Bone morphogenetic proteins (BMPs) are critical players in development and disease, regulating such diverse processes as dorsoventral patterning, palate formation, and ossification. These ligands are classically considered to signal via BMP receptor-specific Smad proteins 1, 5, and 8. To determine the spatiotemporal pattern of Smad1/5/8 activity and thus canonical BMP signaling in the developing zebrafish embryo, we generated a transgenic line expressing EGFP under the control of a BMP-responsive element. EGFP is expressed in many established BMP signaling domains and is responsive to alterations in BMP type I receptor activity and smad1 and smad5 expression. This transgenic Smad1/5/8 reporter line will be useful for determining ligand and receptor requirements for specific domains of BMP activity, as well as for genetic and pharmacological screens aimed at identifying enhancers or suppressors of canonical BMP signaling.

Smad-interacting protein-1 (SIP1) has been implicated in the development of Mowat-Wilson syndrome whose patients exhibit Hirschsprung disease, an aganglionosis of the large intestine, as well as other phenotypes. We have identified and cloned two sip1 orthologues in zebrafish. Both sip1 orthologues are expressed maternally and have dynamic zygotic expression patterns that are temporally and spatially distinct. We have investigated the function of both orthologues using translation and splice-blocking morpholino antisense oligonucleotides. Knockdown of the orthologues causes axial and neural patterning defects consistent with the previously described function of SIP1 as an inhibitor of BMP signaling. In addition, knockdown of both genes leads to a significant reduction/loss of the post-otic cranial neural crest. This results in a subsequent absence of neural crest precursors in the posterior pharyngeal arches and a loss of enteric precursors in the intestine.

Neuropilin-1, a receptor for axon-repellent semaphorins and vascular endothelial growth factor (VEGF), functions both in angiogenesis and axon growth. Here, we show strong expression of neuropilin-1a in primary motor neurons in the trunk of embryonic zebrafish. Reducing the expression of neuropilin-1a using antisense morpholino oligonucleotides induced aberrant branching of motor nerves, additional exit points of motor nerves from the spinal cord, and migration of neurons out of the spinal cord along the motor axon pathway in a dose-dependent manner. These phenotypes could be partially rescued by co-injecting neuropilin-1a mRNA. Other axons in the spinal cord and head appeared unaffected by the morpholino treatment. In addition, neuropilin-1a morpholino treatment disturbed normal formation of blood vessels in the trunk of 24 hours postfertilization embryos, as shown by microangiography. Morpholinos to VEGF also disturbed formation of blood vessels but did not affect motor axons, indicating that correct formation of blood vessels is not needed for the growth of primary motor axons. Morpholinos to the semaphorin 3A homologs semaphorin 3A1 and semaphorin 3A2 also had no effect on motor axon growth. However, combined injections of neuropilin-1a morpholino, at a concentration that did not elicit axonal aberrations when injected alone, with VEGF, semaphorin 3A1, or semaphorin 3A2 morpholinos synergistically increased the proportion of embryos showing aberrant motor axon growth. Thus, neuropilin-1a in primary motor neurons may integrate signals from several ligands and is needed for proper segmental growth of primary motor nerves in zebrafish.

Vertebrate regeneration is a fascinating but poorly understood biological phenomena. Urodele amphibians such as Ambystoma mexicanum (the axolotl) can functionally regenerate complex body structures such as the limb and tail, including the spinal cord, throughout life. So far, molecular studies on regeneration have been limited due to the paucity of tools for knocking-down gene and protein function. In this article, we quantitatively assessed the ability of morpholinos to specifically down-regulate protein expression in both cultured urodele cells and in vivo. We focused on the down-regulation of green fluorescent protein and two axolotl proteins, MSX1 and PAX7. Our data show that the expression of these proteins can be efficiently reduced by morpholinos. MSX1 has been hypothesized to be involved in muscle dedifferentiation based on experiments using cultured myotubes. Our studies in in vivo muscle fibers so far have shown no influence of overexpressing or down-regulating MSX1 on the dedifferentiation process.

The Tbx2 transcription factor is implicated in growth control based on its association with human cancers. In the heart, Tbx2 represses cardiac differentiation to mediate development of the atrioventricular canal (AVC). The zebrafish genome retains two tbx2 genes, and both are required for formation of the AVC. Here, we show that both genes are also expressed earlier in the primitive heart tube, and we describe a previously unrecognized role for Tbx2 in promoting proliferation of presumptive myocardium at the heart tube stage. In contrast to single knockdowns, depletion of both gene products causes chamber defects, resulting in an expanded atrium and a smaller ventricle, associated with decreased proliferation of ventricular cardiomyocytes. The phenotype correlates with changes in the expression for known cardiac growth factors. Therefore, in zebrafish, two tbx2 genes are functionally redundant for regulating chamber development, while each gene is required independently for development of the AVC.

