Pectoral Fin Buds Research Articles

Knockdown of vitamin D receptor affects early stages of pectoral fin development in zebrafish.

The vitamin D receptor (VDR) signalling has been implicated in vertebrate limb or fin formation. However, the involvement of VDR signalling in the early stages of limb/fin development remains to be elucidated. In this study, the role of VDR signalling in pectoral fin development was investigated in zebrafish embryos. Knockdown of vdr induced the severe impairment of pectoral fin development. The zebrafish larvae lacking vdr exhibited reduced pectoral fins with no skeletal elements. Insitu hybridization revealed depletion of vdr downregulated fibroblast growth factor 24 (fgf24), a marker of early pectoral fin bud mesenchyme, in the presumptive fin field even before fin buds were visible. Moreover, a perturbed expression pattern of bone morphogenetic protein 4 (bmp4), a marker of the pectoral fin fold, was observed in the developing fin buds of zebrafish embryos that lost the vdr function. These findings suggest that VDR signalling is crucial in the early stages of fin development, potentially influencing the process by regulating other signalling molecules such as Fgf24 and Bmp4.

The scale of zebrafish pectoral fin buds is determined by intercellular K+ levels and consequent Ca2+-mediated signaling via retinoic acid regulation of Rcan2 and Kcnk5b.

K+ channels regulate morphogens to scale adult fins, but little is known about what regulates the channels and how they control morphogen expression. Using the zebrafish pectoral fin bud as a model for early vertebrate fin/limb development, we found that K+ channels also scale this anatomical structure, and we determined how one K+-leak channel, Kcnk5b, integrates into its developmental program. From FLIM measurements of a Förster Resonance Energy Transfer (FRET)-based K+ sensor, we observed coordinated decreases in intracellular K+ levels during bud growth, and overexpression of K+-leak channels in vivo coordinately increased bud proportions. Retinoic acid, which can enhance fin/limb bud growth, decreased K+ in bud tissues and up-regulated regulator of calcineurin (rcan2). rcan2 overexpression increased bud growth and decreased K+, while CRISPR-Cas9 targeting of rcan2 decreased growth and increased K+. We observed similar results in the adult caudal fins. Moreover, CRISPR targeting of Kcnk5b revealed that Rcan2-mediated growth was dependent on the Kcnk5b. We also found that Kcnk5b enhanced depolarization in fin bud cells via Na+ channels and that this enhanced depolarization was required for Kcnk5b-enhanced growth. Lastly, Kcnk5b-induced shha transcription and bud growth required IP3R-mediated Ca2+ release and CaMKK activity. Thus, we provide a mechanism for how retinoic acid via rcan2 can regulate K+-channel activity to scale a vertebrate appendage via intercellular Ca2+ signaling.

Zebrafish as a model to investigate a biallelic gain-of-function variant in MSGN1, associated with a novel skeletal dysplasia syndrome

