In vivo imaging and quantification of regional adiposity in zebrafish.

Adipose tissues (ATs) are lipid-rich structures that supply and sequester energy-dense lipid in response to the energy status of an organism. As such, ATs provide an organism energetic insurance during periods of adverse physiological burden. ATs are deposited in diverse anatomical locations, and excessive accumulation of particular regional ATs modulates disease risk. Therefore, a model system that facilitates the visualization and quantification of regional adiposity holds significant biomedical promise. The zebrafish (Danio rerio) has emerged as a new model system for AT research in which the entire complement of regional ATs can be imaged and quantified in live individuals. Here we present detailed methods for labeling adipocytes in live zebrafish using fluorescent lipophilic dyes, and for identifying and quantifying regional zebrafish ATs.

Best practices for germ-free derivation and gnotobiotic zebrafish husbandry.

All animals are ecosystems with resident microbial communities, referred to as microbiota, which play profound roles in host development, physiology, and evolution. Enabled by new DNA sequencing technologies, there is a burgeoning interest in animal-microbiota interactions, but dissecting the specific impacts of microbes on their hosts is experimentally challenging. Gnotobiology, the study of biological systems in which all members are known, enables precise experimental analysis of the necessity and sufficiency of microbes in animal biology by deriving animals germ-free (GF) and inoculating them with defined microbial lineages. Mammalian host models have long dominated gnotobiology, but we have recently adapted gnotobiotic approaches to the zebrafish (Danio rerio), an important aquatic model. Zebrafish offer several experimental attributes that enable rapid, large-scale gnotobiotic experimentation with high replication rates and exquisite optical resolution. Here we describe detailed protocols for three procedures that form the foundation of zebrafish gnotobiology: derivation of GF embryos, microbial association of GF animals, and long-term, GF husbandry. Our aim is to provide sufficient guidance in zebrafish gnotobiotic methodology to expand and enrich this exciting field of research.

Transcriptomic analysis of tail regeneration in the lizard Anolis carolinensis reveals activation of conserved vertebrate developmental and repair mechanisms.

Lizards, which are amniote vertebrates like humans, are able to lose and regenerate a functional tail. Understanding the molecular basis of this process would advance regenerative approaches in amniotes, including humans. We have carried out the first transcriptomic analysis of tail regeneration in a lizard, the green anole Anolis carolinensis, which revealed 326 differentially expressed genes activating multiple developmental and repair mechanisms. Specifically, genes involved in wound response, hormonal regulation, musculoskeletal development, and the Wnt and MAPK/FGF pathways were differentially expressed along the regenerating tail axis. Furthermore, we identified 2 microRNA precursor families, 22 unclassified non-coding RNAs, and 3 novel protein-coding genes significantly enriched in the regenerating tail. However, high levels of progenitor/stem cell markers were not observed in any region of the regenerating tail. Furthermore, we observed multiple tissue-type specific clusters of proliferating cells along the regenerating tail, not localized to the tail tip. These findings predict a different mechanism of regeneration in the lizard than the blastema model described in the salamander and the zebrafish, which are anamniote vertebrates. Thus, lizard tail regrowth involves the activation of conserved developmental and wound response pathways, which are potential targets for regenerative medical therapies.

A histological comparison of the original and regenerated tail in the green anole, Anolis carolinensis.

This study provides a histological comparison of the mature regenerated and original tail of the lizard Anolis carolinensis. These data will provide a framework for future studies of this emerging model organism whose genome was recently published. This study demonstrated that the cartilage skeleton of the regenerated tail enclosed a spinal cord with an ependymal core, but there was no evidence that dorsal root ganglia or peripheral nerves are regenerated. The cartilage tube contained foramina that allowed the vasculature to cross, but was otherwise a rigid structure. The original tail has muscle groups arranged in quadrants in a regular pattern that attach to the vertebral column. The regenerated tail has irregular muscle bundles of variable number that form unusual attachments to each other and to the cartilage tube. Furthermore, the data show that there was increased connective tissue within the muscle bundles. Implications for functionality of the regenerated tail and for future biomechanical studies are discussed.

