Temporal and spatial action of tolloid (mini fin) and chordin to pattern tail tissues

In vertebrates, a bone morphogenetic protein (BMP) signaling pathway patterns all ventral cell fates along the embryonic axis. BMP activity is positively regulated by Tolloid, a metalloprotease, that can eliminate the activity of the BMP antagonist Chordin. A tolloid mutant in zebrafish, mini fin (mfn), exhibits a specific loss of ventral tail tissues. Here, we investigate the spatial and temporal requirements for Tolloid (Mfn) in dorsoventral patterning of the tail. Through chimeric analyses, we found that Tolloid (Mfn) functions cell non-autonomously in the ventral-most vegetal cells of the gastrula or their derivatives. We generated a tolloid transgene under the control of the inducible hsp70 promoter and demonstrate that tolloid (mfn) is first required at the completion of gastrulation. Although tolloid is expressed during gastrulation and dorsally and ventrally within the tail bud, our results indicate that Tolloid (Mfn) acts specifically in the ventral tail bud during a approximately 4 h period extending from the completion of gastrulation to early somitogenesis stages to regulate BMP signaling. Examination of the temporal requirements of Chordin activity by overexpression of the hsp70-tolloid transgene indicates that Chordin is required both during and after gastrulation for proper patterning of the tail, contrasting Tld's requirement only during post-gastrula stages. We hypothesize that the gastrula role of Chordin in tail patterning is to generate the proper size domains of cells to enter the ventral and dorsal tail bud, whereas post-gastrula Chordin activity patterns the derivatives of the tail bud. Thus, fine modulation of BMP signaling levels through the negative and positive actions of Chordin and Tolloid, respectively, patterns tail tissues.     

Asymmetric expression of the BMP antagonists chordin and gremlin in the sea anemone Nematostella vectensis: implications for the evolution of axial patterning

The evolutionary origin of the anterior-posterior and the dorsoventral body axes of Bilateria is a long-standing question. It is unclear how the main body axis of Cnidaria, the sister group to the Bilateria, is related to the two body axes of Bilateria. The conserved antagonism between two secreted factors, BMP2/4 (Dpp in Drosophila) and its antagonist Chordin (Short gastrulation in Drosophila) is a crucial component in the establishment of the dorsoventral body axis of Bilateria and could therefore provide important insight into the evolutionary origin of bilaterian axes. Here, we cloned and characterized two BMP ligands, dpp and GDF5-like as well as two secreted antagonists, chordin and gremlin, from the basal cnidarian Nematostella vectensis. Injection experiments in zebrafish show that the ventralizing activity of NvDpp mRNA is counteracted by NvGremlin and NvChordin, suggesting that Gremlin and Chordin proteins can function as endogenous antagonists of NvDpp. Expression analysis during embryonic and larval development of Nematostella reveals asymmetric expression of all four genes along both the oral-aboral body axis and along an axis perpendicular to this one, the directive axis. Unexpectedly, NvDpp and NvChordin show complex and overlapping expression on the same side of the embryo, whereas NvGDF5-like and NvGremlin are both expressed on the opposite side. Yet, the two pairs of ligands and antagonists only partially overlap, suggesting complex gradients of BMP activity along the directive axis but also along the oral-aboral axis. We conclude that a molecular interaction between BMP-like molecules and their secreted antagonists was already employed in the common ancestor of Cnidaria and Bilateria to create axial asymmetries, but that there is no simple relationship between the oral-aboral body axis of Nematostella and one particular body axis of Bilateria.     

Twisted gastrulation is required for forebrain specification and cooperates with Chordin to inhibit BMP signaling during X. tropicalis gastrulation

In the developing vertebrate embryo, proper dorsal-ventral patterning relies on BMP antagonists secreted by the organizer during gastrulation. The BMP antagonist chordin has a complex interaction with BMPs that is governed in part by its interaction with the secreted protein twisted gastrulation (tsg). In different contexts, tsg has activity as either a BMP agonist or as a BMP antagonist. Using morpholino oligonucleotides in Xenopus tropicalis, we show that reducing tsg gene product results in a ventralized embryo, and that tsg morphants specifically lack a forebrain. We provide new evidence that tsg acts as a BMP antagonist during X. tropicalis gastrulation since the tsg depletion phenotype can be rescued in two ways: by chordin overexpression and by BMP depletion. We conclude that tsg acts as a BMP antagonist in the context of the frog gastrula, and that it acts cooperatively with chordin to establish dorsal structures and particularly forebrain tissue during development.     