Background/ObjectivesRare genetic disorders causing specific congenital developmental abnormalities often manifest in single families. Investigation of disease-causing molecular features are most times lacking, although these investigations may open novel therapeutic options for patients. In this study, we aimed to identify the genetic cause in an Iranian patient with severe skeletal dysplasia and to model its molecular function in zebrafish embryos.ResultsThe proband displays short stature and multiple skeletal abnormalities, including mesomelic dysplasia of the arms with complete humero-radio-ulna synostosis, arched clavicles, pelvic dysplasia, short and thin fibulae, proportionally short vertebrae, hyperlordosis and mild kyphosis. Exome sequencing of the patient revealed a novel homozygous c.374G > T, p.(Arg125Leu) missense variant in MSGN1 (NM_001105569). MSGN1, a basic-Helix–Loop–Helix transcription factor, plays a crucial role in formation of presomitic mesoderm progenitor cells/mesodermal stem cells during early developmental processes in vertebrates. Initial in vitro experiments show protein stability and correct intracellular localization of the novel variant in the nucleus and imply retained transcription factor function. To test the pathogenicity of the detected variant, we overexpressed wild-type and mutant msgn1 mRNA in zebrafish embryos and analyzed tbxta (T/brachyury/ntl). Overexpression of wild-type or mutant msgn1 mRNA significantly reduces tbxta expression in the tailbud compared to control embryos. Mutant msgn1 mRNA injected embryos depict a more severe effect, implying a gain-of-function mechanism. In vivo analysis on embryonic development was performed by clonal msgn1 overexpression in zebrafish embryos further demonstrated altered cell compartments in the presomitic mesoderm, notochord and pectoral fin buds. Detection of ectopic tbx6 and bmp2 expression in these embryos hint to affected downstream signals due to Msgn1 gain-of-function.ConclusionIn contrast to loss-of-function effects described in animal knockdown models, gain-of-function of MSGN1 explains the only mildly affected axial skeleton of the proband and rather normal vertebrae. In this context we observed notochord bending and potentially disruption of pectoral fin buds/upper extremity after overexpression of msgn1 in zebrafish embryos. The latter might result from Msgn1 function on mesenchymal stem cells or on chondrogenesis in these regions. In addition, we detected ectopic tbx6 and bmp2a expression after gain of Msgn1 function in zebrafish, which are interconnected to short stature, congenital scoliosis, limb shortening and prominent skeletal malformations in patients. Our findings highlight a rare, so far undescribed skeletal dysplasia syndrome associated with a gain-of-function mutation in MSGN1 and hint to its molecular downstream effectors.

Heterochronic development of pelvic fins in zebrafish: possible involvement of temporal regulation of pitx1 expression.

Anterior and posterior paired appendages of vertebrates are notable examples of heterochrony in the relative timing of their development. In teleosts, posterior paired appendages (pelvic fin buds) emerge much later than their anterior paired appendages (pectoral fin buds). Pelvic fin buds of zebrafish (Danio rerio) appear at 3weeks post-fertilization (wpf) during the larva-to-juvenile transition (metamorphosis), whereas pectoral fin buds arise from the lateral plate mesoderm on the yolk surface at the embryonic stage. Here we explored the mechanism by which presumptive pelvic fin cells maintain their fate, which is determined at the embryonic stage, until the onset of metamorphosis. Expression analysis revealed that transcripts of pitx1, one of the key factors for the development of posterior paired appendages, became briefly detectable in the posterior lateral plate mesoderm at early embryonic stages. Further analysis indicated that the pelvic fin-specific pitx1 enhancer was in the poised state at the larval stage and is activated at the juvenile stage. We discuss the implications of these findings for the heterochronic development of pelvic fin buds.

Augmentation of Pectoral Fin Teratogenicity by Thalidomide in Human Cytochrome P450 3A-Expressing Zebrafish.

The pharmacological and toxicological effects of active metabolites of enzymes including cytochrome P450 (CYP) are important. While it has been believed for a long time that thalidomide causes characteristic limb malformation only in rabbits and primates including humans, the involvement of their CYP3A subtypes (CYP3As) has been suggested. Recently, however, it was reported that zebrafish were sensitive to thalidomide, showing defects of pectoral fins, homologous organs of forelimbs in mammals, as well as other deformities. In this study, we prepared human CYP3A7 (hCYP3A7)-expressing zebrafish (F0) using a transposon system. Thalidomide caused pectoral fin defects and other malformations including pericardial edema in hCYP3A7-expressing embryos/larvae but not in wild-type and hCYP1A1-expressing embryos/larvae. Thalidomide also reduced the expression of fibroblast growth factor 8 in pectoral fin buds in only hCYP3A7-expressing embryos/larvae. The results suggest the involvement of human-type CYP3A in thalidomide teratogenicity.

Early ontogeny of tetra Markiana nigripinnis (Characiformes: Characidae)

Abstract The early development of Markiana nigripinnis is described by morphological characters, pigmentation, and morphometry. Larvae were obtained through semi-natural breeding, collected, fixed, and identified according to their development. Eighty individuals with standard lengths ranging from 3.1 to 24.3 mm were analyzed. Larvae are poorly developed at hatching, with a relatively large yolk sac and finfold. At the preflexion stage, the eyes are pigmented, the mouth and anus are functional, the yolk is completely absorbed, and the pectoral fin bud emerges. At flexion, the first rays of the caudal, anal, and dorsal fins become evident. The pelvic fin bud emerges only at the postflexion stage, in addition to the complete absorption of the finfold. Pigmentation is distributed throughout the body, with a greater concentration on the top of the head, around the mouth, and at the base of the caudal fin. The myomere total number ranged from 34 to 49 (16–23 preanal, and 18–27 postanal). Juveniles show morphological characteristics like adults. The fins ray number are pectoral: 11–13, pelvic: 5–7, dorsal: 8–11, caudal: 16–27, and anal 30–47. The morphometric relationships reveal variations in growth along the early ontogeny of the species.

