Requirement for endoderm and FGF3 in ventral head skeleton formation

The vertebrate head skeleton is derived in part from neural crest cells, which physically interact with head ectoderm, mesoderm and endoderm to shape the pharyngeal arches. The cellular and molecular nature of these interactions is poorly understood, and we explore here the function of endoderm in this process. By genetic ablation and reintroduction of endoderm in zebrafish, we show that it is required for the development of chondrogenic neural crest cells, including their identity, survival and differentiation into arch cartilages. Using a genetic interference approach, we further identify Fgf3 as a critical component of endodermal function that allows the development of posterior arch cartilages. Together, our results reveal for the first time that the endoderm provides differential cues along the anteroposterior axis to control ventral head skeleton development and demonstrate that this function is mediated in part by Fgf3.     

Regulation of Dlx gene expression in the zebrafish pharyngeal arches: from conserved enhancer sequences to conserved activity

The Dlx genes play an important role in the development of the pharyngeal arches and the structures derived from these tissues, including the craniofacial skeleton. They are typically expressed in a nested pattern along the proximo‐distal axis of the branchial arches in mice. This pattern is known as the “Dlx code” and has been proposed to be responsible for an early regional patterning of branchial arches in mammals. A number of cis‐ regulatory elements (CREs) have been identified within the Dlx loci, which target reporter expression to the developing pharyngeal arches of the mouse. Most of these CREs are located in the intergenic regions between the two genes constituting a Dlx bigene cluster. Therefore, the regionalized dlx expression in the branchial arches could be the result of the shared activities of these regulatory regions. Here we analyze previously published and new results showing these CREs are highly conserved in both their sequence and their activity in the pharyngeal arches of two distantly related vertebrates, the zebrafish and the mouse. A better understanding of Dlx gene regulation of the Dlx genes and of the genetic cascades in which they are involved can lead to clues explaining variations in morphology of the craniofacial regions of vertebrates.     

Evolutionary Origins of the Neural Crest and Neural Crest Cells

I evaluate the lines of evidence—cell types, genes, gene pathways, fossils—in putative chordate ancestors—cephalochordates and ascidians—pertaining to the evolutionary origin of the vertebrate neural crest. Given the intimate relationship between the neural crest and the dorsal nervous system during development, I discuss the dorsal nervous system in living (extant) members of the two groups, especially the nature, and genes, and gene regulatory networks of the brain to determine whether any cellular and/or molecular precursors (latent homologues) of the neural may have been present in ancestral cephalochordates or urochordates. I then examine those fossils that have been interpreted as basal chordates or cephalochordates to determine whether they shed any light on the origins of neural crest cell (NCC) derivatives. Do they have, for example, elements of a head skeleton or pharyngeal arches, two fundamental vertebrate characters (synapomorphies)? The third topic recognizes that the origin of the neural crest in the first vertebrates accompanied the evolution of a brain, a muscular pharynx, and paired sensory organs. In a paradigm-breaking hypothesis—often known as the ‘new head hypothesis’—Carl Gans and Glen Northcutt linked these evolutionary innovations to the evolution of the neural crest and ectodermal placodes (Gans and Northcutt Science 220:268-274, 1983. doi:10.1126/science.220.4594.268; Northcutt and Gans The Quarterly Review of Biology 58:1–28, 1983. doi:10.1086/413055). I outline the rationale behind the new head hypothesis before turning to an examination of the pivotal role played by NCCs in the evolution of pharyngeal arches, in the context of the craniofacial skeleton. Integrations between the evolving vertebrate brain, muscular pharynx and paired sensory organs may have necessitated that the pharyngeal arch skeletal system—and subsequently, the skeleton of the jaws and much of the skull (the first vertebrates being jawless)—evolved from NCCs whose developmental connections were to neural ectoderm and neurons rather than to mesoderm and connective tissue; mesoderm produces much of the vertebrate skeleton, including virtually all the skeleton outside the head. The origination of the pharyngeal arch skeleton raises the issue of the group of organisms in which and how cartilage arose as a skeletal tissue. Did cartilage arise in the basal proto-vertebrate from a single germ layer, cell layer or tissue, or were cells and/or genes co-opted from several layers or tissues? Two recent studies utilizing comparative genomics, bioinformatics, molecular fingerprinting, genetic labeling/cell selection, and GeneChip Microarray technologies are introduced as powerful ways to approach the questions that are central to this review.     