HyBMP5-8b, a BMP5-8 orthologue, acts during axial patterning and tentacle formation in hydra

Developmental gradients play a central role in axial patterning in hydra. As part of the effort towards elucidating the molecular basis of these gradients as well as investigating the evolution of the mechanisms underlying axial patterning, genes encoding signaling molecules are under investigation. We report the isolation and characterization of HyBMP5-8b, a BMP5-8 orthologue, from hydra. Processes governing axial patterning are continuously active in adult hydra. Expression patterns of HyBMP5-8b in normal animals and during bud formation, hydra's asexual form of reproduction, were examined. These patterns, coupled with changes in patterns of expression in manipulated tissues during head regeneration, foot regeneration as well as under conditions that alter the positional value gradient indicate that the gene is active in two different processes. The gene plays a role in tentacle formation and in patterning the lower end of the body axis.     

Computational analysis of BMP gradients in dorsal-ventral patterning of the zebrafish embryo

The genetic network controlling early dorsal-ventral (DV) patterning has been extensively studied and modeled in the fruit fly Drosophila. This patterning is driven by signals coming from bone morphogenetic proteins (BMPs), and regulated by interactions of BMPs with secreted factors such as the antagonist short gastrulation (Sog). Experimental studies suggest that the DV patterning of vertebrates is controlled by a similar network of BMPs and antagonists (such as Chordin, a homologue of Sog), but differences exist in how the two systems are organized, and a quantitative comparison of pattern formation in them has not been made. Here, we develop a computational model in three dimensions of the zebrafish embryo and use it to study molecular interactions in the formation of BMP morphogen gradients in early DV patterning. Simulation results are presented on the dynamics BMP gradient formation, the cooperative action of two feedback loops from BMP signaling to BMP and Chordin synthesis, and pattern sensitivity with respect to BMP and Chordin dosage. Computational analysis shows that, unlike the case in Drosophila, synergy of the two feedback loops in the zygotic control of BMP and Chordin expression, along with early initiation of localized Chordin expression, is critical for establishment and maintenance of a stable and appropriate BMP gradient in the zebrafish embryo.     

The ventralized ogon mutant phenotype is caused by a mutation in the zebrafish homologue of Sizzled,a secreted Frizzled-related protein

The BMP signaling pathway plays a key role during dorsoventral pattern formation of vertebrate embryos. In zebrafish, all cloned mutants affecting this process are deficient in members of the BMP pathway. In a search for factors differentially expressed in swirl/bmp2b mutants compared with wild type, we isolated zebrafish Sizzled, a member of the secreted Frizzled-related protein family and putative Wnt inhibitor. The knockdown of sizzled using antisense morpholino phenocopied the ventralized mutant ogon (formerly also known as mercedes and short tail). By sequencing and rescue experiments, we demonstrate that ogon encodes sizzled. Overexpression of sizzled, resulting in strongly dorsalized phenotypes, and the expression domains of sizzled in wild type embryos, localized in the ventral side during gastrulation and restricted to the posterior end during segmentation stages, correlate with its role in dorsoventral patterning. The expanded expression domain of sizzled in ogon and chordino together with its downregulation in swirl suggests a BMP2b-dependent negative autoregulation of sizzled. Indicating a novel role for a secreted Frizzled-related protein, we show that, in addition to the BMP pathway, a component of the Wnt signaling pathway is required for dorsoventral pattern formation in zebrafish.     

