Measuring transcription factor function with cell type-specific somatic transgenesis in chicken embryos.

Retroviral-mediated misexpression in chicken embryos has been a powerful research tool for developmental biologists in the last two decades. In the RCASBP retroviral vectors that are widely used for in vivo somatic transgenesis, a coding sequence of interest is under the transcriptional control of a strong viral promoter in the long terminal repeat. While this has proven to be effective for studying secreted signalling proteins, interpretation of the mechanisms of action of nuclear factors is more difficult using this system since it is not clear whether phenotypic effects are cell-autonomous or not, and therefore whether they represent a function of the endogenous protein. Here, we report the consequences of retroviral expression using the RCANBP backbone, in which the transcription factor Dlx5 is expressed under the control of chondrocyte-specific regulatory sequences from the Col2a1 gene. To our knowledge, this is the first demonstration of a tissue-specific phenotype in the chicken embryo.

DLX gene expression in the developing chick pharyngeal arches and relationship to endothelin signaling and avian jaw patterning.

BACKGROUND: A hinged jaw that articulates with the skull base is a striking feature of the vertebrate head and has been greatly modified between, and within, vertebrate classes. Genes belonging to the DLX homeobox family are conserved mediators of local signaling pathways that distinguish the dorsal and ventral aspects of the first pharyngeal arch. Specifically, a subset of DLX genes are expressed in the cranial neural crest-derived mandibular ectomesenchyme in response to ventral endothelin signaling, an important step that confers the first arch with maxillary and mandibular identities. Downstream targets of DLX genes then execute the morphogenetic processes that lead to functional jaws. Identifying lineage-specific variations in DLX gene expression and the regulatory networks downstream of DLX action is necessary to understand how different kinds of jaws evolved. RESULTS: Here, we describe and compare the expression of all six DLX genes in the chick pharyngeal arches, focusing on the period of active patterning in the first arch. Disruption of endothelin signaling results in the down-regulation of ventral-specific DLX genes and confirms their functional role in avian jaw patterning. CONCLUSIONS: This expression resource will be important for comparative embryology and for identifying synexpression groups of DLX-regulated genes in the chick.

The insufficiency of Dlx5 for ventral patterning in post-migratory neural crest cells reveals a loss of plasticity in early jaw-forming tissue.

The articulated jaws of vertebrates arise from the first pharyngeal arch, the most rostral of several transient ventral structures in pharyngeal stage embryos. Migratory cranial neural crest cells from the caudal midbrain and rostral hindbrain populate the first arch as ectomesenchyme and supply the progenitors of skeletal and soft tissues that form the upper (maxillary) and lower (mandibular) jaws. Dlx genes encode key transcriptional regulators that profoundly influence jaw development through their actions in first pharyngeal arch patterning. The broadly conserved nested expression of Dlx paralogues in vertebrate embryos points to a retained ancestral role in patterning first arch tissue. Loss-of-function experiments consistently highlight the necessity of Dlx gene function for jaw morphogenesis. Specifically, the combined effects of Dlx5 and Dlx6 are required to specify ventral/mandibular fate and forced expression of Dlx5 in migrating neural crest cells results in the ectopic upregulation of ventral markers in the maxillary arch. Here, we ask whether Dlx5 is also sufficient to respecify post-migratory ectomesenchyme in the maxillary branch as mandibular. Unexpectedly, we show that Dlx5 is not sufficient to activate mandibular marker genes in the maxillary branch of PA1, highlighting a loss of plasticity in post-migratory first arch ectomesenchyme.

Dlx5 and Dlx6 can antagonize cell division at the G1/S checkpoint.