The gross anatomy of the original and regenerated tail in the green anole (Anolis carolinensis).

This study investigates the gross anatomy of the original and the regenerated tail in the green anole (Anolis carolinensis). Dissections were conducted on 24 original and 13 regenerated tails. While the extrinsic muscles of the original tail in A. carolinensis are similar to those in other known Anolis lizard species, the extent of the origins of m. caudofemoralis longus and m. caudofemoralis brevis is more restricted. These differences may underlie variation in locomotor performance among anole ecomorphs. The intrinsic muscles of the original tail are also described, confirming previous findings and documenting new details, including muscle origins and insertions and the range of intraspecific variation. A comparison of the intrinsic muscles of the original tail and the regenerated tail muscles reveals key differences, such as the lack of interdigitating muscle segments and intramuscular septa in the regenerated tail. These findings, along with the replacement of interlocking vertebrae with a stiff, cartilaginous rod, suggest that important functional differences exist between the original and regenerated tail. In particular, the regenerated tail is predicted to be less capable of coordinated, fine movements. Studies of the physical properties and range of motion of the original and regenerated tail are required to test this hypothesis. This atlas of tail anatomy in A. carolinensis represents a key resource for developmental and genetic studies of tail regeneration in lizards, as well as studies of anole evolution and biomechanics.

Numb regulates Notch1, but not Notch3, during myogenesis.

In the vertebrate embryo, skeletal muscle is derived from the myotome of the somites. Notch1-3 demonstrate overlapping and distinct expression patterns in mouse somites. Notch1 and Notch2 have been shown to be inhibitors of skeletal myogenesis. The current data demonstrate that Notch3 also is an effective inhibitor of MyoD induced myogenesis. Numb, an adaptor protein that promotes Notch degradation by recruiting the E3 ubiquitin ligase, Itch, is limited in expression to dividing cells of the dorsal medial lip of the dermomyotome and the myotome itself. Here the specificity of the four protein isoforms of Numb for the Notch receptors was examined. In transcription and myogenic differentiation assays, Notch1 was consistently negatively regulated by all four Numb isoforms, and Notch3 was not a target for Numb. Notch2 however was variably affected. Subsequent analyses showed that unlike Notch1, that Notch3 was not polyubiquitinated, nor degraded when co-expressed in cells with Numb. These data provide the first observations that Notch receptors are variably affected by Numb and will be important for the interpretation of the function of Notch and Numb interactions during the development of many different cells and tissues.

A deficiency of lunatic fringe is associated with cystic dilation of the rete testis.

Lunatic fringe belongs to a family of beta1-3 N-acetyltransferases that modulate the affinity of the Notch receptors for their ligands through the elongation of O-fucose moieties on their extracellular domain. A role for Notch signaling in vertebrate fertility has been predicted by the intricate expression of the Notch receptors and their ligands in the oocyte and granulosa cells of the ovary and the spermatozoa and Sertoli cells of the testis. It has been demonstrated that disruption of Notch signaling by inactivation of lunatic fringe led to infertility associated with pleiotropic defects in follicle development and meiotic maturation of oocytes. Lunatic fringe null males were found to be subfertile. Here, we report that gene expression data demonstrate that fringe and Notch signaling genes are expressed in the developing testis and the intratesticular ductal tract, predicting roles for this pathway during embryonic gonadogenesis and spermatogenesis. Spermatogenesis was not impaired in the majority of the lunatic fringe null males; however, spermatozoa were unilaterally absent in the epididymis of many mice. Histological and immunohistochemical analysis of these testes revealed the development of unilateral cystic dilation of the rete testis. Tracer dye experiments confirm a block in the connection between the rete testis and the efferent ducts. Further, the dye studies demonstrated that many lunatic fringe mutant males had partial blocks of the connection between the rete testis and the efferent ducts bilaterally.

