NBP, a zebrafish homolog of human Kank3, is a novel Numb interactor essential for epidermal integrity and neurulation.

Numb is an adaptor protein implicated in diverse basic cellular processes. Using the yeast-two hybrid system we isolated a novel Numb interactor in zebrafish called NBP which is an ortholog of human renal tumor suppressor Kank. NBP interacts with the PTB domain of Numb through a region well conserved among vertebrate Kanks containing the NGGY sequence. Similar NBP and Numb morphant phenotype such as impaired convergence and extension movements during gastrulation, neurulation and epidermis defects and enhanced phenotypic aberrations in double morphants suggest that the genes interact genetically. We demonstrate that the expression of NBP undergoes quantitative and qualitative changes during embryogenesis and that the protein accumulates at the cell periphery to sites of cell-cell contact during gastrulation and later in development it concentrates at the basal poles of differentiated cells. These findings imply a possible role of NBP in establishing and maintaining cell adhesion and tissue integrity.

Hedgehog signalling controls zebrafish neural keel morphogenesis via its level-dependent effects on neurogenesis.

We investigated the role of hedgehog (Hh) signalling on zebrafish neurulation, focusing on the intimate relationship between neurogenesis and morphogenesis during the neural keel stage. Through the analyses of Hh loss- and gain-of-function phenotypes, we found that Hh signalling controls the neural keel morphogenesis. To investigate underlying mechanisms, we examined cellular elongation polarity in the neural keel of Hh loss- and gain-of-function phenotypes and compared this with the deficient phenotype of a planar cell polarity (PCP) molecule, Trilobite/Strabismus. We found that Hh signalling controls cell elongation polarity of the neuroepithelium at least in part by means of PCP pathway; however, its effects are not strong enough per se to affect keel morphogenesis; instead Hh signalling mainly controls keel morphogenesis by means of affecting both medial and lateral neurogenesis. We devised a method for precise evaluation of neurogenesis in loss- and gain-of-Hh phenotypes that compensates for its delay caused by disturbed morphogenesis. We present a model that Hh signalling exerts level-dependent and binary-opposite effects on medial neurogenesis, whose modification to explain lateral neurogenesis reveals regional differences of underlying mechanisms between the two proneural domains. Such differences seem to be created in part by regional effector signalling; the effects of high Hh-signalling on medial neurogenesis can be reversed in accordance to medial Tri/Stbm level, in a polarity independent manner.

Apical localization of ASIP/PAR-3:EGFP in zebrafish neuroepithelial cells involves the oligomerization domain CR1, the PDZ domains, and the C-terminal portion of the protein.

Neurulation in zebrafish (Danio rerio) embryos is characterized by oriented cell divisions and the progressive establishment of cellular polarity. Mitoses in the neural plate and neural tube are planar, but in the neural keel/rod stage, the mitotic spindle rotates by 90 degrees, causing cell divisions to occur perpendicular to the plane of the neuroepithelium. The mechanisms and molecules that establish cellular polarity and cause the stereotypic orientation of the mitotic spindle during neurulation are largely unknown. In Caenorhabditis elegans and Drosophila, the PAR/aPKC complex has been shown to be involved in both establishment of cellular polarity and spindle orientation. Here, we show that the conserved N-terminal oligomerization domain (CR1) and the PDZ domains of ASIP/PAR-3:EGFP are involved in its localization to the apical membrane in zebrafish neuroepithelial cells. We further show that the C-terminal part of ASIP/PAR-3 contributes to proper localization and that the apical localization signals in ASIP/PAR-3 prevent the basolateral localization of a Numb:PAR-3 fusion protein. The parallel orientation of the mitotic spindle in the neural tube, however, is only weakly impaired upon overexpression of various ASIP/PAR-3:EGFP constructs.

Asymmetric localization of Numb:EGFP in dividing neuroepithelial cells during neurulation in Danio rerio.

