SUMMARYBone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a [Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1. This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning, but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.

Organogenesis requires coordinated regulation of cellular differentiation and morphogenesis. Cartilage cells in the vertebrate skeleton form polarized stacks, which drive the elongation and shaping of skeletal primordia. Here we show that an atypical cadherin, Fat3, and its partner Dachsous-2 (Dchs2), control polarized cell-cell intercalation of cartilage precursors during craniofacial development. In zebrafish embryos deficient in Fat3 or Dchs2, chondrocytes fail to stack and misregulate expression of sox9a. Similar morphogenetic defects occur in rerea/atr2a −/− mutants, and Fat3 binds REREa, consistent with a model in which Fat3, Dchs2 and REREa interact to control polarized cell-cell intercalation and simultaneously control differentiation through Sox9. Chimaeric analyses support such a model, and reveal long-range influences of all three factors, consistent with the activation of a secondary signal that regulates polarized cell-cell intercalation. This coordinates the spatial and temporal morphogenesis of chondrocytes to shape skeletal primordia and defects in these processes underlie human skeletal malformations. Similar links between cell polarity and differentiation mechanisms are also likely to control organ formation in other contexts.

The hindbrain and pharyngeal arch-derived structures of vertebrates are determined, at least in part, by Hox paralog group 2 genes. In sarcopterygians, the Hoxa2 gene alone appears to specify structures derived from the second pharyngeal arch (PA2), while in zebrafish (Danio rerio), either of the two Hox PG2 genes, hoxa2b or hoxb2a, can specify PA2-derived structures. We previously reported three Hox PG2 genes in striped bass (Morone saxatilis), including hoxa2a, hoxa2b, and hoxb2a and observed that only HoxA cluster genes are expressed in PA2, indicative that they function alone or together to specify PA2. In this paper, we present the cloning and expression analysis of Nile tilapia (Oreochromis niloticus) Hox PG2 genes and show that all three genes are expressed in the hindbrain and in PA2. The expression of hoxb2a in PA2 was unexpected given the close phylogenetic relationship of Nile tilapia and striped bass, both of which are members of the order Perciformes. A reanalysis of striped bass hoxb2a expression demonstrated that it is expressed in PA2 with nearly the same temporal and spatial expression pattern as its Nile tilapia ortholog. Further, we determined that Nile tilapia and striped bass hoxa2a orthologs are expressed in PA2 well beyond the onset of chondrogenesis whereas neither hoxa2b nor hoxb2a expression persist until this stage, which, according to previous hypotheses, suggests that hoxa2a orthologs in these two species function alone as selector genes of PA2 identity.

Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC) cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D) and ventral (V) elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3), (Tg(hsp70I:tcf3-GFP), or the canonical Wnt inhibitor dickkopf1 (dkk1), (Tg(hsp70i:dkk1-GFP) after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe), and ventral cartilage differentiation (e.g. lower jaws). These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.

Background: Endochondral ossification is a major bone forming mechanism in vertebrates, defects in which can result in skeletal dysplasia or craniofacial anomalies in humans. The zebrafish holds great potential to advance our understanding of endochondral growth zone development and genetics, yet several important aspects of its biology remain unexplored. Here we provide a comprehensive description of endochondral growth zones in the pharyngeal skeleton, including their developmental progression, cellular activity, and adult fates. Results: Postembryonic growth of the pharyngeal skeleton is supported by endochondral growth zones located either at skeletal epiphyses or synchondroses. Col2a1a and col10a1a in situ hybridization and anti-PCNA immunostaining identify resting-, hypertrophic-and proliferative zones, respectively, in pharyngeal synchondroses. Cellular hypertrophy and matrix deposition contribute little, if at all, to axial growth in most skeletal elements. Zebrafish endochondral growth zones develop during metamorphosis and arrest in adults. Conclusions: Two endochondral growth zone configurations in the zebrafish pharyngeal skeleton produce either unidirectional (epiphyses) or bidirectional (synchondroses) growth. Cell proliferation drives endochondral growth and its modulation, in contrast to mammalian long bones in which bone length depends more on cell enlargement during hypertrophy and intramembranous ossification is the default mechanism of bone growth in zebrafish adults.