Wnt3a links left-right determination with segmentation and anteroposterior axis elongation.

The alignment of the left-right (LR) body axis relative to the anteroposterior (AP) and dorsoventral (DV) axes is central to the organization of the vertebrate body plan and is controlled by the node/organizer. Somitogenesis plays a key role in embryo morphogenesis as a principal component of AP elongation. How morphogenesis is coupled to axis specification is not well understood. We demonstrate that Wnt3a is required for LR asymmetry. Wnt3a activates the Delta/Notch pathway to regulate perinodal expression of the left determinant Nodal, while simultaneously controlling the segmentation clock and the molecular oscillations of the Wnt/beta-catenin and Notch pathways. We provide evidence that Wnt3a, expressed in the primitive streak and dorsal posterior node, acts as a long-range signaling molecule, directly regulating target gene expression throughout the node and presomitic mesoderm. Wnt3a may also modulate the symmetry-breaking activity of mechanosensory cilia in the node. Thus, Wnt3a links the segmentation clock and AP axis elongation with key left-determining events, suggesting that Wnt3a is an integral component of the trunk organizer.

Notch pathway genes are expressed in mammalian ovarian follicles.

Folliculogenesis is the process of development of ovarian follicles that ultimately results in the release of fertilizable oocytes at ovulation. This is a complex program that involves the proliferation and differentiation of granulosa cells. Granulosa cells are necessary for follicle growth and support the oocyte during folliculogenesis. Genes that regulate the proliferation and differentiation of granulosa cells are beginning to be elucidated. In this study, the expression patterns of Notch receptor genes and their ligands, which have been shown to regulate cell-fate decisions in many systems during development, were examined in the mammalian ovary. In situ hybridization data showed that Notch2, Notch3, and Jagged2 were expressed in an overlapping pattern in the granulosa cells of developing follicles. Jagged1 was expressed in oocytes exclusively. Downstream target genes of Notch also were expressed in granulosa cells. These data implicate the Notch signaling pathway in the regulation of mammalian folliculogenesis.

Developmental history of the mammalian oocyte: insight from mouse mutations.

Growth and differentiation of the mammalian oocyte is regulated with the coordinate development of the granulosa cells. The complex signaling pathways that regulate the growth and development of mammalian oocytes are beginning to be elucidated through the use of gene targeting. These technologies have provided new insight into the roles of specific genes during the development of the germ cells and gonads, as well as post-pubertal development of oocytes. In many cases, these studies have resulted in a new understanding of the function of certain genes, in others they have provided new genes and pathways to be studied in mammalian reproductive biology. Ultimately, these studies will shed light on human genetic disease and infertility.

Requirements for the kit receptor tyrosine kinase during regeneration of zebrafish fin melanocytes.

Embryonic neural crest-derived melanocytes and their precursors express the kit receptor tyrosine kinase and require its function for their migration and survival. However, mutations in kit also cause deficits in melanocytes that make up adult pigment patterns, including melanocytes that re-establish the zebrafish fin stripes during regeneration. As adult melanocytes in mice and zebrafish are generated and maintained by stem cell populations that are presumably established during embryonic development, it has been proposed that adult phenotypes in kit mutants result from embryonic requirements for kit. We have used a temperature-sensitive zebrafish kit mutation to show that kit is required during adult fin regeneration to promote melanocyte differentiation, rather than during embryonic stages to establish their stem cell precursors. We also demonstrate a transient role for kit in promoting the survival of newly differentiated regeneration melanocytes.

How the zebrafish gets its stripes.