Fgf signalling is required for formation of cartilage in the head

Characterisation of human craniofacial syndromes and studies in transgenic mice have demonstrated the requirement for Fgf signalling during morphogenesis of membrane bone of the cranium. Here, we report that Fgf activity is also required for development of the oro-pharyngeal skeleton, which develops first as cartilage with some elements subsequently becoming ossified. We show that inhibition of FGF receptor activity in the zebrafish embryo following neural crest emigration from the neural tube results in complete absence of neurocranial and pharyngeal cartilages. Moreover, this Fgf signal is required during a 6-h period soon after initiation of neural crest migration. The spatial and temporal expression of Fgf3 and Fgf8 in pharyngeal endoderm and ventral forebrain and its correlation with patterns of Fgf signalling activity in migrating neural crest makes them candidate regulators of cartilage development. Inhibition of Fgf3 results in the complete absence of cartilage elements that normally form in the third, fourth, fifth, and sixth pharyngeal arches, while those of the first, second, and seventh arches are largely unaffected. Inhibition of Fgf8 alone has variable, but mild, effects. However, inhibition of both Fgf3 and Fgf8 together causes a complete absence of pharyngeal cartilages and the near-complete loss of the neurocranial cartilage. These data implicate Fgf3 and Fgf8 as key regulators of cartilage formation in the vertebrate head.     

Expression of five frizzleds during zebrafish craniofacial development

Wnt/Planar Cell Polarity (PCP) signaling is critical for proper animal development. While initially identified in Drosophila, this pathway is also essential for the proper development of vertebrates. Zebrafish mutants, defective in the Wnt/PCP pathway, frequently display defects in convergence and extension gastrulation movements and additional later abnormalities including problems with craniofacial cartilage morphogenesis. Although multiple Frizzled (Fzd) homologues, Wnt receptors, were identified in zebrafish, it is unknown which Fzd plays a role in shaping the early larvae head skeleton. In an effort to determine which Frizzleds are involved in this process, we analyzed the expression of five zebrafish frizzled homologues fzd2, 6, 7a, 7b, and 8a from 2–4 days post-fertilization (dpf). During the analyzed developmental time points fzd2 and fzd6 are broadly expressed throughout the head, while the expression of fzd7a, 7b and 8a is much more restricted. Closer examination revealed that fzd7b is expressed in the neural crest and the mesodermal core of the pharyngeal arches and in the chondrocytes of newly stacked craniofacial cartilage elements. However, fzd7a is only expressed in the neural crest of the pharyngeal arches and fzd8a is expressed in the pharyngeal endoderm.     

Ectodermal P2X receptor function plays a pivotal role in craniofacial development of the zebrafish

P2X receptors are non-selective cation channels operated by extracellular ATP. Currently, little is known concerning the functions of these receptors during development. Previous work from our lab has shown that zebrafish have two paralogs of the mammalian P2X3 receptor subunit. One paralog, p2rx3.1, is expressed in subpopulations of neural and ectodermal cells in the embryonic head. To investigate the role of this subunit in early cranial development, we utilized morpholino oligonucleotides to disrupt its translation. Loss of this subunit resulted in craniofacial defects that included malformation of the pharyngeal skeleton. During formation of these structures, there was a marked increase in cell death within the branchial arches. In addition, the epibranchial (facial, glossopharyngeal, and vagal) cranial sensory ganglia and their circuits were perturbed. These data suggest that p2rx3.1 function in ectodermal cells is involved in purinergic signaling essential for proper craniofacial development and sensory circuit formation in the embryonic and larval zebrafish.     

