Gerrit Begemann

Induction and prepatterning of the zebrafish pectoral fin bud requires axial retinoic acid signaling

Vertebrate forelimbs arise as bilateral appendages from the lateral plate mesoderm (LPM). Mutants in aldh1a2 (raldh2), an embryonically expressed gene encoding a retinoic acid (RA)-synthesizing enzyme, have been used to show that limb development and patterning of the limb bud are crucially dependent on RA signaling. However, the timing and cellular origin of RA signaling in these processes have remained poorly resolved. We have used genetics and chemical modulators of RA signaling to resolve these issues in the zebrafish. By rescuing pectoral fin induction in the aldh1a2/neckless mutant with exogenous RA and by blocking RA signaling in wild-type embryos, we find that RA acts as a permissive signal that is required during the six- to eight-somite stages for pectoral fin induction. Cell-transplantation experiments show that RA production is not only crucially required from flanking somites, but is sufficient to permit fin bud initiation when the trunk mesoderm is genetically ablated. Under the latter condition, intermediate mesoderm alone cannot induce the pectoral fin field in the LPM. We further show that induction of the fin field is directly followed by a continued requirement for somite-derived RA signaling to establish a prepattern of anteroposterior fates in the condensing fin mesenchyme. This process is mediated by the maintained expression of the transcription factor hand2, through which the fin field is continuously posteriorized, and lasts up to several hours prior to limb-budding. Thus, RA signaling from flanking somites plays a dual early role in the condensing limb bud mesenchyme.

Developmental regulation of Tbx5 in zebrafish embryogenesis.

T-box (tbx) genes constitute a large family of transcriptional regulators involved in developmental patterning processes. In tetrapods, tbx5 has been implicated in specifying forelimb type identity. Here, we report the cloning of the zebrafish tbx5.1 gene and characterise its expression during zebrafish embryogenesis and early larval development of wild type and mutant embryos that affect pectoral fin patterning. tbx5.1 is expressed during development of the heart, the pectoral fins and the eye. Notably, its expression in the lateral plate mesoderm defines a single and continuous region of heart and pectoral fin precursor cells, and constitutes the earliest specific marker for pectoral fin development in the zebrafish.

Retinoic acid signaling controls the formation, proliferation and survival of the blastema during adult zebrafish fin regeneration

Adult teleosts rebuild amputated fins through a proliferation-dependent process called epimorphic regeneration, in which a blastema of cycling progenitor cells replaces the lost fin tissue. The genetic networks that control formation of blastema cells from formerly quiescent stump tissue and subsequent blastema function are still poorly understood. Here, we investigated the cellular and molecular consequences of genetically interfering with retinoic acid (RA) signaling for the formation of the zebrafish blastema. We show that RA signaling is upregulated within the first few hours after fin amputation in the stump mesenchyme, where it controls Fgf, Wnt/β-catenin and Igf signaling. Genetic inhibition of the RA pathway at this stage blocks blastema formation by inhibiting cell cycle entry of stump cells and impairs the formation of the basal epidermal layer, a signaling center in the wound epidermis. In the established blastema, RA signaling remains active to ensure the survival of the highly proliferative blastemal population by controlling expression of the anti-apoptotic factor bcl2. In addition, RA signaling maintains blastema proliferation through the activation of growth-stimulatory signals mediated by Fgf and Wnt/β-catenin signaling, as well as by reducing signaling through the growth-inhibitory non-canonical Wnt pathway. The endogenous roles of RA in adult vertebrate appendage regeneration are uncovered here for the first time. They provide a mechanistic framework to understand previous observations in salamanders that link endogenous sources of RA to the regeneration process itself and support the hypothesis that the RA signaling pathway is an essential component of vertebrate tissue regeneration.

Cloning of zebrafish T-box genes tbx15 and tbx18 and their expression during embryonic development

Members of the T-box (tbx) gene family encode developmentally regulated transcription factors, several of which are implicated in human hereditary diseases. We have cloned the paralogous genes tbx15 and tbx18 in zebrafish and have characterised their expression in detail. tbx15 is expressed in paraxial head mesenchyme and its derivatives, the extraocular and jaw musculature and the posterior neurocranium. Further areas of tbx15 expression are in the anterior somitic mesoderm, in periocular mesenchyme and in the pectoral fin mesenchyme throughout larval development. Areas of strong tbx18 expression are found in the developing somitic and presomitic mesoderm, in the heart and in pectoral fin mesenchyme, as well as the ventral neuroectoderm and the developing palate. Both genes exhibit particular differences in expression compared to their murine orthologs.