The study of vertebrate pigment patterns is a classic and enduring field of developmental biology. Knowledge of pigment pattern development comes from a variety of systems, including avians, mouse, and more recently, the zebrafish (Danio rerio). Recent analyses of the mechanisms underlying the development of the neural crest-derived pigment cell type common to all vertebrates, the melanocyte, have revealed remarkable similarities and several surprising differences between amniotes and zebrafish. Here, we summarize recent advances in the study of melanocyte development in zebrafish, with reference to human, mouse, and avian systems. We first review melanocyte development in zebrafish and mammals, followed by a summary of the molecules known to be required for their development. We then discuss several relatively unaddressed issues in vertebrate pigment pattern development that are being investigated in zebrafish. These include determining the relationships between genetically distinct classes of melanocytes, characterizing and dissecting melanocyte stem cell development, and understanding how pigment cells organize into a patterned tissue. Further analysis of zebrafish pigment pattern mutants as well as new generations of directed mutant screens promise to extend our understanding of pigment pattern morphogenesis.

Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio.

Pigment patterns of fishes are a tractable system for studying the genetic and cellular bases for postembryonic phenotypes. In the zebrafish Danio rerio, neural crest-derived pigment cells generate different pigment patterns during different phases of the life cycle. Whereas early larvae exhibit simple stripes of melanocytes and silver iridophores in a background of yellow xanthophores, this pigment pattern is transformed at metamorphosis into that of the adult, comprising a series of dark melanocyte and iridophore stripes, alternating with light stripes of iridophores and xanthophores. Although several genes have been identified in D. rerio that contribute to the development of both early larval and adult pigment patterns, comparatively little is known about genes that are essential for pattern formation during just one or the other life cycle phase. In this study, we identify the gene responsible for the rose mutant phenotype in D. rerio. rose mutants have wild-type early larval pigment patterns, but fail to develop normal numbers of melanocytes and iridophores during pigment pattern metamorphosis and exhibit a disrupted pattern of these cells. We show that rose corresponds to endothelin receptor b1 (ednrb1), an orthologue of amniote Ednrb genes that have long been studied for their roles in neural crest and pigment cell development. Furthermore, we demonstrate that D. rerio ednrb1 is expressed both during pigment pattern metamorphosis and during embryogenesis, and cells of melanocyte, iridophore, and xanthophore lineages all express this gene. These analyses suggest a phylogenetic conservation of roles for Ednrb signaling in the development of amniote and teleost pigment cell precursors. As murine Ednrb is essential for the development of all neural crest derived melanocytes, and D. rerio ednrb1 is required only by a subset of adult melanocytes and iridophores, these analyses also reveal variation among vertebrates in the cellular requirements for Ednrb signaling, and suggest alternative models for the cellular and genetic bases of pigment pattern metamorphosis in D. rerio.

Zebrafish kit mutation reveals primary and secondary regulation of melanocyte development during fin stripe regeneration.

Fin regeneration in adult zebrafish is accompanied by re-establishment of the pigment stripes. To understand the mechanisms underlying fin stripe regeneration and regulation of normal melanocyte stripe morphology, we investigated the origins of melanocytes in the regenerating fin and their requirement for the kit receptor tyrosine kinase. Using pre-existing melanin as a lineage tracer, we show that most fin regeneration melanocytes develop from undifferentiated precursors, rather than from differentiated melanocytes. Mutational analysis reveals two distinct classes of regeneration melanocytes. First, an early regeneration class develops dependent on kit function. In the absence of kit function and kit-dependent melanocytes, a second class of melanocytes develops at later stages of regeneration. This late kit-independent class of regeneration melanocytes has little or no role in wild-type fin stripe development, thus revealing a secondary mode for regulation of fin stripes. Expression of melanocyte markers in regenerating kit mutant fins suggests that kit normally acts after mitf and before dct to promote development of the primary kit-dependent melanocytes. kit-dependent and kit-independent melanocytes are also present during fin stripe ontogeny in patterns similar to those observed during regeneration.

Requirements for the mitochondrial import and localization of dihydroorotate dehydrogenase.