Tbx5a and Tbx5b paralogues act in combination to control separate vectors of migration in the fin field of zebrafish

The T-box containing family member, TBX5, has been shown to play important functional roles in the pectoral appendages of a variety of vertebrate species. While a single TBX5 gene exists in all tetrapods studied to date, the zebrafish genome retains two paralogues, designated as tbx5a and tbx5b, resulting from a whole genome duplication in the teleost lineage. Zebrafish deficient in tbx5a lack pectoral fin buds, whereas zebrafish deficient in tbx5b exhibit misshapen pectoral fins, showing that both paralogues function in fin development. The mesenchymal cells of the limb/fin bud are derived from the Lateral Plate Mesoderm (LPM). Previous fate mapping work in zebrafish has shown that wildtype (wt) fin field cells are initially located adjacent to somites (s)1–4. The wt fin field cells migrate in opposing diagonal directions placing the limb bud between s2-3 and lateral to the main body. To better characterize tbx5 paralogue functions in zebrafish, time-lapse analyses of the migrations of fin bud precursors under conditions of tbx5a knock-down, tbx5b knock-down and double-knock-down were performed. Our data suggest that zebrafish tbx5a and tbx5b have functionally separated migration direction vectors, that when combined recapitulate the migration of the wt fin field. We and others have shown that loss of Tbx5a function abolishes an fgf24 signaling cue resulting in fin field cells failing to converge in an Antero-Posterior (AP) direction and migrating only in a mediolateral (ML) direction. We show here that loss of Tbx5b function affects initial ML directed movements so that fin field cells fail to migrate laterally but continue to converge along the AP axis. Furthermore, fin field cells in the double Tbx5a/Tbx5b knock-down zebrafish do not engage in directed migrations along either the ML or AP axis. Therefore, these two paralogues may be acting to instruct separate vectors of fin field migration in order to direct proper fin bud formation.

Developmental hourglass and heterochronic shifts in fin and limb development.

How genetic changes are linked to morphological novelties and developmental constraints remains elusive. Here, we investigate genetic apparatuses that distinguish fish fins from tetrapod limbs by analyzing transcriptomes and open-chromatin regions (OCRs). Specifically, we compared mouse forelimb buds with the pectoral fin buds of an elasmobranch, the brown-banded bamboo shark (Chiloscyllium punctatum). A transcriptomic comparison with an accurate orthology map revealed both a mass heterochrony and hourglass-shaped conservation of gene expression between fins and limbs. Furthermore, open-chromatin analysis suggested that access to conserved regulatory sequences is transiently increased during mid-stage limb development. During this stage, stage-specific and tissue-specific OCRs were also enriched. Together, early and late stages of fin/limb development are more permissive to mutations than middle stages, which may have contributed to major morphological changes during the fin-to-limb evolution. We hypothesize that the middle stages are constrained by regulatory complexity that results from dynamic and tissue-specific transcriptional controls.

Development of fin‐innervating motor neurons after peripheral target removal in medaka fish