In the neural plate and tube of the zebrafish embryo, cells divide with their mitotic spindles oriented parallel to the plane of the neuroepithelium, whilst in the neural keel and rod, the spindle is oriented perpendicular to it. This change is achieved by a 90 degrees rotation of the mitotic spindle. We cloned zebrafish homologues of the gene for the Drosophila cell fate determinant Numb, and analyzed the localization of EGFP fusion proteins in vivo in dividing neuroepithelial cells during neurulation. Whereas Numb isoform 3 and the related protein Numblike are localized in the cytoplasm, Numb isoform 1 is localized to the cell membrane. Time-lapse analyses showed that Numb 1 is distributed uniformly around the cell cortex in dividing cells during plate and keel stages, but becomes localized at the basolateral membrane of some dividing cells during the transition from neural rod to tube. Using in vitro mutagenesis and Numb:EGFP deletion constructs, we showed that the first 196 amino acids of Numb are sufficient for this localization. Furthermore, we found that an 11-amino acid insertion in the PTB domain is essential for localization to the cortex, whereas amino acids 2-12 mediate the basolateral localization in the neural tube stage.

her3, a zebrafish member of the hairy-E(spl) family, is repressed by Notch signalling.

her3 encodes a zebrafish bHLH protein of the Hairy-E(Spl) family. During embryogenesis, the gene is transcribed exclusively in the developing central nervous system, according to a fairly simple pattern that includes territories in the mesencephalon/rhombencephalon and the spinal cord. In all territories, the her3 transcription domain encompasses regions in which neurogenin 1 (neurog1) is not transcribed, suggesting regulatory interactions between the two genes. Indeed, injection of her3 mRNA leads to repression of neurog1 and to a reduction in the number of primary neurones, whereas her3 morpholino oligonucleotides cause ectopic expression of neurog1 in the rhombencephalon. Fusions of Her3 to the transactivation domain of VP16 and to the repression domain of Engrailed show that Her3 is indeed a transcriptional repressor. Dissection of the Her3 protein reveals two possible mechanisms for transcriptional repression: one mediated by the bHLH domain and the C-terminal WRPW tetrapeptide; and the other involving the N-terminal domain and the orange domain. Gel retardation assays suggest that the repression of neurog1 transcription occurs by binding of Her3 to specific DNA sequences in the neurog1 promoter. We have examined interrelationships of her3 with members of the Notch signalling pathway by the Gal4-UAS technique and mRNA injections. The results indicate that Her3 represses neurog1 and, probably as a consequence of the neurog1 repression, deltaA, deltaD and her4. Moreover, Her3 represses its own transcription as well. Surprisingly, and in sharp contrast to other members of the E(spl) gene family, transcription of her3 is repressed rather than activated by Notch signalling.

A 90-degree rotation of the mitotic spindle changes the orientation of mitoses of zebrafish neuroepithelial cells.

In the neural plate and neural tube in the trunk region of the zebrafish embryo, dividing cells are oriented parallel to the plane of the neuroepithelium, while in neural keel/rod, cells divide perpendicular to it. This change in the orientation of mitosis is brought about by a 90 degrees rotation of the mitotic spindle. As the two halves of the neural primordium in keel/rod stage are in apposition, the perpendicular orientation of mitoses in this stage determines that daughter cells become allocated to both sides of the neural tube. To assess the role played by cell junctions in controlling the orientation of dividing cells, we studied the expression of components of adherens and tight junctions in the neuroepithelial cells. We find that these proteins are distributed irregularly at the neural plate stage and become polarised apically in the cell membrane only during the keel/rod stage. The stereotypic orientation of mitoses is perturbed only weakly upon loss of function of the cell junction components ASIP and aPKClambda, suggesting that mitotic orientation depends in part on the integrity of cell junctions and the polarity of the epithelium as a whole. However, the 90-degree rotation of the spindle does not require perfectly polarised cell junctions between the neuroepithelial cells.