In animals, dihydroorotate dehydrogenase (DHODH) is a mitochondrial protein that carries out the fourth step in de novo pyrimidine biosynthesis. Because this is the only enzyme of this pathway that is localized to mitochondria and because the enzyme is cytosolic in some bacteria and fungi, we carried out studies to understand the mode of targeting of animal DHODH and its submitochondrial localization. Analysis of fractionated rat liver mitochondria revealed that DHODH is an integral membrane protein exposed to the intermembrane space. In vitro-synthesized Drosophila, rat and human DHODH proteins were efficiently imported into the intermembrane space of isolated yeast mitochondria. Import did not alter the size of the in vitro synthesized protein, nor was there a detectable size difference when compared to the DHODH protein found in vivo. Thus, there is no apparent proteolytic processing of the protein during import either in vitro or in vivo. Import of rat DHODH into isolated yeast mitochondria required inner membrane potential and was at least partially dependent upon matrix ATP, indicating that its localization uses the well described import machinery of the mitochondrial inner membrane. The DHODH proteins of animals differ from the cytosolic proteins found in some bacteria and fungi by the presence of an N-terminal segment that resembles mitochondrial-targeting presequences. Deletion of the cationic portion of this N-terminal sequence from the rat DHODH protein blocked its import into isolated yeast mitochondria, whereas deletion of the adjacent hydrophobic segment resulted in import of the protein into the matrix. Thus, the N-terminus of the DHODH protein contains a bipartite signal that governs import and correct insertion into the mitochondrial inner membrane.

Genetic regulation of somite formation.

Segmentation of the paraxial mesoderm into somites requires a strategy distinct from the division of a preexisting field of cells, as seen in the segmentation of the vertebrate hindbrain into rhombomeres and the formation of the body plan of invertebrates. Each new somite forms from the anterior end of the segmental plate; therefore, the conditions for establishing the anterior-posterior boundary must be re-created prior to the formation of the next somite. It has been established that regulation of this process is native to the anterior end of the segmental plate, however, the components of a genetic pathway are poorly understood. A growing library of candidate genes has been generated from hybridization screens and sequence homology searches, which include cell adhesion molecules, cell surface receptors, growth factors, and transcription factors. With the increasing accessibility of gene knockout technology, many of these genes have been tested for their role in regulating somitogenesis. In this chapter, we will review the significant advances in our understanding of segmentation based on these experiments.

Differential regulation of epaxial and hypaxial muscle development by paraxis.

In vertebrates, skeletal muscle is derived from progenitor cell populations located in the epithelial dermomyotome compartment of the each somite. These cells become committed to the myogenic lineage upon delamination from the dorsomedial and dorsolateral lips of the dermomyotome and entry into the myotome or dispersal into the periphery. Paraxis is a developmentally regulated transcription factor that is required to direct and maintain the epithelial characteristic of the dermomyotome. Therefore, we hypothesized that Paraxis acts as an important regulator of early events in myogenesis. Expression of the muscle-specific myogenin-lacZ transgene was used to examine the formation of the myotome in the paraxis-/- background. Two distinct types of defects were observed that mirrored the different origins of myoblasts in the myotome. In the medial myotome, where the expression of the myogenic factor Myf5 is required for commitment of myoblasts, the migration pattern of committed myoblasts was altered in the absence of Paraxis. In contrast, in the lateral myotome and migratory somitic cells, which require the expression of MyoD, expression of the myogenin-lacZ transgene was delayed by several days. This delay correlated with an absence of MyoD expression in these regions, indicating that Paraxis is required for commitment of cells from the dorsolateral dermomyotome to the myogenic lineage. In paraxis-/-/myf5-/- neonates, dramatic losses were observed in the epaxial and hypaxial trunk muscles that are proximal to the vertebrae in the compound mutant, but not those at the ventral midline or the non-segmented muscles of the limb and tongue. In this genetic background, myoblasts derived from the medial (epaxial) myotome are not present to compensate for deficiencies of the lateral (hypaxial) myotome. Our data demonstrate that Paraxis is an important regulator of a subset of the myogenic progenitor cells from the dorsolateral dermomyotome that are fated to form the non-migratory hypaxial muscles.