Peripheral targets regulate the development and survival of the nerve centers that serve them, because the elimination of the target normally results in massive death of the developing neurons that innervate it. This widely accepted theory appears to be well supported by developing limbs and their innervation in tetrapods, but it is unclear whether this concept applies to primitive vertebrates that have paired appendages. In this study, we examined the development of spinal motor neurons following pectoral fin bud removal (FBR) in medaka fish. After FBR, motor axons initially extended to the plexus region in a morphologically normal pattern. During the period of fin innervation, motor axons in the FBR-medaka failed to form the normal brachial plexus and elongated ventrally toward the abdominal region. In the ventral horn that would normally innervate the pectoral fin, however, neurons did not undergo cell death following FBR. There were no differences in the numbers of axons in the ventral roots between the FBR and control sides. Motor neuron markers, RALDH2 and FOXP1, that are expressed in limb-innervating motor neurons in the lateral motor column in tetrapods, were also expressed in the ventral horns of both the control and FBR sides in medaka fish. These results suggest that, although both tetrapod and medaka motor neurons share the same molecular characteristics for innervating paired appendages, the fates of neurons differ following the removal of their peripheral target. Therefore, the relationship between the peripheral target and its nerve center may be altered among vertebrates.

The Cdx transcription factors and retinoic acid play parallel roles in antero-posterior position of the pectoral fin field during gastrulation

The molecular regulators that determine the precise position of the vertebrate limb along the anterio-posterior axis have not been identified. One model suggests that a combination of hox genes in the lateral plate mesoderm (LPM) promotes formation of the limb field, however redundancy among duplicated paralogs has made this model difficult to confirm. In this study, we identify an optimal window during mid-gastrulation stages when transient mis-regulation of retinoic acid signaling or the caudal related transcription factor, Cdx4, both known regulators of hox genes, can alter the position of the pectoral fin field. We show that increased levels of either RA or Cdx4 during mid-gastrulation are sufficient to rostrally shift the position of the pectoral fin field at the expense of surrounding gene expression in the anterior lateral plate mesoderm (aLPM). Alternatively, embryos deficient for both Cdx4 and Cdx1a (Cdx-deficient) form pectoral fins that are shifted towards the posterior and reveal an additional effect on size of the pectoral fin buds. Prior to formation of the pectoral fin buds, the fin field in Cdx-deficient embryos is visibly expanded into the posterior LPM (pLPM) region at the expense of surrounding gene expression. The effects on gene expression immediately post-gastrulation and during somitogenesis support a model where RA and Cdx4 act in parallel to regulate the position of the pectoral fin. Our transient method is a potentially useful model for studying the mechanisms of limb positioning along the AP axis.

Spatio-temporal expression and distribution of collagen VI during zebrafish development

Collagen VI (ColVI) is an extracellular matrix (ECM) protein involved in a range of physiological and pathological conditions. Zebrafish (Danio rerio) is a powerful model organism for studying vertebrate development and for in vivo analysis of tissue patterning. Here, we performed a thorough characterization of ColVI gene and protein expression in zebrafish during development and adult life. Bioinformatics analyses confirmed that zebrafish genome contains single genes encoding for α1(VI), α2(VI) and α3(VI) ColVI chains and duplicated genes encoding for α4(VI) chains. At 1 day post-fertilization (dpf) ColVI transcripts are expressed in myotomes, pectoral fin buds and developing epidermis, while from 2 dpf abundant transcript levels are present in myosepta, pectoral fins, axial vasculature, gut and craniofacial cartilage elements. Using newly generated polyclonal antibodies against zebrafish α1(VI) protein, we found that ColVI deposition in adult fish delineates distinct domains in the ECM of several organs, including cartilage, eye, skin, spleen and skeletal muscle. Altogether, these data provide the first detailed characterization of ColVI expression and ECM deposition in zebrafish, thus paving the way for further functional studies in this species.

Involvement of HGF/MET signaling in appendicular muscle development in cartilaginous fish.

In amniotes, limb muscle precursors de-epithelialize from the ventral dermomyotome and individually migrate into limb buds. In catsharks, Scyliorhinus, fin muscle precursors are also derived from the ventral dermomyotome, but shortly after de-epithelialization, they reaggregate within the pectoral fin bud and differentiate into fin muscles. Delamination of muscle precursors has been suggested to be controlled by hepatocyte growth factor (HGF) and its tyrosine kinase receptor (MET) in amniotes. Here, we explore the possibility that HGF/MET signaling regulates the delamination of appendicular muscle precursors in embryos of the catshark Scyliorhinus canicula. Our analysis reveals that Hgf is expressed in pectoral fin buds, whereas c-Met expression in fin muscle precursors is rapidly downregulated. We propose that alteration of the duration of c-Met expression in appendicular muscle precursors might underlie the evolution of individually migrating muscle precursors, which leads to the emergence of complex appendicular muscular systems in amniotes.

