Andrew J. Bendall

Role of Dlx genes in craniofacial morphogenesis: Dlx2 influences skeletal patterning by inducing ectomesenchymal aggregation in ovo

SUMMARY Dlx homeodomain transcription factors are expressed in neural crest‐derived mesenchyme of the pharyngeal arches and are required for patterning of the craniofacial skeleton. However, the cellular and molecular mechanisms by which Dlx factors control skeletogenesis in the facial primordia are unclear. We have investigated the function of Dlx2 and Dlx5 by sustained misexpression in ovo. We find that RCAS‐Dlx2‐ and RCAS‐Dlx5‐infected avian embryos exhibit very similar patterns of local, stereotypical changes in skeletal development in the upper jaw. The changes include ectopic dermal bone along the jugal arch, and ectopic cartilages that develop between the quadrate and the trabecula. The ectopic cartilage associated with the trabecula is reminiscent of a normally occurring element in this region in some bird taxa. Analysis of the distribution of RCAS‐Dlx2‐infected cells suggests that Dlx2 induces aggregation of undifferentiated mesenchyme, which subsequently develops into the ectopic skeletal elements. Comparison of infected embryos with restricted or widespread misexpression, and of embryos in which Dlx genes were delivered to migratory or postmigratory neural crest, indicate that there are limited regions of competence in which the ectopic elements can arise. The site‐specific differentiation program that the aggregates undergo may be dependent on local environmental signals. Our results suggest that Dlx factors mediate localization of ectomesenchymal subpopulations within the pharyngeal arches and in doing so define where skeletogenic condensations will arise. Consequently, variation in Dlx expression or activity may have influenced the morphology of jaw elements during vertebrate evolution.

Dlx5 Is a Cell Autonomous Regulator of Chondrocyte Hypertrophy in Mice and Functionally Substitutes for Dlx6 during Endochondral Ossification

The axial and appendicular skeleton of vertebrates develops by endochondral ossification, in which skeletogenic tissue is initially cartilaginous and the differentiation of chondrocytes via the hypertrophic pathway precedes the differentiation of osteoblasts and the deposition of a definitive bone matrix. Results from both loss-of-function and misexpression studies have implicated the related homeobox genes Dlx5 and Dlx6 as partially redundant positive regulators of chondrocyte hypertrophy. However, experimental perturbations of Dlx expression have either not been cell type specific or have been done in the context of endogenous Dlx5 expression. Thus, it has not been possible to conclude whether the effects on chondrocyte differentiation are cell autonomous or whether they are mediated by Dlx expression in adjacent tissues, notably the perichondrium. To address this question we first engineered transgenic mice in which Dlx5 expression was specifically restricted to immature and differentiating chondrocytes and not the perichondrium. Col2a1-Dlx5 transgenic embryos and neonates displayed accelerated chondrocyte hypertrophy and mineralization throughout the endochondral skeleton. Furthermore, this transgene specifically rescued defects of chondrocyte differentiation characteristic of the Dlx5/6 null phenotype. Based on these results, we conclude that the role of Dlx5 in the hypertrophic pathway is cell autonomous. We further conclude that Dlx5 and Dlx6 are functionally equivalent in the endochondral skeleton, in that the requirement for Dlx5 and Dlx6 function during chondrocyte hypertrophy can be satisfied with Dlx5 alone.

Expression patterns of ShcD and Shc family adaptor proteins during mouse embryonic development

The Src homology and collagen (Shc) proteins function as molecular adaptors in signaling pathways mediated by a variety of cell surface receptors. Of the four mammalian Shc proteins, ShcD is the least characterized. To this end, ShcD expression was documented and compared to that of other Shc family proteins. In the developing mouse embryo, expression of ShcD overlaps with that of other Shc proteins in the central nervous system, with specific distribution in post‐mitotic neurons. In addition, robust ShcD expression is seen within differentiated epithelial cells of several organs, as well as in skeletal and cardiac muscle, and various tissues of neural crest origin. Interestingly, all Shc family members are expressed in hypertrophic chondrocytes, the first report of Shc protein expression in the developing skeleton. The unique tissue distribution patterns of Shc proteins likely contribute to their complex tissue‐specific signaling functions during embryogenesis. Developmental Dynamics, 2011.

Dlx5- and Dlx6-mediated chondrogenesis: Differential domain requirements for a conserved function.