Differential regulation of msx genes in the development of the gonopodium, an intromittent organ, and of the ''sword,'' a sexually selected trait of swordtail fishes (Xiphophorus)

Summary The possession of a conspicuous extension of colored ventral rays of the caudal fin in male fish of swordtails (genus Xiphophorus) is a prominent example for a trait that evolved by sexual selection. To understand the evolutionary history of this so‐called sword molecularly, it is of interest to unravel the developmental pathways responsible for extended growth of sword rays during development of swordtail males. We isolated two msx genes and showed that they are differentially regulated during sword outgrowth. During sword growth in juvenile males, as well as during testosterone‐induced sword development and fin ray regeneration in the sword after amputation, expression of msxC is markedly up‐regulated in the sword forming fin rays. In contrast, msxE/1 is not differentially expressed in ventral and dorsal male fin rays, suggesting a link between the development of male secondary sexual characters in fins and up‐regulation of msxC expression. In addition, we showed that msx gene expression patterns differ significantly between Xiphophorus and zebrafish. We also included in our study the gonopodium, a testosterone‐dependent anal fin modification that serves as a fertilization organ in males of live‐bearing fishes. Our finding that increased levels of msxC expression are associated with the testosterone‐induced outgrowth of the gonopodium might suggest either that at least parts of the signaling pathways that pattern the evolutionary older gonopodium have been coopted to evolve a sexually selected innovation such as the sword or that increased msxC expression may be inherent to the growth process of long fin rays in general.

Expression of zebrafish aldh1a3 (raldh3) and absence of aldh1a1 in teleosts

The vitamin A-derived morphogen retinoic acid (RA) plays important roles during the development of chordate animals. The Aldh1a-family of RA-synthesizing enzymes consists of three members, Aldh1a1-3 (Raldh1-3), that are dynamically expressed throughout development. We have searched the known teleost genomes for the presence of Raldh family members and have found that teleost fish possess orthologs of Aldh1a2 and Aldh1a3 only. Here we describe the expression of aldh1a3 in the zebrafish, Danio rerio. Whole mount in situ hybridization shows that aldh1a3 is expressed during eye development in the retina flanking the optic stalks and later is expressed ventrally, opposite the expression domain of aldh1a2. During inner ear morphogenesis, aldh1a3 is expressed in developing sensory epithelia of the cristae and utricular macula and is specifically up-regulated in epithelial projections throughout the formation of the walls of the semicircular canals and endolymphatic duct. In contrast to the mouse inner ear, which expresses all three Raldhs, we find that only aldh1a3 is expressed in the zebrafish otocyst, while aldh1a2 is present in the periotic mesenchyme. During larval stages, additional expression domains of aldh1a3 appear in the anterior pituitary and the swim bladder. Our analyses provide a starting point for genetic studies to examine the role of RA in these organs and emphasize the suitability of the zebrafish inner ear in dissecting the contribution of RA signaling to the development of the vestibular system.

The origin of bmp16, a novel Bmp2/4 relative, retained in teleost fish genomes

BackgroundWhole genome sequences have allowed us to have an overview of the evolution of gene repertoires. The target of the present study, the TGFβ superfamily, contains many genes involved in vertebrate development, and provides an ideal system to explore the relationships between evolution of gene repertoires and that of developmental programs.ResultsAs a result of a bioinformatic survey of sequenced vertebrate genomes, we identified an uncharacterized member of the TGFβ superfamily, designated bmp16, which is confined to teleost fish species. Our molecular phylogenetic study revealed a high affinity of bmp16 to the Bmp2/4 subfamily. Importantly, further analyses based on the maximum-likelihood method unambiguously ruled out the possibility that this teleost-specific gene is a product of teleost-specific genome duplication. This suggests that the absence of a bmp16 ortholog in tetrapods is due to a secondary loss. In situ hybridization showed embryonic expression of the zebrafish bmp16 in the developing swim bladder, heart, tail bud, and ectoderm of pectoral and median fin folds in pharyngula stages, as well as gut-associated expression in 5-day embryos.ConclusionComparisons of expression patterns revealed (1) the redundancy of bmp16 expression with its homologs in presumably plesiomorphic expression domains, such as the fin fold, heart, and tail bud, which might have permitted its loss in the tetrapod lineage, and (2) the loss of craniofacial expression and gain of swim bladder expression of bmp16 after the gene duplication between Bmp2, -4 and -16. Our findings highlight the importance of documenting secondary changes of gene repertoires and expression patterns in other gene families.