BACKGROUND: Dlx5 and Dlx6 stimulate differentiation of diverse progenitors during embryonic development. Their actions as pro-differentiation transcription factors includes the up-regulation of differentiation markers but the extent to which differentiation may also be stimulated by regulation of the cell cycle has not been addressed. RESULTS: We document that expression of Dlx5 and Dlx6 antagonizes cell proliferation in a variety of cell types without inducing apoptosis or promoting cell cycle exit. Rather, a variety of evidence indicates that elevated Dlx5 and Dlx6 expression reduces the proportion of cells in S phase and affects the length of the cell cycle. CONCLUSIONS: Antagonism of S-phase entry by Dlx5 and Dlx6 proteins likely represents a lineage-independent function to effect Dlx-mediated differentiation in multiple progenitor cell types.

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.

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. © 2016 Wiley Periodicals, Inc.

Small interfering RNA-mediated knockdown of chicken interferon-γ expression.

Interferon (IFN)-γ is a cytokine with a variety of functions, including direct antiviral activities and the capacity to polarize T-cells. However, there is limited information available about the function of this cytokine in the avian immune system. To gain a better understanding of the biological relevance of IFN-γ in chicken immunity, gain-of-function (upregulation) and loss-of-function (downregulation) studies need to be conducted. RNA interference (RNAi), a technique employed for downregulating gene expression, is mediated by small interfering RNA (siRNA), which can trigger sequence-specific gene silencing. In this regard, sequence specificity and delivery of siRNA molecules remain critical issues, especially to cells of the immune system. Various direct and indirect approaches have been employed to deliver siRNA, including the use of viral vectors. The objectives of the present study were to determine whether RNAi could effectively downregulate expression of chicken IFN-γ in vitro, and investigate the feasibility of recombinant adeno-associated virus to deliver siRNA in vitro as well. Three 27-mer Dicer substrate RNAs were selected based on the chicken IFN-γ coding sequence and transfected into cells or delivered using a recombinant avian adeno-associated virus (rAAAV) into a chicken fibroblast cell line expressing chIFN-γ. The expression of chIFN-γ transcripts was significantly downregulated when a cocktail containing all three siRNAs was used. Expression of endogenous IFN-γ was also significantly downregulated in primary cells after stimulation with a peptide. Further, significant suppression of IFN-γ transcript was also observed in vitro in cells that were treated with rAAAV, expressing siRNA targeting IFN-γ. Off-target effects in the form of triggering IFN responses by RNAi, including expression of chicken 2',5'-oligoadenylate synthetase and IFN-α, were also examined. Our results suggest that siRNAs selected were effective at downregulating IFN-γ in vitro both when delivered directly as well as when expressed by an rAAAV-based vector.

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.

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

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.

A novel mutation of the CLCN1 gene associated with myotonia hereditaria in an Australian cattle dog.

BACKGROUND: Heritable myotonia is a genetic muscle disorder characterized by slow relaxation of skeletal muscles. The main clinical signs are skeletal muscle stiffness, especially after vigorous contraction, and muscle hypertrophy. Muscle stiffness may be enhanced by inactivity, and often is relieved by exercise. Myotonia can be inherited in an autosomal dominant or recessive manner (Thomsen- or Becker-type myotonia, respectively). In mice, goats, Miniature Schnauzer dogs, and most affected humans, the disorder is caused by mutations in CLCN1, which encodes the skeletal muscle voltage-gated chloride channel, Cl1C-1. HYPOTHESIS: We hypothesized that an Australian Cattle Dog with generalized muscle stiffness and hypertrophy examined at the Ontario Veterinary College would have a mutation in the CLCN1 gene. ANIMALS: A pure-bred Australian Cattle Dog from Ontario, Canada, was used. METHODS: Based on clinical signs and electromyographic test results, a diagnosis of myotonia hereditaria was made, and a muscle biopsy was collected for genetic analysis. RESULTS: Sequence data obtained from the affected dog confirmed that it was homozygous for a single base insertion in the CLCN1 coding sequence. This mutation would result in a truncated ClC-1 protein being expressed, which, based on molecular evidence from other studies, would result in functionally compromised chloride conduction in the skeletal muscles of the animal. CONCLUSIONS AND CLINICAL IMPORTANCE: To the authors' knowledge, this report describes the Ist case of myotonia in an Australian Cattle Dog and represents the 1st non-Schnauzer canine myotonia to be genetically characterized. In addition, we developed a polymerase chain reaction-based genetic screen to detect heterozygotes with this mutation in the at-large Australian Cattle Dog population.

