Charles B. Kimmel

Organization of hindbrain segments in the zebrafish embryo

To learn how neural segments are structured in a simple vertebrate, we have characterized the embryonic zebrafish hindbrain with a library of monoclonal antibodies. Two regions repeat in an alternating pattern along a series of seven segments. One, the neuromere centers, contains the first basal plate neurons to develop and the first neuropil. The other region, surrounding the segment boundaries, contains the first neurons to develop in the alar plate. The projection patterns of these neurons differ: those in the segment centers have descending axons, while those in the border regions form ventral commissures. A row of glial fiber bundles forms a curtain-like structure between each center and border region. Specific features of the individual hindbrain segments in the series arise within this general framework. We suggest that a cryptic simplicity underlies the eventual complex structure that develops from this region of the CNS.

Genetics and early development of zebrafish

Zebrafish genes and development are being studied in a growing number of laboratories. Given that many other organisms are already being exploited by large numbers of investigators, and that our general knowledge about the zebrafish embryo and genome is at present rather sketchy, why should we now concern ourselves with how this tropical fish develops? Whereas the zebrafish embryo is similar in important ways to other vertebrate embryos, it is relatively simple and unusually accessible for both cellular and genetic analyses.

A pair of Sox: distinct and overlapping functions of zebrafish sox9 co-orthologs in craniofacial and pectoral fin development

Understanding how developmental systems evolve after genome amplification is important for discerning the origins of vertebrate novelties, including neural crest, placodes, cartilage and bone. Sox9 is important for the development of these features, and zebrafish has two co-orthologs of tetrapod SOX9 stemming from an ancient genome duplication event in the lineage of ray-fin fish. We have used a genotype-driven screen to isolate a mutation deleting sox9b function, and investigated its phenotype and genetic interactions with a sox9a null mutation. Analysis of mutant phenotypes strongly supports the interpretation that ancestral gene functions partitioned spatially and temporally between Sox9 co-orthologs. Distinct subsets of the craniofacial skeleton, otic placode and pectoral appendage express each gene, and are defective in each single mutant. The double mutant phenotype is additive or synergistic. Ears are somewhat reduced in each single mutant but are mostly absent in the double mutant. Loss-of-function animals from mutations and morpholino injections, and gain-of-function animals injected with sox9a and sox9b mRNAs showed that sox9 helps regulate other early crest genes, including foxd3, sox10, snai1b and crestin, as well as the cartilage gene col2a1 and the bone gene runx2a; however, tfap2a was nearly unchanged in mutants. Chondrocytes failed to stack in sox9a mutants, failed to attain proper numbers in sox9b mutants and failed in both morphogenetic processes in double mutants. Pleiotropy can cause mutations in single copy tetrapod genes, such as Sox9, to block development early and obscure later gene functions. By contrast, subfunction partitioning between zebrafish co-orthologs of tetrapod genes, such as sox9a and sox9b, can relax pleiotropy and reveal both early and late developmental gene functions.

MicroRNA Mirn140 modulates Pdgf signaling during palatogenesis.

Disruption of signaling pathways such as those mediated by sonic hedgehog (Shh) or platelet-derived growth factor (Pdgf) causes craniofacial abnormalities, including cleft palate. The role that microRNAs play in modulating palatogenesis, however, is completely unknown. We show that, in zebrafish, the microRNA Mirn140 negatively regulates Pdgf signaling during palatal development, and we provide a mechanism for how disruption of Pdgf signaling causes palatal clefting. The pdgf receptor alpha (pdgfra) 3′ UTR contained a Mirn140 binding site functioning in the negative regulation of Pdgfra protein levels in vivo. pdgfra mutants and Mirn140-injected embryos shared a range of facial defects, including clefting of the crest-derived cartilages that develop in the roof of the larval mouth. Concomitantly, the oral ectoderm beneath where these cartilages develop lost pitx2 and shha expression. Mirn140 modulated Pdgf-mediated attraction of cranial neural crest cells to the oral ectoderm, where crest-derived signals were necessary for oral ectodermal gene expression. Mirn140 loss of function elevated Pdgfra protein levels, altered palatal shape and caused neural crest cells to accumulate around the optic stalk, a source of the ligand Pdgfaa. These results suggest that the conserved regulatory interactions of mirn140 and pdgfra define an ancient mechanism of palatogenesis, and they provide candidate genes for cleft palate.