Generation of a Tg(cyp1a-12DRE:EGFP) transgenic zebrafish line as a rapid in vivo model for detecting dioxin-like compounds

Dioxin-like compounds (DLCs) are extremely stable toxic organic compounds and can cause serious health risks. To develop a convenient biomonitoring tool for the detection of DLCs in the environment, we generated a transgenic line—Tg(cyp1a-12DRE:EGFP)—with a zebrafish cyp1a promoter recombined with multiple dioxin-responsive elements (DREs) that drive EGFP expression. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced EGFP expression was observed in the head cartilage (most sensitive), gut, otic vesicle, pectoral fin bud and eye of larvae. The lowest observed effect concentration of TCDD was estimated to be approximately 1 ng/L. Compared with existing zebrafish lines, our transgenic fish displayed comparable or even higher detection sensitivity to DLCs and could serve as an improved and rapid assay in an in vivo context. The Tg(cyp1a-12DRE:EGFP) transgenic zebrafish line also had higher stability for inducing EGFP expression (nearly 100% of our zebrafish induced EGFP at approximately 1 ng/L TCDD) than other lines. In addition, Tg(cyp1a-12DRE:EGFP) zebrafish could serve as a convenient and straightforward tool to assess potential cranial malformations and related health effects.

Prolongation of somitogenesis in two anguilliform species, the Japanese eel Anguilla japonica and pike eel Muraenesox cinereus, with refined descriptions of their early development.

The embryonic development of the Japanese eel Anguilla japonica and pike eel Muraenesox cinereus was morphologically investigated with laboratory-reared specimens to clarify the characteristics of somitogenesis. In A. japonica, somites were first observed at 18 h post fertilization (hpf) when epiboly reached 90%. Somitogenesis progressed at a rate of 1·6 h-1 at mean ± s.d. 22·6 ± 0·7° C and completed at 107 hpf (3 days post hatching; dph) when total number of somites (ST) reached 114, which corresponds to the species' number of vertebrae (112-119). In M. cinereus, somites were first observed at 14 hpf when epiboly completed. Somitogenesis progressed at a rate of 1·9 h-1 at mean ± s.d. 24·4 ± 0·2° C and completed at 90 hpf (2 dph) with 149 ± 4 ST, which corresponds to the species' number of vertebrae (142-158). Both species hatched before somitogenesis was completed, at 37 hpf with 47 ST and 42 hpf with 82 ± 4 ST, respectively. The formation of other organs such as the heart, mouth and pectoral fin bud occurred during somitogenesis. Comparison with the development of zebrafish Danio rerio indicates a prolongation of somitogenesis in A. japonica and M. cinereus. Their somitogenesis rates, however, correspond well with that of D. rerio estimated at the same temperature and their developmental stages at hatching are almost equivalent to other fishes having similar yolk sizes. Therefore, the prolongation of somitogenesis in A. japonica and M. cinereus may be accounted for solely by the increased numbers of somites to be formed, not by a slow somitogenesis rate or an acceleration in organogenesis.

Functional and Comparative Genomics of Hoxa2 Gene cis-Regulatory Elements: Evidence for Evolutionary Modification of Ancestral Core Element Activity.

Hoxa2 is an evolutionarily conserved developmental regulatory gene that functions to specify rhombomere (r) and pharyngeal arch (PA) identities throughout the Osteichthyes. Japanese medaka (Oryzias latipes) hoxa2a, like orthologous Hoxa2 genes from other osteichthyans, is expressed during embryogenesis in r2–7 and PA2-7, whereas the paralogous medaka pseudogene, ψhoxa2b, is expressed in noncanonical Hoxa2 domains, including the pectoral fin buds. To understand the evolution of cis-regulatory element (CRE) control of gene expression, we conducted eGFP reporter gene expression studies with extensive functional mapping of several conserved CREs upstream of medaka hoxa2a and ψhoxa2b in transient and stable-line transgenic medaka embryos. The CREs tested were previously shown to contribute to directing mouse Hoxa2 gene expression in r3, r5, and PA2-4. Our results reveal the presence of sequence elements embedded in the medaka hoxa2a and ψhoxa2b upstream enhancer regions (UERs) that mediate expression in r4 and the PAs (hoxa2a r4/CNCC element) or in r3–7 and the PAs ψhoxa2b r3–7/CNCC element), respectively. Further, these elements were shown to be highly conserved among osteichthyans, which suggests that the r4 specifying element embedded in the UER of Hoxa2 is a deeply rooted rhombomere specifying element and the activity of this element has been modified by the evolution of flanking sequences that redirect its activity to alternative developmental compartments.