Impaired nuclear import of mammalian Dlx4 proteins as a consequence of rapid sequence divergence.

Dlx genes encode a developmentally important family of transcription factors with a variety of functions and sites of action during vertebrate embryogenesis. The murine Dlx4 gene is an enigmatic member of the family; little is known about the normal developmental function(s) of Dlx4. Here, we show that Dlx4 is expressed in the murine placenta and in a trophoblast cell line where the protein localizes to both the nucleus and cytoplasm. Despite the presence of several leucine/valine-rich motifs that match known nuclear export sequences, cytoplasmic Dlx4 is not due to CRM-1-mediated nuclear export. Rather, nuclear import of Dlx4 is compromised by specific residues that flank the nuclear localization signal. One of these residues represents a novel conserved feature of the Dlx4 protein in placental mammals, and the second represents novel variation within mouse Dlx4 isoforms. Comparison of orthologous protein sequences reveals a particularly high rate of non-synonymous change in the coding regions of mammalian Dlx4 genes. Since impaired nuclear localization is unlikely to enhance the function of a nuclear transcription factor, these data point to reduced selection pressure as the basis for the rapid divergence of the Dlx4 gene within the mammalian clade.

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.

Dlx3 is expressed in the ventral forebrain of chicken embryos: implications for the evolution of the Dlx gene family.

The archetypal genomic arrangement of vertebrate Dlx genes is as three bigene clusters (Dlx1/2, Dlx3/4, Dlx5/6). Phylogenetic sequence analysis of mouse and zebrafish Dlx clusters supports the notion that the Dlx3/4 cluster is more derived and the absence of expression of either Dlx3 or Dlx4 in the central nervous system, as reported to date, is consistent with this. Together, these observations have prompted a model in which cis-regulatory elements, responsible for directing Dlx gene transcription in the forebrain, were lost from the Dlx3/4 bigene cluster prior to the divergence of tetrapods from fish. Here, we describe Dlx3 expression in the forebrain of chicken embryos; this constitutes the first documented evidence of expression of either Dlx3 or Dlx4 in the central nervous system of a vertebrate. Our observations have implications for models of the evolutionary history of the Dlx gene family, for the genomic organization of Dlx genes in birds and for functional redundancy of Dlx gene function during avian forebrain development.

Dlx5 regulates chondrocyte differentiation at multiple stages.

Endochondral ossification, in which cartilaginous templates are progressively replaced by marrow and bone, represents the dominant mode of development of the axial and appendicular skeleton of vertebrates. Chondrocyte differentiation within the cartilaginous core of these skeletal elements is tightly regulated, both spatially and temporally. Here, we describe the expression of Dlx5 in the cartilaginous core of limb skeletal elements in chicken and mouse embryos. We find that Dlx5 is one of the earliest genes expressed in condensing limb mesenchyme that will give rise to the limb skeleton. Later, when proliferating and differentiating chondrocytes are found in spatially distinct regions of the cartilaginous model, Dlx5 is expressed in the zone of hypertrophy and in proliferating chondrocytes that are poised to differentiate. Consistent with this pattern of expression, we show that forced expression of Dlx5 potentiates early and late chondrocyte differentiation and inhibits proliferation in cultured cells. Examination of the limbs of mutant Dlx5 mouse embryos revealed that they displayed a delay in chondrocyte maturation compared with wild type littermates. Taken together, our data reveal a positive role for Dlx5 during multiple stages of chondrocyte differentiation and, along with previous studies of Dlx5 and osteogenesis, identify Dlx5 as a general regulator of differentiation in the mouse skeleton.