Cell lineage of zebrafish blastomeres: I. Cleavage pattern and cytoplasmic bridges between cells☆

Patterns of cleavage and cytoplasmic connections between blastomeres in the embryo of the zebrafish, Brachydanio rerio have been described. The cell division pattern is often very regular; in many embryos a blastomeres lineage may be ascertained from its position in the cluster through the 64-cell stage. At the 5th cleavage, however, significant variability in pattern is observed, and alternative patterns of the 5th cleavage are described. The early cleavages are partial, incompletely separating blastomeres from the giant yolk cell. The tracer fluorescein-dextran (FD) was injected into blastomeres to learn the extent of the cytoplasmic bridging. It was observed that until the 10th cleavage, blastomeres located along the blastoderm margin maintain cytoplasmic bridges to the yolk cell. Beginning with the 5th cleavage, FD injected into a nonmarginal blastomere either remains confined to the injected cell, or if the injection was early in the cell cycle, the tracer spreads to the cells sibling, through a bridge persisting from the previous cleavage. On the other hand, injected Lucifer yellow spreads, presumably via gap junctions, widely among blastomeres in a pattern unrelated to lineage.

Two endothelin 1 effectors, hand2 and bapx1,pattern ventral pharyngeal cartilage and the jaw joint

A conserved endothelin 1 signaling pathway patterns the jaw and other pharyngeal skeletal elements in mice, chicks and zebrafish. In zebrafish, endothelin 1 (edn1 or sucker) is required for formation of ventral cartilages and joints in the anterior pharyngeal arches of young larvae. Here we present genetic analyses in the zebrafish of two edn1 downstream targets, the bHLH transcription factor Hand2 and the homeobox transcription factor Bapx1, that mediate dorsoventral (DV) patterning in the anterior pharyngeal arches. First we show that edn1-expressing cells in the first (mandibular) and second (hyoid) pharyngeal arch primordia are located most ventrally and surrounded by hand2-expressing cells. Next we show that along the DV axis of the early first arch primordia, bapx1 is expressed in an intermediate domain, which later marks the jaw joint, and this expression requires edn1 function. bapx1 function is required for formation of the jaw joint, the joint-associated retroarticular process of Meckels cartilage, and the retroarticular bone. Jaw joint expression of chd and gdf5 also requires bapx1 function. Similar to edn1, hand2 is required for ventral pharyngeal cartilage formation. However, the early ventral arch edn1-dependent expression of five genes (dlx3, EphA3, gsc, msxe and msxb) are all present in hand2 mutants. Further, msxe and msxb are upregulated in hand2 mutant ventral arches. Slightly later, an edn1-dependent ventral first arch expression domain of gsc is absent in hand2 mutants, providing a common downstream target of edn1 and hand2. In hand2 mutants, bapx1 expression is present at the joint region, and expanded ventrally. In addition, expression of eng2, normally restricted to first arch dorsal mesoderm, expands ventrally in hand2 and edn1 mutants. Thus, ventral pharyngeal specification involves repression of dorsal and intermediate (joint region) fates. Together our results reveal two critical edn1 effectors that pattern the vertebrate jaw: hand2 specifies ventral pharyngeal cartilage of the lower jaw and bapx1 specifies the jaw joint.

Shaping the zebrafish notochord.

Promptly after the notochord domain is specified in the vertebrate dorsal mesoderm, it undergoes dramatic morphogenesis. Beginning during gastrulation, convergence and extension movements change a squat cellular array into a narrow, elongated one that defines the primary axis of the embryo. Convergence and extension might be coupled by a highly organized cellular intermixing known as mediolateral intercalation behavior (MIB). To learn whether MIB drives early morphogenesis of the zebrafish notochord, we made 4D recordings and quantitatively analyzed both local cellular interactions and global changes in the shape of the dorsal mesodermal field. We show that MIB appears to mediate convergence and can account for extension throughout the dorsal mesoderm. Comparing the notochord and adjacent somitic mesoderm reveals that extension can be regulated separately from convergence. Moreover, mutational analysis shows that extension does not require convergence. Hence, a cellular machine separate from MIB that can drive dorsal mesodermal extension exists in the zebrafish gastrula. The likely redundant control of morphogenesis may provide for plasticity at this critical stage of early development.

