W. Browder (Ed.), Developmental Biology, <http://www.ucalgary.ca/~browder>.
Copyright © 1996 Joanne Archer, Trisha Armishaw, Cindi Corbett.This material may be reproduced for educational purposes only provided credit is given to the original source.
December 3, 1996