Background Melanic patterns such as horizontal stripes, vertical bars and spots are common among teleost fishes and often serve roles in camouflage or mimicry. Extensive research in the zebrafish model has shown that the development of horizontal stripes depends on complex cellular interactions between melanophores, xanthophores and iridophores. Little is known about the development of horizontal stripes in other teleosts, and even less is known about bar or spot development. Here, we compare chromatophore composition and development of stripes, bars and spots in two cichlid species of sand-dwellers from Lake Malawi— Copadichromis azureus and Dimidiochromis compressiceps . Results (1) In D. compressiceps, stripes are made of dense melanophores underlaid by xanthophores and overlaid by iridophores. Melanophores and xanthophores are either loose or absent in interstripes, and iridophores are dense. In C. azureus, spots and bars are composed of a chromatophore arrangement similar to that of stripes but are separated by interbars where density of melanophores and xanthophores is only slightly lower than in stripes and iridophore density appears slightly greater. (2) Stripe, bar and spot chromatophores appear in the skin at metamorphosis. Stripe melanophores directly differentiate along horizontal myosepta into the adult pattern. In contrast, bar number and position are dynamic throughout development. As body length increases, new bars appear between old ones or by splitting of old ones through new melanophore appearance, not migration. Xanthophore and iridophore distributions follow melanophore patterns. (3) Metamorphic pigmentation arises in cichlids in a fashion similar to that described in zebrafish: melanophore progenitors derived from the medial route of neural crest migration migrate from the vicinity of the neural tube to the skin during metamorphosis. Conclusion The three pigment cell types forming stripes, bars and spots arise in the skin at metamorphosis. Stripes develop by differentiation of melanophores along horizontal myosepta, while bars do not develop along patent anatomical boundaries and increase in number in relation with body size. We propose that metamorphic melanophore differentiation and migratory arrest upon arrival to the skin lead to stripe formation, while bar formation must be supported by extensive migration of undifferentiated melanophores in the skin.

The evolution of a specialized pharyngeal jaw apparatus (PJA) has been argued to be the key evolutionary innovation that allowed the explosive adaptive radiation of cichlid fishes in East African lakes. Subsequent studies together with recent molecular phylogenies have shown that similar innovations evolved independently several times within the teleosts, which poses the questions: (1) how similar are the developmental mechanisms responsible for these changes in divergent taxa and (2) how did such complex features arise independently in evolution? A detailed knowledge of PJA development in cichlids and other teleosts is needed to address these questions. Here, we provide a detailed account of the development of the PJA in one species of cichlid, the Nile tilapia (Oreochromis niloticus), from the early segmentation and patterning of its embryonic precursors -pharyngeal arches 3 to 7 -to its ossification. We find that pharyngeal segmentation occurs sequentially from anterior to posterior during early segmentation stages through the mid-pharyngula period. We show a clear combinatorial code of Hox gene expression such that each posterior arch is defined by a distinctive Hox signature. Posterior arch chondrogenesis in tilapia is essentially complete by the end of the hatching period, and most elements become ossified over the next two days. Our results reveal that both the fusion of lower jaw bones and articulation between the neurocranium and upper jaws occur during post-embryonic development. Abstract end data should be: Anat Rec, 292:1780Rec, 292: -1800Rec, 292: , 2009. V V C 2009 Wiley-Liss, Inc.

BackgroundCichlid fishes from the Rift Lakes of East Africa have undergone the most spectacular adaptive radiations in vertebrate history. Eco-morphological adaptations in lakes Victoria, Malawi and Tanganyika have resulted in a vast array of skull shapes and sizes, yet primary axes of morphological variation are conserved in all three radiations, prominently including the size of the preorbital region of the skull. This conserved pattern suggests that development may constrain the trajectories of cichlid head morphological evolution.ResultsHere, we (1) present a comparative analysis of adult head morphology in two sand-dweller cichlids from Lake Malawi with preorbital size differences representative of the main axis of variation among the three lakes and (2) analyze the ontogeny of shape and size differences by focusing on known developmental modules throughout the head. We find that (1) developmental differences between the two species correlate with known developmental modules; (2) differences in embryonic cartilage development result in phenotypically integrated changes among all bones derived from a single cartilage, while differences in dermal bone development tend to influence isolated regions within a bone; and lastly (3) species-specific morphologies appear in the embryo as subtle differences, which become progressively amplified throughout ontogeny. We propose that this amplification takes place at skeletal growth zones, the locations and shapes of which are patterned during embryogenesis.ConclusionsThis study is the most anatomically comprehensive analysis of the developmental differences underlying cichlid skull evolution in the Rift Lakes of East Africa. The scale of our analysis reveals previously unnoticed correlations between developmental modules and patterns of phenotypic integration. We propose that the primary axes of morphological variation among East African cichlid adaptive radiations are constrained by the hierarchical modularity of the teleost head skeleton.