During endochondral ossification in the vertebrate limb, multipotent mesenchymal cells first differentiate into chondroblasts (chondrogenesis) that further differentiate (via chondrocyte hypertrophy) to a terminal cellular phenotype. Dlx5 and Dlx6 are functionally redundant regulators of chondrocyte hypertrophy. We now show that Dlx5 and Dlx6 also regulate the earlier step of chondrogenesis in the limb. Limb bud mesenchymal cells from Dlx5/6(-/-) embryos show reduced chondrogenesis compared to wild-type littermates, and expression of either Dlx5 or Dlx6 stimulated differentiation of limb bud mesenchymal cells to chondroblasts. The functional overlap between Dlx5 and Dlx6 occurs despite the fact that the amino- and carboxyl-terminal domains of the encoded proteins are dissimilar. In order to reconcile the disparity between the divergent structures of Dlx5 and Dlx6 with their overlapping biological functions, we investigated the domain requirements and transcriptional activities associated with Dlx5- and Dlx6-mediated chondrogenesis. We find distinct domain requirements for the chondrogenic function of these related homeoproteins, indicating divergent molecular mechanisms of action.

Direct evidence of allele equivalency at the Dlx5/6 locus

The retention of paralogous regulatory genes is a vertebrate hallmark and likely underpinned vertebrate origins. Dlx genes belong to a family of paralogous transcription factors whose evolutionary history of gene expansion and divergence is apparent from the gene synteny, shared exon–intron structure, and coding sequence homology found in extant vertebrate genomes. Dlx genes are expressed in a nested combination within the first pharyngeal arch and knockout studies in mice clearly point to a “Dlx code” that operates to define maxillary and mandibular position in the first arch. The nature of that code is not yet clear; an important goal for understanding Dlx gene function in both patterning and differentiation lies in distinguishing functional inputs that are paralog‐specific (a qualitative model) versus Dlx family‐generic (a quantitative model) and, in the latter case, the relative contribution made by each paralog. Here, multiple developmental deficiencies were identified in derivatives of the first pharyngeal arch in neonatal Dlx5/6+/− mice that resembled those seen in either paralog‐specific null mutants. These data clearly demonstrate a substantial degree of allele equivalency and support a quantitative model of Dlx function during craniofacial morphogenesis. genesis 54:272–276, 2016.

Dlx5 functions as a negative regulator of the G1/S transition

Extracellular repetitive fine structures have various crucial functions for organismal survival such as super-water repellency of lotus leaves and structural coloration of peacock feathers. Extracellular pore structures of nanometer level are one of such functional fine structures essential for regulating small molecule transport between inside and outside of our body, or between tissues. However, morphogenetic mechanisms of this type of porous extracellular structures are not well understood. To address this issue, we focused on morphogenesis of porous exoskeleton on Drosophila olfactory sensilla. In olfactory sensilla on maxillary palps, arrays of pore structures of ~50 nm in size with regular alignment of 150 200 nm intervals are formed on the surface exoskeleton of an olfactory sensillum to facilitate odorant inhalation, and to prevent outflow of inner lymph with surface tension of water at the same time. Minute electron microscopic observations of morphogenetic processes revealed that the porous cuticle was formed on the surface of a single shaft cell protruded on the epidermis, and that electrondense plasma membrane plaques (PMPs) were associated with the forming electron-dense nanopore precursor structure in the nascent cuticular envelope layer. F-actin meshwork and phosphatidylinositol (4, 5)-bisphosphate (PIP2)-positive clusters of 200-500 nm were also colocalized at the cell cortex. These observations implied that a hypothetical transmembrane proteins accumulated in the PMPs regulate nanopore formation. We searched for such transmembrane proteins specifically expressed in the shaft cell through RNAseq analysis using mutants without shaft cells, and identified that a transmembrane protein gene that we named gore-tex is necessary for nanopore formation. Gore-tex protein has a predicted signal peptide, a conserved domain of unknown function (DUF1676/Osiris) and two transmembrane domains, suggestive of its morphogenetic function on the shaft cell surface. We will discuss the role of PMPs and Gore-tex molecules on cell surface in alignment and formation of nanopores.

Measuring inputs to a common function: The case of Dlx5 and Dlx6.

Physically linked Dlx5 and Dlx6 paralogs are co-expressed in vertebrates and various combinations of null alleles in mice demonstrate not only functional redundancy between the paralogous factors but a similar quantitative contribution to craniofacial functions during development. While it is not possible to rule out that the bigene pair contributes some paralog-specific functions it is clear that, for many functions in the head, Dlx5 and Dlx6 are interchangeable. To assess the relative quantitative contribution made by each paralog to bigene function, we have made comparisons of the expression of Dlx5 and Dlx6 in chick embryos and quantitated the transcriptional properties of the encoded proteins in a variety of regulatory and cellular contexts. Our data indicate that the transcriptional activities of both Dlx5 and Dlx6 are very much context dependent; isolated domains fused to a heterologous DNA binding domain have little intrinsic activity, while individual domains are more active when contiguous with their own homeodomain. We find Dlx5 and Dlx6 to be quantitatively indistinguishable on a variety of natural cis-regulatory sequences in a heterologous cellular context but observed quantitatively different transcriptional outputs in cells that normally express these genes, suggesting differential interactions with co-evolved co-activators.