Unlike mammalian GRIFIN, the zebrafish homologue (DrGRIFIN) represents a functional carbohydrate-binding galectin

Galectins, a family of β-galactoside-binding proteins, participate in a variety of biological processes, such as early development, tissue organization, immune regulation, and tumor evasion and metastasis. Although as many as fifteen bona fide galectins have been identified in mammals, but the detailed mechanisms of their biological roles still remain unclear for most. This fragmentary knowledge extends to galectin-like proteins such as the rat lens crystallin protein GRIFIN (Galectin-related inter fiber protein) and the galectin-related protein GRP (previously HSPC159; hematopoietic stem cell precursor) that lack carbohydrate-binding activity. Their inclusion in the galectin family has been debated, as they are considered products of evolutionary co-option. We have identified a homologue of the GRIFIN in zebrafish (Danio rerio) (designated DrGRIFIN), which like the mammalian equivalent is expressed in the lens, particularly in the fiber cells, as revealed by whole mount in situ hybridization and immunostaining of 2 dpf (days post fertilization) embryos. As evidenced by RT-PCR, it is weakly expressed in the embryos as early as 21 hpf (hour post fertilization) but strongly at all later stages tested (30 hpf and 3, 4, 6, and 7 dpf). In adult zebrafish tissues, however, DrGRIFIN is also expressed in oocytes, brain, and intestine. Unlike the mammalian homologue, DrGRIFIN contains all amino acids critical for binding to carbohydrate ligands and its activity was confirmed as the recombinant DrGRIFIN could be purified to homogeneity by affinity chromatography on a lactosyl-Sepharose column. Therefore, DrGRIFIN is a bona fide galectin family member that in addition to its carbohydrate-binding properties, may also function as a crystallin.     

Fin-mutant female zebrafish (Danio rerio) exhibit differences in association preferences for male fin length

Females often choose to associate with males that have exaggerated traits. In fishes, this may reflect an overall preference for larger size in a potential mate. Female zebrafish (Danio rerio) prefer males with larger bodies but not longer fins. The availability of mutant and transgenic strains of zebrafish make this a unique model system in which to study the role of phenotypic variation in social and sexual behavior. We used mutant strains of zebrafish with truncated (short fin) and exaggerated (long fin) fins to further examine female preferences for fin length in dichotomous association tests. Wild type females showed no preferences between wild type males and short fin mutant males or between wild type males and long fin mutant males. short fin females also showed no preference for short fin males or wild type males while long fin females preferred to associate with long fin males over wild type males. These results suggest that the single gene long fin mutation that results in altered fin morphological may also be involved in a related female association preference.     

Zebrafish chordin-like and chordin are functionally redundant in regulating patterning of the dorsoventral axis

Chordin is the prototype of a group of cysteine-rich domain-containing proteins that bind and modulate signaling of various TGFβ-like ligands. Chordin-like 1 and 2 (CHL1 and 2) are two members of this group that have been described in human, mouse, and chick. However, in vivo roles for CHL1 and 2 in early development are unknown due to lack of loss-of-function analysis. Here we identify and characterize zebrafish, Danio rerio, CHL (Chl). The chl gene is on a region of chromosome 21 syntenic with the area of murine chromosome 7 bearing the CHL2 gene. Inability to identify a separate zebrafish gene corresponding to the mammalian CHL1 gene suggests that Chl may serve roles in zebrafish distributed between CHL1 and CHL2 in other species. Chl is a maternal factor that is also zygotically expressed later in development and has spatiotemporal expression patterns that differ from but overlap those of zebrafish chordin (Chd), suggesting differences but also possible overlap in developmental roles of the two proteins. Chl, like Chd, dorsalizes embryos upon overexpression and is cleaved by BMP1, which antagonizes this activity. Loss-of-function experiments demonstrate that Chl serves as a BMP antagonist with functions that overlap and are redundant with those of Chd in forming the dorsoventral axis.     