Zebrafish sparse corresponds to an orthologue of c-kit and is required for the morphogenesis of a subpopulation of melanocytes, but is not essential for hematopoiesis or primordial germ cell development.

The relative roles of the Kit receptor in promoting the migration and survival of amniote melanocytes are unresolved. We show that, in the zebrafish, Danio rerio, the pigment pattern mutation sparse corresponds to an orthologue of c-kit. This finding allows us to further elucidate morphogenetic roles for this c-kit-related gene in melanocyte morphogenesis. Our analyses of zebrafish melanocyte development demonstrate that the c-kit orthologue identified in this study is required both for normal migration and for survival of embryonic melanocytes. We also find that, in contrast to mouse, the zebrafish c-kit gene that we have identified is not essential for hematopoiesis or primordial germ cell development. These unexpected differences may reflect evolutionary divergence in c-kit functions following gene duplication events in teleosts.

Activated notch inhibits myogenic activity of the MADS-Box transcription factor myocyte enhancer factor 2C.

Skeletal muscle gene expression is dependent on combinatorial associations between members of the MyoD family of basic helix-loop-helix (bHLH) transcription factors and the myocyte enhancer factor 2 (MEF2) family of MADS-box transcription factors. The transmembrane receptor Notch interferes with the muscle-inducing activity of myogenic bHLH proteins, and it has been suggested that this inhibitory activity of Notch is directed at an essential cofactor that recognizes the DNA binding domains of the myogenic bHLH proteins. Given that MEF2 proteins interact with the DNA binding domains of myogenic bHLH factors to cooperatively regulate myogenesis, we investigated whether members of the MEF2 family might serve as targets for the inhibitory effects of Notch on myogenesis. We show that a constitutively activated form of Notch specifically blocks DNA binding by MEF2C, as well as its ability to cooperate with MyoD and myogenin to activate myogenesis. Responsiveness to Notch requires a 12-amino-acid region of MEF2C immediately adjacent to the DNA binding domain that is unique to this MEF2 isoform. Two-hybrid assays and coimmunoprecipitations show that this region of MEF2C interacts directly with the ankyrin repeat region of Notch. These findings reveal a novel mechanism for Notch-mediated inhibition of myogenesis and demonstrate that the Notch signaling pathway can discriminate between different members of the MEF2 family.

A mutation that uncouples allosteric regulation of carbamyl phosphate synthetase in Drosophila.

In animals, UTP feedback inhibition of carbamyl phosphate synthetase II (CPSase) controls pyrimidine biosynthesis. Suppressor of black (Su(b) or rSu(b)) mutants of Drosophila melanogaster have elevated pyrimidine pools, and this mutation has been mapped to the rudimentary locus. We report that rSu(b) is a missense mutation resulting in a glutamate to lysine substitution within the second ATP binding site (i.e. CPS.B2 domain) of CPSase. This residue corresponds to Glu780 in the Escherichia coli enzyme (Glu1153 in hamster CAD) and is universally conserved among CPSases. When a transgene expressing the Glu-->Lys substitution was introduced into Drosophila lines homozygous for the black mutation, the resulting flies exhibited the Su(b) phenotype. Partially purified CPSase from rSu(b) and transgenic flies carrying this substitution exhibited a dramatic reduction in UTP feedback inhibition. The slight UTP inhibition observed with the Su(b) enzyme in vitro was due mainly to chelation of Mg2+ by UTP. However, the Km values for glutamate, bicarbonate, and ATP obtained from the Su(b) enzyme were not significantly different from wild-type values. From these experiments, we conclude that this residue plays an essential role in the UTP allosteric response, probably in propagating the response between the effector binding site and the ATP binding site. This is the first CPSase mutation found to abolish feedback inhibition without significantly affecting other enzyme catalytic parameters.