Conservation and co-option in developmental programmes: the importance of homology relationships

One of the surprising insights gained from research in evolutionary developmental biology (evo-devo) is that increasing diversity in body plans and morphology in organisms across animal phyla are not reflected in similarly dramatic changes at the level of gene composition of their genomes. For instance, simplicity at the tissue level of organization often contrasts with a high degree of genetic complexity. Also intriguing is the observation that the coding regions of several genes of invertebrates show high sequence similarity to those in humans. This lack of change (conservation) indicates that evolutionary novelties may arise more frequently through combinatorial processes, such as changes in gene regulation and the recruitment of novel genes into existing regulatory gene networks (co-option), and less often through adaptive evolutionary processes in the coding portions of a gene. As a consequence, it is of great interest to examine whether the widespread conservation of the genetic machinery implies the same developmental function in a last common ancestor, or whether homologous genes acquired new developmental roles in structures of independent phylogenetic origin. To distinguish between these two possibilities one must refer to current concepts of phylogeny reconstruction and carefully investigate homology relationships. Particularly problematic in terms of homology decisions is the use of gene expression patterns of a given structure. In the future, research on more organisms other than the typical model systems will be required since these can provide insights that are not easily obtained from comparisons among only a few distantly related model species.

MicroRNAs and RNA interference in zebrafish development.

Posttranscriptional regulation of gene activity has been a somewhat neglected subject in developmental genetics. With the discovery of RNA-mediated silencing mechanisms, however, insights into how targeted transcript inactivation integrates with developmental processes have changed radically. The number of studies in zebrafish that take advantage of techniques to manipulate the activity of microRNAs (miRNAs)--a group of short, noncoding RNAs that suppress translation of target genes--is on a steady rise, and the studies are starting to provide unique insights into the diversity of developmental processes that are controlled by transcript inhibition. Here I review recent studies in the zebrafish that demonstrate roles for miRNAs in the fine-tuning of neural crest cell migration, regulation of neural Hox gene expression, and regeneration after tissue amputation. New discoveries on the involvement of miRNAs in regulating red blood cell maturation also shed light on how miRNA gene activity itself is controlled. Because experimental suppression of single miRNAs often results in surprisingly specific phenotypes, it will have to be considered whether novel mutants identified in genetic screens should also be assayed for lesions in miRNA genes or their target sequences, rather than in protein-coding genes alone.

Formation of oral and pharyngeal dentition in teleosts depends on differential recruitment of retinoic acid signaling

One of the goals of evolutionary developmental biology is to link specific adaptations to changes in developmental pathways. The dentition of cypriniform fishes, which in contrast to many other teleost fish species possess pharyngeal teeth but lack oral teeth, provides a suitable model to study the development of feeding adaptations. Here, we have examined the involvement of retinoic acid (RA) in tooth development and show that RA is specifically required to induce the pharyngeal tooth developmental program in zebrafish. Perturbation of RA signaling at this stage abolished tooth induction without affecting the development of tooth‐associated ceratobranchial bones. We show that this inductive event is dependent on RA synthesis from aldh1a2 in the ventral posterior pharynx. Fibroblast growth factor (FGF) signaling has been shown to be critical for tooth induction in zebrafish, and its loss has been associated with oral tooth loss in cypriniform fishes. Pharmacological treatments targeting the RA and FGF pathways revealed that both pathways act independently during tooth induction. In contrast, we find that in Mexican tetra and medaka, species that also possess oral teeth, both oral and pharyngeal teeth are induced independently of RA. Our analyses suggest an evolutionary scenario in which the gene network controlling tooth development obtained RA dependency in the lineage leading to the cypriniforms. The loss of pharyngeal teeth in this group was cancelled out through a shift in aldh1a2 expression, while oral teeth might have been lost ultimately due to deficient RA signaling in the oral cavity.—Gibert, Y., Bernard, L., Debiais‐Thibaud, M., Bourrat, F., Joly, J.‐S., Pottin, K., Meyer, A., Retaux, S., Stock, D. W., Jackman, W. R., Seritrakul, P., Begemann, G., Laudet, V. Formation of oral and pharyngeal dentition in teleosts depends on differential recruitment of retinoic acid signaling. FASEB J. 24, 3298–3309 (2010). www.fasebj.org