On the organisation of the regulatory region of the zebrafish deltaD gene.

DeltaD is one of the four zebrafish Delta homologues presently known. Experimental evidence indicates that deltaD participates in a number of important processes during embryogenesis, including early neurogenesis and somitogenesis, whereby the protein it encodes acts as a ligand for members of the Notch receptor family. In accordance with its functional role, deltaD is transcribed in several domains of mesodermal and ectodermal origin during embryogenesis. We have analysed the organisation of the regulatory region of the deltaD gene using fusions to the reporter gene gfp and germline transgenesis. Cis-regulatory sequences are dispersed over a stretch of 12.5 kb of genomic DNA, and are organised in a similar manner to those in the regulatory region of the Delta-like 1 gene of mouse. Germline transformation using a minigene comprising 10.5 kb of this genomic DNA attached to the 3' end of a full-length cDNA clone rescues the phenotype of embryos homozygous for the amorphic deltaD mutation after eight(AR33). Several genomic regions that drive transcription in mesodermal and neuroectodermal domains have been identified. Transcription in all the neural expression domains, with one exception, is controlled by two relatively small genomic regions, which are regulated by the proneural proteins neurogenin 1 and zash1a/b acting as transcriptional activators that bind to so-called E-boxes. Transcriptional control of deltaD by proneural proteins therefore represents a molecular target for the regulatory feedback loop mediated by the Notch pathway in lateral inhibition.

A quantitative analysis of the kinetics of Gal4 activator and effector gene expression in the zebrafish.

Using a temperature-inducible hsp70:Gal4 activator and UAS:myc-notch1a-intra as effector, we determined quantitatively the kinetics of expression of both transgenes and analysed the effects of varying their expressivity on several phenotypic traits in the developing zebrafish. hsp70:Gal4 is transcribed within 15 min after temperature-mediated induction, but Gal4 RNA decays rapidly. The Gal4 protein was found to be quite stable, as functional Gal4, which was detectable 1.5 h after heat shock (HS), persisted for at least 13 h. myc-notch1a-intra RNA is expressed approximately 1.5 h after HS, but unlike the Gal4 RNA, it was found to be very stable; it continues to accumulate during the succeeding 17 h after HS. Fully penetrant phenotypic effects are obtained after a relatively long activator induction with a 30-min HS.

A clonal analysis of spinal cord development in the zebrafish.

We describe the results of a clonal analysis of spinal cord development in the zebrafish. The data were obtained from embryos in which fluorescent lineage tracer was injected into single cells in the neural plate at the two-somite stage. Injected animals were allowed to survive until either 4 days or 2 weeks postfertilization. In other experiments, bromodeoxy uridine (BrdU) was injected intraperitoneally at 30 h postfertilization (hpf) after lineage tracer injection in the neural plate at the two-somite stage, and the embryos fixed at 38 hpf. We restricted our experiments to the thoracic region of the spinal cord. Our experiments were aimed at answering questions regarding the proliferative abilities of neuroepithelial cells during embryonic development (as deduced from the size of the clones), the modes of cell division (as deduced from the uptake of BrdU into clone cells), positional differences in the proliferation of cells within the neural plate itself, the cellular composition of the clones, and cell dispersion (deduced from the regional distribution of clone cells).

Autonomous and non-autonomous phenotypic expression of Notch mutant cells in Drosophila embryogenesis.

Transplantation of single homozygous Notch - cells into the ventral neuroectoderm of Drosophila wild-type embryos reveals a non-autonomous behaviour of these cells. However, mitotic recombination events induced in heterozygous cells in imaginal discs lead to generation of homozygous Notch - cells which differentiate cell autonomously. We have tested various possible explanations for the non-autonomous behaviour of Notch mutant cells following transplantation. We confirm the previous results using different Notch alleles. Moreover, we find that increasing the number of wild-type Notch copies in a cell increases probability that it will take on an epidermal fate. However, single Notch mutant cells behave differently depending on whether they are placed in the ventral neuroectoderm, the procephalic neuroectoderm or the proctodeal anlage. Following transplantation into host embryos devoid of mesoderm, almost all single Notch mutant cells behave autonomously. These results suggest an influence of the mesoderm on ectodermal development. Finally, we provide further evidence that DELTA acts as the signal source in lateral inhibition.