The endoderm plays an important role in patterning the segmented pharyngeal region in zebrafish (Danio rerio)

The development of the vertebrate head is a highly complex process involving tissues derived from all three germ layers. The endoderm forms pharyngeal pouches, the paraxial mesoderm gives rise to endothelia and muscles, and the neural crest cells, which originate from the embryonic midbrain and hindbrain, migrate ventrally to form cartilage, connective tissue, sensory neurons, and pigment cells. All three tissues form segmental structures: the hindbrain compartmentalizes into rhombomeres, the mesoderm into somitomeres, and the endoderm into serial gill slits. It is not known whether the different segmented tissues in the head develop by the same molecular mechanism or whether different pathways are employed. It is also possible that one tissue imposes segmentation on the others. Most recent studies have emphasized the importance of neural crest cells in patterning the head. Neural crest cells colonize the segmentally arranged arches according to their original position in the brain and convey positional information from the hindbrain into the periphery. During the screen for mutations that affect embryonic development of zebrafish, one mutant, called van gogh (vgo), in which segmentation of the pharyngeal region is absent, was isolated. In vgo, even though hindbrain segmentation is unaffected, the pharyngeal endoderm does not form reiterated pouches and surrounding mesoderm is not patterned correctly. Accordingly, migrating neural crest cells initially form distinct streams but fuse when they reach the arches. This failure to populate distinct pharyngeal arches is likely due to the lack of pharyngeal pouches. The results of our analysis suggest that the segmentation of the endoderm occurs without signaling from neural crest cells but that tissue interactions between the mesendoderm and the neural crest cells are required for the segmental appearance of the neural crest-derived cartilages in the pharyngeal arches. The lack of distinct patches of neural crest cells in the pharyngeal region is also seen in mutants of one-eyed pinhead and casanova, which are characterized by a lack of endoderm, as well as defects in mesodermal structures, providing evidence for the important role of the endoderm and mesoderm in governing head segmentation.     

The role of neural crest during cardiac development in a mouse model of DiGeorge syndrome

The velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a genetic disorder characterized by phenotypic abnormalities of the derivatives of the pharyngeal arches, including cardiac outflow tract defects. Neural crest cells play a major role in the development of the pharyngeal arches, and defects in these cells are likely responsible for the syndrome. Most patients are hemizygous for a 1.5- to 3.0-Mb region of 22q11, that is suspected to be critical for normal pharyngeal arch development. Mice hemizygous for a 1.5-Mb homologous region of chromosome 16 (Lgdel/+) exhibit conotruncal cardiac defects similar to those seen in affected VCFS/DGS patients. To investigate the role of Lgdel genes in neural crest development, we fate mapped neural crest cells in Lgdel/+ mice and we performed hemizygous neural crest-specific inactivation of Lgdel. Hemizygosity of the Lgdel region does not eliminate cardiac neural crest migration to the forming aortic arches. However, neural crest cells do not differentiate appropriately into smooth muscle in both fourth and sixth aortic arches and the affected aortic arch segments develop abnormally. Tissue-specific hemizygous inactivation of Lgdel genes in neural crest results in normal cardiovascular development. Based on our studies, we propose that Lgdel genes are required for the expression of soluble signals that regulate neural crest cell differentiation.     