Zebrafish fgf24 functions with fgf8 to promote posterior mesodermal development.

Fibroblast growth factor (Fgf) signaling plays an important role during development of posterior mesoderm in vertebrate embryos. Blocking Fgf signaling by expressing a dominant-negative Fgf receptor inhibits posterior mesoderm development. In mice, Fgf8 appears to be the principal ligand required for mesodermal development, as mouse Fgf8 mutants do not form mesoderm. In zebrafish, Fgf8 is encoded by the acerebellar locus, and, similar to its mouse otholog, is expressed in early mesodermal precursors during gastrulation. However, zebrafish fgf8 mutants have only mild defects in posterior mesodermal development, suggesting that it is not the only Fgf ligand involved in the development of this tissue. We report here the identification of an fgf8-related gene in zebrafish, fgf24, that is co-expressed with fgf8 in mesodermal precursors during gastrulation. Using morpholino-based gene inactivation, we have analyzed the function of fgf24 during development. We found that inhibiting fgf24 function alone has no affect on the formation of posterior mesoderm. Conversely, inhibiting fgf24 function in embryos mutant for fgf8 blocks the formation of most posterior mesoderm. Thus, fgf8 and fgf24 are together required to promote posterior mesodermal development. We provide both phenotypic and genetic evidence that these Fgf signaling components interact with no tail and spadetail, two zebrafish T-box transcription factors that are required for the development of all posterior mesoderm. Last, we show that fgf24 is expressed in early fin bud mesenchyme and that inhibiting fgf24 function results in viable fish that lack pectoral fins.

Tissue-Specific Cell Lineages Originate in the Gastrula of the Zebrafish

In the zebrafish, cells of a clone derived from a single blastomere migrate away from one another during gastrulation. Later in development their descendants are usually found scattered within several different types of tissues of embryo. The divisions and migrations of individual cells were monitored during early development, revealing that in most cases the lineal descendants of single cells present at gastrula stage exclusively populate only single tissues, and may have stereotyped positional relationships within these tissues. Thus the gastrula stage is the first stage when heritable restrictions in cell type might arise in the zebrafish.

An essential role for Fgfs in endodermal pouch formation influences later craniofacial skeletal patterning

Fibroblast growth factor (Fgf) proteins are important regulators of pharyngeal arch development. Analyses of Fgf8 function in chick and mouse and Fgf3 function in zebrafish have demonstrated a role for Fgfs in the differentiation and survival of postmigratory neural crest cells (NCC) that give rise to the pharyngeal skeleton. Here we describe, in zebrafish, an earlier, essential function for Fgf8 and Fgf3 in regulating the segmentation of the pharyngeal endoderm into pouches. Using time-lapse microscopy, we show that pharyngeal pouches form by the directed lateral migration of discrete clusters of endodermal cells. In animals doubly reduced for Fgf8 and Fgf3, the migration of pharyngeal endodermal cells is disorganized and pouches fail to form. Transplantation and pharmacological experiments show that Fgf8 and Fgf3 are required in the neural keel and cranial mesoderm during early somite stages to promote first pouch formation. In addition, we show that animals doubly reduced for Fgf8 and Fgf3 have severe reductions in hyoid cartilages and the more posterior branchial cartilages. By examining early pouch and later cartilage phenotypes in individual animals hypomorphic for Fgf function, we find that alterations in pouch structure correlate with later cartilage defects. We present a model in which Fgf signaling in the mesoderm and segmented hindbrain organizes the segmentation of the pharyngeal endoderm into pouches. Moreover, we argue that the Fgf-dependent morphogenesis of the pharyngeal endoderm into pouches is critical for the later patterning of pharyngeal cartilages.