The dlx5a/dlx6a genes play essential roles in the early development of zebrafish median fin and pectoral structures.

The Dlx5 and Dlx6 genes encode homeodomain transcription factors essential for the proper development of limbs in mammalian species. However, the role of their teleost counterparts in fin development has received little attention. Here, we show that dlx5a is an early marker of apical ectodermal cells of the pectoral fin buds and of the median fin fold, but also of cleithrum precursor cells during pectoral girdle development. We propose that early median fin fold establishment results from the medial convergence of dlx5a-expressing cells at the lateral edges of the neural keel. Expression analysis also shows involvement of dlx5a during appendage skeletogenesis. Using morpholino-mediated knock down, we demonstrate that disrupted dlx5a/6a function results in pectoral fin agenesis associated with misexpression of bmp4, fgf8a, and1 and msx genes. In contrast, the median fin fold presents defects in mesenchymal cell migration and actinotrichia formation, whereas the initial specification seems to occur normally. Our results demonstrate that the dlx5a/6a genes are essential for the induction of pectoral fin outgrowth, but are not required during median fin fold specification. The dlx5a/6a knock down also causes a failure of cleithrum formation associated with a drastic loss of runx2b and col10a1 expression. The data indicate distinct requirements for dlx5a/6a during median and pectoral fin development suggesting that initiation of unpaired and paired fin formation are not directed through the same molecular mechanisms. Our results refocus arguments on the mechanistic basis of paired appendage genesis during vertebrate evolution.

Abstract 531: Mutagenic Gene Trapping to Study Novel Genes in Zebrafish Cardiovascular Development

The zebrafish has emerged as an excellent model for cardiovascular research thanks to its ex-utero and rapid embryonic development, its embryonic transparent nature, and its capacity to survive in the absence of a functional cardiovascular system during the first week of development, which enables functional characterization of mutations that would otherwise induce lethality in traditional murine models. The aim of this project is to identify and characterize novel genes involved in zebrafish cardiovascular development using mutagenic gene trapping, a technique that generates random insertional mutations across the genome. We use the well-defined RP2 gene-breaking transposon system, which not only mutates, but also fluorescently tags the trapped gene product(s) (Clark, Nat Methods, 2011). RP2 also introduces loxP sites into the mutant locus, which can be used for Cre-mediated phenotype rescue by microinjection of Cre recombinase, or by crossing to tissue-specific Cre lines. From over 3000 RP2-injected embryos, 141 fish showed germline transmission. Among them, 51 expressed strong fluorescence in different tissues, including the heart, vessels, notochord, central nervous system (CNS), and eyes. Three cardiovascular lines were selected for phenotypic characterization and functional studies. RP2#C2 strain expressed fluorescence in the heart valves, CNS, eyes and pectoral fin buds. Inverse PCR in RP2#C2 demonstrated a trapped gene at meis4.1a, which encodes a homeobox transcription factor that has not been previously studied in zebrafish. RP2#121 strain expressed strong fluorescence in cardiac and skeletal muscles. RP2#91 strain showed expression in the vasculature and demonstrated a trapped gene at pdgfra. Homozygous RP2#91 mutants showed severe defects in the heart, blood flow, and other body parts including the head and musculature. We are currently creating a panel of tissue-specific Cre lines targeting tissues such as cardiomyocytes, endothelial cells and smooth muscle cells for spatiotemporal rescuing of the mutant phenotype. The generated zebrafish protein-trap lines are invaluable tools to annotate gene function, dissect the molecular mechanisms of cardiovascular development, and potentially serve as disease models.