On the formation of the neural keel and neural tube in the zebrafishDanio (Brachydanio) rerio.

Morphogenetic movements accompanying formation of the neural keel and neural tube in the zebrafishDanino (Brachydanio) rerio were studied by labelling single neural plate cells with fluoresceinated dextran (FDA) during late gastrula stages (95-100% epiboly) and localizing their progeny with an anti-fluorescein antibody on histological sections throughout neurulation. The mediolateral extent of the neural plate correlates directly with the dorso-ventral extent of the neural tube. That is to say, the progeny of cells located medially in the neural plate come to lie ventrally in the neural tube; cells located laterally in the neural plate give rise to progeny that populate dorsal levels in the neural tube. Fixation of labelled cells at various stages reveals that neural keel and nerve rod are organized as monostratified epithelia and that they maintain this organization during neurulation. These observations strongly suggest that the neural keel in the zebrafish forms by way of infolding of the neural plate and, therefore, utilizes a mechanism similar to primary neurulation in other vertebrates. The folding process juxtaposes the apical surfaces of both flanks of the neural plate at the midline. Mitoses occur preferentially in this zone, leading very frequently to formation of bilaterally symmetrical clones of progeny cells. The size of the clones that develop from injected cells suggests that neural plate cells divide an 1.5 times on average between late gastrula and the end of neurulation.

Dorso-ventral polarity of the zebrafish embryo is distinguishable prior to the onset of gastrulation.

We describe a set of observations on developing zebrafish embryos and discuss the main conclusions they allow:(1) the embryonic dorso-ventral polarity axis is morphologically distinguishable prior to the onset of gastrulation; and (2) the involution of deep layer cells starts on the prospective dorsal side of the embryo. An asymmetry can be distinguished in the organization of the blastomeres in the zebrafish blastula at the 30% epiboly stage, in that one sector of the blastoderm is thicker than the other. Dye-labelling experiments with DiI and DiO and histological analysis allow us to conclude that the embryonic shield will form on the thinner side of the blastoderm. Therefore, this side corresponds to the prospective dorsal side of the embryo. Simultaneous injections of dyes on the thinner side of the blastoderm and on the opposite side show that involution of deep layer cells during gastrulation starts at the site at which the embryonic shield will form and extends from here to the prospective ventral regions of the germ ring.

A functional analysis of the genes Enhancer of split and HLH-m5 during early neurogenesis in Drosophila melanogaster.

To assess the functional domains of the proteins encoded by E(spl) and HLH-m5, two genes of the Enhancer of split complex [E(SPL)-C] of Drosophila melanogaster, a number of variants have been made by in vitro mutagenesis, transformed into the germ line of the wild-type, and genetically combined with a chromosomal deletion lacking four of the genes of the E(SPL)-C. All constructs used attenuated the neurogenic phenotype associated with this deletion. However, constructs encoding proteins with truncated carboxy-termini exibited in all cases a higher activity than constructs encoding the full length version of the protein. Neutralization of the basic domain severely reduced, but did not completely abolish the rescuing activity of E(spl), while proteins in which a proline residue within the basic domain had been changed to either threonine or asparagine were slightly less efficient in their rescuing activity than the corresponding wild-type versions. We discuss the possible significance of these results for the function of the protein domains.

Neurulation in the anterior trunk region of the zebrafish Brachydanio rerio.