Specification and morphogenesis of the zebrafish larval head skeleton

Forward genetic analyses can reveal important developmental regulatory genes and how they function to pattern morphology. This is because a mutated gene can produce a novel, sometimes beautiful, phenotype that, like the normal phenotype, immediately seems worth understanding. Generally the loss-of-function mutant phenotype is simplified from the wild-type one, and often the nature of the pattern simplification allows one to deduce how the wild-type gene contributes to patterning the normal, more complex, morphology. This truism seems no less valid for the vertebrate head skeleton than for other and simpler cases of patterning in multicellular plants and animals. To show this, we review selected zebrafish craniofacial mutants. "Midline group" mutations, in genes functioning in one of at least three signal transduction pathways, lead to neurocranial pattern truncations that are primarily along the mediolateral axis. Mutation of lazarus/pbx4, encoding a hox gene partner, and mutation of valentino/kreisler, a hox gene regulator, produce anterior-posterior axis disruptions of pharyngeal cartilages. Dorsoventral axis patterning of the same cartilages is disrupted in sucker/endothelin-1 mutants. We infer that different signal transduction pathways pattern cartilage development along these three separate axes. Patterning of at least the anterior-posterior and dorsoventral axes have been broadly conserved, e.g., reduced Endothelin-1 signaling similarly perturbs cartilage specification in chick, mouse, and zebrafish. We hypothesize that Endothelin-1 also is an upstream organizer of the patterns of cellular interactions during cartilage morphogenesis.     

The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans

The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.     

Zebrafish hox paralogue group 2 genes function redundantly as selector genes to pattern the second pharyngeal arch

The pharyngeal arches are one of the defining features of the vertebrates, with the first arch forming the mandibles of the jaw and the second forming jaw support structures. The cartilaginous elements of each arch are formed from separate migratory neural crest cell streams, which derive from the dorsal aspect of the neural tube. The second and more posterior crest streams are characterized by specific Hox gene expression. The zebrafish has a larger overall number of Hox genes than the tetrapod vertebrates, as the result of a duplication event in its lineage. However, in both zebrafish and mouse, there are just two members of Hox paralogue group 2 (PG2): Hoxa2 and Hoxb2. Here, we show that morpholino-mediated "knock-down" of both zebrafish Hox PG2 genes results in major defects in second pharyngeal arch cartilages, involving replacement of ventral elements with a mirror-image duplication of first arch structures, and accompanying changes to pharyngeal musculature. In the mouse, null mutants of Hoxa2 have revealed that this single Hox gene is required for normal second arch patterning. By contrast, loss-of-function of either zebrafish Hox PG2 gene individually has no phenotypic consequence, showing that these two genes function redundantly to confer proper pattern to the second pharyngeal arch. We have also used hoxb1a mis-expression to induce localized ectopic expression of zebrafish Hox PG2 genes in the first arch; using this strategy, we find that ectopic expression of either Hox PG2 gene can confer second arch identity onto first arch structures, suggesting that the zebrafish Hox PG2 genes act as "selector genes."     

lazarus is a novel pbx gene that globally mediates hox gene function in zebrafish

Individual vertebrate Hox genes specify aspects of segment identity along the anterior-posterior axis. The exquisite in vivo specificity of Hox proteins is thought to result from their interactions with members of the Pbx/Exd family of homeodomain proteins. Here, we report the identification and cloning of a zebrafish gene, lazarus, which is required globally for segmental patterning in the hindbrain and anterior trunk. We show that lazarus is a novel pbx gene and provide evidence that it is the primary pbx gene required for the functions of multiple hox genes during zebrafish development. lazarus plays a critical role in orchestrating the corresponding segmentation of the hindbrain and the pharyngeal arches, a key step in the development of the vertebrate body plan.     

Dlx5/6-enhancer directed expression of Cre recombinase in the pharyngeal arches and brain

Dlx5 and Dlx6, two members of the Distalless gene family, are required for development of numerous tissues during embryogenesis, including facial and limb development. This gene pair is expressed in tandem, transcribed toward each other and separated by a short intergenic region containing multiple putative enhancers. Targeted inactivation of Dlx5 and Dlx6 in mice results in multiple developmental defects in craniofacial and limb structures, suggesting that these genes are crucial for aspects of both neural crest and nonneural crest development. To further investigate potential developmental roles of Dlx5 and Dlx6, we used one of the Dlx5/6 intergenic enhancers to drive Cre recombinase expression in transgenic mice. Crossing Dlx5/6-Cre transgenic mice with mice from the R26R strain results in beta-galactosidase staining in the apical ectodermal ridge, brain, and neural crest-derived mesenchyme of the pharyngeal arches, with staining in term embryos observed in the facial skeleton and specific brain structures. However, in contrast to endogenous expression patterns of Dlx5 and Dlx6, Cre expression within the pharyngeal arches occurs during a very narrow window in early development. Our studies suggest that Dlx5/6-Cre mice may prove useful both in further understanding the function and regulation of Distalless genes during development and in studies of gene function in conditional knockout mice.     

Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development

Background  

The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC). Genetic studies in zebrafish and mice have established that the Hedgehog (Hh)-signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in CNCC development. Disruption of the Hh-signaling pathway in humans can lead to the spectral disorder of Holoprosencephaly (HPE), which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia [1, 2]. Previous work has uncovered a role for Hh-signaling in zebrafish dorsal neurocranium patterning and chondrogenesis, however Hh-signaling mutants have not been described with respect to the ventral pharyngeal arch (PA) skeleton. Lipid-modified Hh-ligands require the transmembrane-spanning receptor Dispatched 1 (Disp1) for proper secretion from Hh-synthesizing cells to the extracellular field where they act on target cells. Here we study chameleon mutants, lacking a functional disp1(con/disp1).     

moz regulates Hox expression and pharyngeal segmental identity in zebrafish

In vertebrate embryos, streams of cranial neural crest (CNC) cells migrate to form segmental pharyngeal arches and differentiate into segment-specific parts of the facial skeleton. To identify genes involved in specifying segmental identity in the vertebrate head, we screened for mutations affecting cartilage patterning in the zebrafish larval pharynx. We present the positional cloning and initial phenotypic characterization of a homeotic locus discovered in this screen. We show that a zebrafish ortholog of the human oncogenic histone acetyltransferase MOZ (monocytic leukemia zinc finger) is required for specifying segmental identity in the second through fourth pharyngeal arches. In moz mutant zebrafish, the second pharyngeal arch is dramatically transformed into a mirror-image duplicated jaw. This phenotype resembles a similar but stronger transformation than that seen in hox2 morpholino oligo (hox2-MO) injected animals. In addition, mild anterior homeotic transformations are seen in the third and fourth pharyngeal arches of moz mutants. moz is required for maintenance of most hox1-4 expression domains and this requirement probably at least partially accounts for the moz mutant homeotic phenotypes. Homeosis and defective Hox gene expression in moz mutants is rescued by inhibiting histone deacetylase activity with Trichostatin A. Although we find early patterning of the moz mutant hindbrain to be normal, we find a late defect in facial motoneuron migration in moz mutants. Pharyngeal musculature is transformed late, but not early, in moz mutants. We detect relatively minor defects in arch epithelia of moz mutants. Vital labeling of arch development reveals no detectable changes in CNC generation in moz mutants, but later prechondrogenic condensations are mispositioned and misshapen. Mirror-image hox2-dependent gene expression changes in postmigratory CNC prefigure the homeotic phenotype in moz mutants. Early second arch ventral expression of goosecoid (gsc) in moz mutants and in animals injected with hox2-MOs shifts from lateral to medial, mirroring the first arch pattern. bapx1, which is normally expressed in first arch postmigratory CNC prefiguring the jaw joint, is ectopically expressed in second arch CNC of moz mutants and hox2-MO injected animals. Reduction of bapx1 function in wild types causes loss of the jaw joint. Reduction of bapx1 function in moz mutants causes loss of both first and second arch joints, providing functional genetic evidence that bapx1 contributes to the moz-deficient homeotic pattern. Together, our results reveal an essential embryonic role and a crucial histone acetyltransferase activity for Moz in regulating Hox expression and segmental identity, and provide two early targets, bapx1 and gsc, of moz and hox2 signaling in the second pharyngeal arch.