Expression patterns of dnmt3aa, dnmt3ab, and dnmt4 during development and fin regeneration in zebrafish

Epigenetic modifications such as DNA methylation and chromatin modifications are critical for regulation of spatiotemporal gene expression during development. In mammals, the de novo-type DNA methyltransferases (Dnmts), Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns during development. In addition to developmental processes, we recently showed that DNA methylation levels are dynamically changed during zebrafish fin regeneration, suggesting that the de novo-type Dnmts might play roles in the regulation of gene expression during regeneration processes. Here, we showed the detailed expression profiles of three zebrafish dnmt genes (dnmt3aa, dnmt3ab, and dnmt4), which were identified as the orthologues of mammalian dnmt3a and dnmt3b, during embryonic and larval development, as well as fin regeneration processes. dnmt3aa and dnmt3ab are expressed in the brain, pharyngeal arches, pectoral fin buds, intestine, and swim bladder; the specific expression of dnmt3aa is observed in the pronephric duct during larval development. dnmt4 expression is observed in the zona limitans intrathalamica, midbrain–hindbrain boundary, ciliary marginal zone, pharyngeal arches, auditory capsule, pectoral fin buds, intestine, pancreas, liver, and hematopoietic cells in the aorta–gonad–mesonephros and caudal hematopoietic tissue from 48 to 72h post-fertilization. Furthermore, during fin regeneration, strong dnmt3aa expression, and faint dnmt3ab and dnmt4 expression are detected in blastema cells at 72h post-amputation. Taken together, our results suggest that zebrafish Dnmt3aa, Dnmt3ab, and Dnmt4 may play roles in the formation of various organs, such as the brain, kidney, digestive organs, and/or hematopoietic cells, as well as in the differentiation of blastema cells.

Characterization of zebrafish Pax1b and Pax9 in fin bud development.

Both Pax1 and Pax9 belong to the important paired box gene family (PAX), which mainly participates in animal development and sclerotome differentiation. To date, the precise molecular mechanism and related signaling pathway of Pax1 remain unclear. In our study, microinjection of morpholino- (MO-) modified antisense oligonucleotides against pax1b induced pectoral fin bud defects. Furthermore, we demonstrate that the phenotypes caused by the knockdown of Pax1b in zebrafish could not be phenocopied by pax9 MO and could not be rescued by either Pax1a or Pax9 overexpression. We further find that Pax1b affects the expression of col2a1, Uncx4.1, Noggin3, and aggrecan, confirming the role of Pax1b in chondrocyte differentiation and bone maturation. Moreover, we identify an interaction between PAX1 and FOXO1 and find that the interaction was enhanced under hypoxia stress. Together, this evidence for cell death caused by pax1b knockdown provides new insight into the role of the Pax protein family in cell fate determination and tissue specification.

Odd‐skipped related 2 is required for fin chondrogenesis in zebrafish

odd-skipped related 2 (osr2) encodes a vertebrate ortholog of the Drosophila odd-skipped zinc-finger transcription factor. Osr2 in mouse is required for proper palate, eyelid, and bone development. Zebrafish knock-down experiments have also suggested a role for osr2, along with its paralog osr1, in early pectoral fin specification and pronephric development. We show here that osr2 has a specific function later in development, independent of osr1, in the regulation of sox9a expression and promoting fin chondrogenesis. mRNA in situ hybridization demonstrated osr2 expression in the developing floorplate and later during organogenesis in the pronephros and gut epithelium. In the pectoral fin buds, osr2 was specifically expressed in fin mesenchyme. osr2 knock down in zebrafish embryos disrupted both three and five zinc finger alternatively spliced osr2 isoforms and eliminated wild-type osr2 mRNA. osr2 morphants exhibited normal pectoral fin bud specification but exhibited defective fin chondrogenesis, with loss of differentiated chondrocytes. Defects in chondrogenesis were paralleled by loss of sox9a as well as subsequent col2a1 expression, linking osr2 function to essential regulators of chondrogenesis. The zebrafish odd-skipped related 2 gene regulates sox9a and col2a1 expression in chondrocyte development and is specifically required for zebrafish fin morphogenesis.