We have studied the process of neurulation within the anterior trunk region of the zebrafish by means of serial sectioning of staged embryos and labelling cells by applications of the dye Dil and intracellular injections of fluoresceine dextran amine. The first morphological manifestation of the prospective neural plate is a dorsomedial ectodermal thickening which becomes visible immediately after gastrulation. Within 1-2 h, by the time somatogenesis begins, two bilaterally symmetrical thickenings have appeared more laterally, which eventually fuse with the medial thickening to form the neural keel. The central canal forms next by separation of the cells on either side of the midline of the neural keel, beginning ventrally at the 17-somite stage and progressing towards dorsal levels. By means of fluorescent dye labelling in the late gastrula, we found that both the medial and lateral thickenings contribute to the nerve cord. The medial thickening was found to contain, exclusively, neural progenitor cells from the 90-100% epiboly stage on, whereas the adjacent regions contained a mixture of neural and epidermal progenitor cells, as well as prospective neural crest cells. Between the 90-100% epiboly and 2-somite stages, this heterogeneity of developmental capabilities is resolved into territories, with epidermogenic and neurogenic cells clearly separated from each other. To achieve this segregation into neural and epidermal anlagen, cells from the lateral thickenings have to move over a distance of roughly 400 µm within 1-2 h. Epidermal overgrowth of the nerve cord occurs during the morphogenetic movements that accompany nerve cord formation.

Regulatory signals and signal molecules in early neurogenesis of Drosophila melanogaster.

The ectodermal germ layer of Drosophila melanogaster gives rise to two major cell lineages, the neural and the epidermal. Progenitor cells for each of these lineages arise from groups of cells, whose elements must decide between taking on either fate. Commitment of the progenitor cells to one of the developmental fates implies two factors. One is intrinsic to the ectodermal cells and determines a propensity to take on neural fate; this factor is probably represented by the products of the so-called proneural genes, which are differentially distributed throughout the ectoderm. The other factor in the cells' decision to adopt one of the two alternative fates is intercellular communication, which is mediated by the products of the so-called neurogenic genes. Two types of interactions, one inhibiting and the other stimulating neural development, have been inferred. We discuss here the assumed role of various neurogenic genes, in particular Notch and Delta, in these processes.

Second-site modifiers of the Delta wing phenotype in Drosophila melanogaster.

We have screened for dominant enhancers and suppressors of the wing phenotype associated with two Delta alleles: Dl 9P39, an amorphic allele, and Dl FE32, an antimorphic allele. The interactions of some of the modifiers with Delta are due to haplo-insufficient expression of the corresponding genes. Although not explicitly shown for the remaining cases, we assume that haploin-sufficiency is also the basis for the relationships of these genes to Delta, since no allele specific interactions were observed. The modifiers found define 22 genes with pleiotropic expression, which can be classified into two groups: genes required for wing vein pattern formation and for neurogenesis, and genes which are not required for neurogenesis. Among the genes of the first group, Hairless and Star were previously known to participate in neural development. One further modifier was found which may correspond to a new neurogenic gene. The second group of genes is larger and includes already known loci, e.g., Plexate, blistered, plexus, etc, as well as other previously unidentified genes, which function during wing morphogenesis.

Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster.

The larval and early pupal development of the optic lobes in Drosophila is described qualitatively and quantitatively using [3H]thymidine autoradiography on 2-µm plastic sections. The optic lobes develop from 30-40 precursor cells present in each hemisphere of the freshly hatched larva. During the first and second larval instars, these cells develop to neuroblasts arranged in two epithelial optic anlagen. In the third larval instar and in the early pupa these neuroblasts generate the cells of the imaginal optic lobes at discrete proliferation zones, which can be correlated with individual visual neuropils.The different neuropils as well as the repetitive elements of each neuropil are generated in a defined temporal sequence. Cells of the medulla are the first to become postmitotic with the onset of the third larval instar, followed by cells of the lobula complex and finally of the lamina at about the middle of the third instar. The elements of each neuropil connected to the most posterior part of the retina are generated first, elements corresponding to the most anterior retina are generated last.The proliferation pattern of neuroblasts into ganglion mother cells and ganglion cells is likely to include equal as well as unequal divisions of neuroblasts, followed by one or two generations of ganglion mother cells. For the lamina the proliferation pattern and its temporal coordination with the differentiation of the retina are shown.