Role of BMP signaling and the homeoprotein Iroquois in the specification of the cranial placodal field

Different types of placodes originate at the anterior border of the neural plate but it is still an unresolved question whether individual placodes arise as distinct ectodermal specializations in situ or whether all or a subset of the placodes originate from a common preplacodal field. We have analyzed the expression and function of the homeoprotein Iro1 in Xenopus and zebrafish embryos, and we have compared its expression with several preplacodal and placodal markers. Our results indicate that the iro1 genes are expressed in the preplacodal region, being one of the earliest markers for this area. We show that an interaction between the neural plate and the epidermis is able to induce the expression of several preplacodal markers, including Xiro1, by a similar mechanism to that previously shown for neural crest induction. In addition, we analyzed the role of BMP in the specification of the preplacodal field by studying the expression of the preplacodal markers Six1, Xiro1, and several specific placodal markers. We experimentally modified the level of BMP activity by three different methods. First, we implanted beads soaked with noggin in early neurula stage Xenopus embryos; second, we injected the mRNA that encodes a dominant negative of the BMP receptor into Xenopus and zebrafish embryos; and third, we grafted cells expressing chordin into zebrafish embryos. The results obtained using all three methods show that a reduction in the level of BMP activity leads to an expansion of the preplacodal and placodal region similar to what has been described for neural crest regions. By using conditional constructs of Xiro1, we performed gain and loss of function experiments. We show that Xiro1 play an important role in the specification of both the preplacodal field as well as individual placodes. We have also used inducible dominant negative and activator constructs of Notch signaling components to analyze the role of these factors on placodal development. Our results indicate that the a precise level of BMP activity is required to induce the neural plate border, including placodes and neural crest cells, that in this border the iro1 gene is activated, and that this activation is required for the specification of the placodes.     

Development of dorsal-ventral polarity in the optic vesicle and its presumptive role in eye morphogenesis as shown by embryonic transplantation and in ovo explant culturing

Dorsal and ventral specification in the early optic vesicle appears to play a crucial role in the proper development of the eye. In the present study, we performed embryonic transplantation and organ culturing of the chick optic vesicle in order to investigate how the dorsal-ventral (D-V) polarity is established in the optic vesicle and what role this polarity plays in proper eye development. The left optic vesicle was cut and transplanted inversely in the right eye cavity of host chick embryos. This method ensured that the D-V polarity was reversed while the anteroposterior axis remained normal. The results showed that the location of the choroid fissure was altered from the normal (ventral) to ectopic positions as the embryonic stage of transplantation progressed from 6 to 18 somites. At the same time, the shape of the optic vesicle and the expression patterns of Pax2 and Tbx5, marker genes for ventral and dorsal regions of the optic vesicle, respectively, changed concomitantly in a similar way. The crucial period was between the 8- and 14-somite stages, and during this period the polarity seemed to be gradually determined. In ovo explant culturing of the optic vesicle showed that the D-V polarity and choroid fissure formation were already specified by the 10-somite stage. These results indicate that the D-V polarity of the optic vesicle is established gradually between 8- and 14-somite stages under the influence of signals derived from the midline portion of the forebrain. The presumptive signal(s) appeared to be transmitted from proximal to distal regions within the optic vesicle. A severe anomaly was observed in the development of optic vesicles reversely transplanted around the 10-somite stage: the optic cup formation was disturbed and subsequently the neural retina and pigment epithelium did not develop normally. We concluded that establishment of the D-V polarity in the optic vesicle plays an essential role in the patterning and differentiation of the neural retina and pigment epithelium.     

Rhythmic patterns in phagocytosis and the production of reactive oxygen species by zebrafish leukocytes

Multiple components of vertebrate immune systems have been shown to exhibit circadian fluctuations. While the zebrafish is currently generating a wealth of information on the molecular pacemakers that may control circadian rhythms, there have been no reports of rhythmic activity in zebrafish leukocytes. In this study, we found that phagocytosis and the production of reactive oxygen species by zebrafish leukocytes varied significantly throughout twenty-four hour periods. A distinct peak in cellular ROS levels occurred before dawn, while the kinetics of respiratory burst responses were least rapid at this time of day. Phagocytosis of E. coli peaked late in the day, whereas there was no daily variation in phagocytosis of S. aureus. As seen in other species, the number of bacteria ingested per cell peaked during the night. These data provide direct evidence of rhythmic immune system activity, and demonstrate that zebrafish can be a valuable model in which to study the relationships between circadian gene expression, systemic pacemakers, and the activity of vertebrate immune system cells.     

