In vivo mechanical loading rapidly activates ß-catenin signaling in osteocytes through a prostaglandin mediated mechanism.

The response of the skeleton to loading appears to be mediated through the activation of the Wnt/ß-catenin signaling pathway and osteocytes have long been postulated to be the primary mechanosensory cells in bone. To examine the kinetics of the mechanoresponse of bone and cell types involved in vivo, we performed forearm loading of 17-week-old female TOPGAL mice. ß-catenin signaling was observed only in embedded osteocytes, not osteoblasts, at 1h post-loading, spreading to additional osteocytes and finally to cells on the bone surface by 24h. This early activation at 1h appeared to be independent of receptor (Lrp5/6) mediated activation as it occurred in the presence of the inhibitors sclerostin and/or Dkk1. The COX-2 inhibitor, Carprofen, blocked the activation of ß-catenin signaling and decline in sclerostin positive osteocytes post-loading implying an important role for prostaglandin. In vitro, PI3K/Akt activation was shown to be required for ß-catenin nuclear translocation downstream from prostaglandin in MLO-Y4 osteocyte-like cells supporting this mechanism. Downstream targets of ß-catenin signaling, sclerostin and Dkk1, were also examined and found to be significantly downregulated in osteocytes in vivo at 24h post-loading. The pattern of initially activated osteocytes appeared random and in order to understand this heterogeneous expression, a novel finite element model of the strain field in the ulna was developed, which predicts highly variable local magnitudes of strain experienced by osteocytes. In summary, both in vivo and in vitro models show the rapid activation of ß-catenin in response to load through the early release of prostaglandin and that strain fields in the bone are extremely heterogeneous resulting in heterogeneous activation of the ß-catenin pathway in osteocytes in vivo.

The WNT antagonist cSFRP2 modulates programmed cell death in the developing hindbrain.

In the avian hindbrain, the loss of premigratory neural crest cells from rhombomeres 3 and 5 (r3, r5) through programmed cell death contributes to the patterning of emigrant crest cells into three discrete streams. Programmed cell death is induced by the upregulation of Bmp4 and Msx2 in r3 and r5. We show that cSFRP2, a WNT antagonist, is expressed in the even-numbered rhombomeres and that over-expression of cSfrp2 inhibits Bmp4 expression in r3 and r5, preventing programmed cell death. By contrast, depleting cSFRP2 function in r4 results in elevated levels of Msx2 expression and ectopic programmed cell death, as does overexpression of Wnt1. We propose that programmed cell death in the rhombencephalic neural crest is modulated by pre-patterned cSfrp2 expression and a WNT-BMP signalling loop.

Specific craniofacial cartilage dysmorphogenesis coincides with a loss of dlx gene expression in retinoic acid-treated zebrafish embryos.

Treatments of zebrafish embryos with retinoic acid (RA), a substance known to cause abnormal craniofacial cartilage development in other vertebrates, result in dose- and stage-dependent losses of dlx homeobox gene expression in several regions of the embryo. Dlx expression in neural crest cells migrating from the hindbrain and in the visceral arch primordia is particularly sensitive to RA treatment. The strongest effects are observed when RA is administered prior to or during crest cell migration but effects can also be observed if RA is applied when the cells have entered the primordia of the arches. Losses of dlx expression correlate either with the loss of cartilage elements originating from hindbrain neural crest cells or with abnormal morphology of these elements. Cartilage elements that originate from midbrain neural crest cells, which do not express dlx genes, are less affected. Taken together with the observation that the normal patterns of visceral arch dlx expression just prior to cartilage condensation resemble the morphology of the cartilage elements that are about to differentiate, our results suggest that dlx genes are an important part of a multi-step process in the development of a subset of craniofacial cartilage elements.

Relationship between the genomic organization and the overlapping embryonic expression patterns of the zebrafish dlx genes.

To understand the relationship between the expression and the genomic organization of the zebrafish dlx genes, we have determined the genomic structure of the dlx2 and dlx4 loci. This led to the identification of the zebrafish dlx1 and dlx6 genes, which are closely linked to dlx2 and dlx4, respectively. Therefore, the inverted convergent configuration of Dlx genes is conserved among vertebrates. Analysis of the expression patterns of dlx1 and dlx6 showed striking similarities to those of dlx2 and dlx4, respectively, the genes to which they are linked. Furthermore, the expression patterns of dlx3 and dlx7, which likely constitute a third pair of convergently transcribed genes, are indistinguishable. Thus, the overlapping expression patterns of linked Dlx genes during embryonic development suggest that they share cis-acting sequences that control their spatiotemporal expression. The evolutionary conservation of the genomic organization and combinatorial expression of Dlx genes in distantly related vertebrates suggest tight control mechanisms that are essential for their function during development.

The evolution of the vertebrate Dlx gene family.

The vertebrate Dlx gene family consists of homeobox-containing transcription factors distributed in pairs on the same chromosomes as the Hox genes. To investigate the evolutionary history of Dlx genes, we have cloned five new zebrafish family members and have provided additional sequence information for two mouse genes. Phylogenetic analyses of Dlx gene sequences considered in the context of their chromosomal arrangements suggest that an initial tandem duplication produced a linked pair of Dlx genes after the divergence of chordates and arthropods but prior to the divergence of tunicates and vertebrates. This pair of Dlx genes was then duplicated in the chromosomal events that led to the four clusters of Hox genes characteristic of bony fish and tetrapods. It is possible that a pair of Dlx genes linked to the Hoxc cluster has been lost from mammals. We were unable to distinguish between independent duplication and retention of the ancestral state of bony vertebrates to explain the presence of a greater number of Dlx genes in zebrafish than mammals. Determination of the linkage relationship of these additional zebrafish Dlx genes to Hox clusters should help resolve this issue.