The separation and specification of mesoderm into the notochord and somites involves members of the non-clustered δ-protocadherins. Axial (AXPC) and paraxial (PAPC) protocadherins are expressed in the early dorsal mesoderm and later become refined to the developing notochordal and somitic mesoderm, respectively. The role of PAPC in this process has been studied extensively, but the role of AXPC is poorly understood. Partial knockdown of AXPC causes a specific bent-axis phenotype, while more severe knockdown results in the loss of notochord formation. The inability of these embryos to develop a notochord is not due to a cell-sorting event via changes in cell adhesion during gastrulation, but rather this defect is manifested through the loss of axial mesoderm specification, but not general mesoderm induction. The results presented here show that AXPC functions in notochord morphogenesis by directing cell-fate decisions rather than cell-cell adhesion.

Neural crest cells emerge by delamination from the dorsal neural tube and give rise to various components of the peripheral nervous system in vertebrate embryos. These cells change from non-motile into highly motile cells migrating to distant areas before further differentiation. Mechanisms controlling delamination and subsequent migration of neural crest cells are not fully understood. Slit2, a chemorepellant for axonal guidance that repels and stimulates motility of trunk neural crest cells away from the gut has recently been suggested to be a tumor suppressor molecule. The goal of this study was to further investigate the role of Slit2 in trunk neural crest cell migration by constitutive expression in neural crest cells.

The inner ear is a complex organ containing sensory tissue, including hair cells, the development of which is not well understood. Our long-term goal is to discover genes critical for the correct formation and function of the inner ear and its sensory tissue. A novel gene, transmembrane inner ear (Tmie), was found to cause hearing-related disorders when defective in mice and humans. A homologous tmie gene in zebrafish was cloned and its expression characterized between 24 and 51 hours post-fertilization. Embryos injected with morpholinos (MO) directed against tmie exhibited circling swimming behavior (approximately 37%), phenocopying mice with Tmie mutations; semicircular canal formation was disrupted, hair cell numbers were reduced, and maturation of electrically active lateral line neuromasts was delayed. As in the mouse, tmie appears to be required for inner ear development and function in the zebrafish and for hair cell maturation in the vestibular and lateral line systems as well.

PTEN is a tumor suppressor gene associated with multiple tumor types. PTEN function is essential for early embryonic development and is involved in the regulation of cell size, number, and survival. By dephosphorylating PIP(3), PTEN normally acts to inhibit the PI3-Kinase/AKT pathway. Here we have identified two zebrafish orthologs, ptena and ptenb, of the single mammalian PTEN gene and analyzed the role of these genes in zebrafish development. Ptena transcripts were expressed throughout the embryo at early somitogenesis. By 24 hpf, expression was predominant in the central nervous system, axial vasculature, retina, branchial arches, ear, lateral line primordium, and pectoral fin bud. Ptenb was also ubiquitously expressed early in somitogenesis, but transcripts became more restricted to the somites and central nervous system as development progressed. By 48 hpf, ptena and ptenb were expressed predominantly in the central nervous system, branchial arches, pectoral fins, and eye. Antisense morpholinos were used to knock down translation of ptena and ptenb mRNA in zebrafish embryos. Knockdown of either pten gene caused increased levels of phosphorylated Akt in morphant embryos, indicating that Ptena and Ptenb each possess PIP(3) lipid phosphatase activity. Ptena morphants had irregularities in notochord shape (73%), vasculogenesis (83%), head shape (72%), and inner ear development (59%). The most noticeable defects in ptenb morphants were upward hooked tails (73%), domed heads (83%), and reduced yolk extensions (90%). These results indicate that ptena and ptenb encode functional enzymes and that each pten gene plays a distinct role during zebrafish embryogenesis.

Rfx winged-helix transcription factors, best known as key regulators of core ciliogenesis, also play ciliogenesis-independent roles during neural development. Mammalian Rfx4 controls neural tube morphogenesis via both mechanisms.

Adult zebrafish spontaneously regenerate their retinas after damage. Although a number of genes and signaling pathways involved in regeneration have been identified, the exact mechanisms regulating various aspects of regeneration are unclear. microRNAs (miRNAs) were examined for their potential roles in regulating zebrafish retinal regeneration.

The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)-2α is associated with neural crest stem cell appearance and aggressiveness in tumors. However, little is known about its role in normal neural crest development.