zen and the art of phenotypic maintenance: Canalization of embryonic dorsal-ventral patterning in Drosophila

We recently uncovered a novel genetic mechanism that generates the phenotypic uniformity, or canalization, of BMP signaling and cell fate specification during patterning of the dorsal-ventral (D/V) axis in D. melanogaster embryos. We went on to show that other wild-type Drosophila species lack this canalizing genetic circuitry and, consequently, have non-robust D/V patterning. In this review, we propose molecular mechanisms that may give rise to stereotyped BMP signaling, and we identify an additional species that could have decanalized D/V patterning. Extension of these analyses could in turn help explain why canalization is not a universal necessity for species survival.     

Saltatory control of isometric growth in the zebrafish caudal fin is disrupted in long fin and rapunzel mutants

Zebrafish fins grow by sequentially adding new segments of bone to the distal end of each fin ray. In wild type zebrafish, segment addition is regulated such that an isometric relationship is maintained between fin length and body length over the lifespan of the growing fish. Using a novel, surrogate marker for fin growth in conjunction with cell proliferation assays, we demonstrate here that segment addition is not continuous, but rather proceeds by saltation. Saltation is a fundamental growth mechanism shared by disparate vertebrates, including humans. We further demonstrate that segment addition proceeds in conjunction with cyclic bursts of cell proliferation in the distal fin ray mesenchyme. In contrast, cells in the distal fin epidermis proliferate at a constant rate throughout the fin ray growth cycle. Finally, we show that two separate fin overgrowth mutants, long fin and rapunzel, bypass the stasis phase of the fin ray growth cycle to develop asymmetrical and symmetrical fin overgrowth, respectively.     

crm-1 facilitates BMP signaling to control body size in Caenorhabditis elegans

We have identified in Caenorhabditis elegans a homologue of the vertebrate Crim1, crm-1, which encodes a putative transmembrane protein with multiple cysteine-rich (CR) domains known to have bone morphogenetic proteins (BMPs) binding activity. Using the body morphology of C. elegans as an indicator, we showed that attenuation of crm-1 activity leads to a small body phenotype reminiscent of that of BMP pathway mutants. We showed that the crm-1 loss-of-function phenotype can be rescued by constitutive supply of sma-4 activity. crm-1 can enhance BMP signaling and this activity is dependent on the presence of the DBL-1 ligand and its receptors. crm-1 is expressed in neurons at the ventral nerve cord, where the DBL-1 ligand is produced. However, ectopic expression experiments reveal that crm-1 gene products act outside the DBL-1 producing cells and function non-autonomously to facilitate dbl/sma pathway signaling to control body size.     

Characterisation of Fmrp in zebrafish: evolutionary dynamics of the <Emphasis Type="Italic">fmr1</Emphasis> gene

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by the lack of the Fragile X Mental Retardation Protein (FMRP), which is encoded by the FMR1 gene. Although Fmr1 knockout mice display some characteristics also found in fragile X patients, it is a complex animal model to study brain abnormalities, especially during early embryonic development. Interestingly, the ortholog of the FMR1 gene has been identified not only in mouse, but also in zebrafish (Danio rerio). In this study, an amino acid sequence comparison of FMRP orthologs was performed to determine the similar regions of FMRP between several species, including human, mouse, frog, fruitfly and zebrafish. Further characterisation of Fmrp has been performed in both adults and embryos of zebrafish using immunohistochemistry and western blotting with specific antibodies raised against zebrafish Fmrp. We have demonstrated a strong Fmrp expression in neurons of the brain and only a very weak expression in the testis. In brain tissue, a different distribution of the isoforms of Fmrp, compared to human and mouse brain tissue, was shown using western blot analysis. Due to the high similarity between zebrafish Fmrp and human FMRP and their similar expression pattern, the zebrafish has great potential as a complementary animal model to study the pathogenesis of the fragile X syndrome, especially during embryonic development.Edited by D. Tautz