Second-site modifiers of the split mutation of Notch define genes involved in neurogenesis in Drosophila melanogaster.

We have searched for dominant modifiers, i.e., enhancers and suppressors, of the compound eye phenotype of split, a recessive viable allele of Notch. Among the spl modifiers found, we have detected mutations in loci whose functions were previously known to cooperate with Notch in embryonic neurogenesis, such as daughterless, master mind, Delta and Hairless. In addition, other spl modifier mutations have been found in loci that were not previously known to interact with Notch, such as scabrous, glass, roughened eye, and several other genes that have not yet been assigned to known loci. The phenotypes associated with mutations in some of these latter loci suggest the participation of the corresponding genes in embryonic neurogenesis. We show that in some cases the observed interactions are due to genetic haplo-insufficent expression of the genes, whereas allele-specific interactions with spl are observed in master mind and Delta alleles. From this observation, we propose a direct functional association between the proteins encoded by Notch, Delta and master mind.

Defective ommatidial cell assembly leads to defective morphogenesis: a phenotypic analysis of the E(spl) D mutation of Drosophila melanogaster.

The spl mutation of the N gene causes, among other phenotypic traits, the lack of a few ommatidia, roughness and a general reduction in the size of the compound eye; these defects are drastically enhanced by the dominant mutation E(spl) D. We have studied cellular and developmental aspects of the phenotypic interaction between spl and E(spl) D. We found that the initial clustering of photoreceptor cells is affected in eye imaginal discs of spl larvae causing the defects visible in the adult eye. The degree of disorganization of the spl/Y; E(spl) D/ + eye disc is much higher, only a few photoreceptor cells are able to group with representatives of the other cell types and differentiate normally. BrdU incorporation shows that the proliferation pattern of the spl/Y; E(spl) D/ + disc cells during the third instar is normal. Abundant cell death occurs posteriorly in the mutant discs, which accounts for their small size. Finally, we found that in the eye imaginal disc the transcription of m8, the E(spl) gene, responsible for the enhancement of the spl phenotype caused by the E(spl) D mutation, is restricted to the morphogenetic furrow, where the ommatidial cells start grouping with each other to take on their future developmental fates; the m8 transcription rate is highly increased in E(spl) D eye discs. All these observations indicate that the assembly of the ommatidial cells is affected in the spl/Y; E(spl) D/ + disc and that the other abnormalities are morphogenetic consequences of the defective cell grouping.

Two groups of interrelated genes regulate early neurogenesis in Drosophila melanogaster.

In Drosophila melanogaster the neuroblasts separate from epidermoblasts to give rise to the neural primordium. This process is under the control of several genes. The group of the so-called neurogenic genes is required for epidermal development; other genes, comprising those of the achaete-scute complex and daughterless, are required for neural development. We have studied the relationships between both groups of genes in two different ways. We have analyzed the phenotype of double-mutant embryos and our results show that the neural hyperplasia caused by neurogenic mutations can be partially prevented if a mutation in one of the other genes is present in the same genome. Only the neural cells that do not require the function of a particular gene of the achaete-scute complex in the wild-type seem to develop to a neural fate in the double mutant embryos. At least some of the genetic interactions affect the transcriptional level, as shown by in situ hybridization, since the territories of transcription of the achaetescute genes are expanded in neurogenic mutants. All cells of the neurogenic region of the double mutants apparently initiate neural development. However, during later development some of these cells switch their fate either to epidermogenesis or to cell death and this leads to the final phenotype of the double mutants. We discuss these results with respect to the events of early neurogenesis.