Fgf signals from a novel signaling center determine axial patterning of the prospective neural retina

Axial eye patterning determines the positional code of retinal ganglion cells (RGCs), which is crucial for their topographic projection to the midbrain. Several asymmetrically expressed determinants of retinal patterning are known, but it is unclear how axial polarity is first established. We find that Fgf signals, including Fgf8, determine retinal patterning along the nasotemporal (NT) axis during early zebrafish embryogenesis: Fgf8 induces nasal and/or suppresses temporal retinal cell fates; and inhibition of all Fgf-receptor signaling leads to complete retinal temporalization and concomitant loss of all nasal fates. Misprojections of RGCs with Fgf-dependent alterations in retinal patterning to the midbrain demonstrate the importance of this early patterning process for late topographic map formation. The crucial period of Fgf-dependent patterning is at the onset of eye morphogenesis. Fgf8 expression, the restricted temporal requirement for Fgf-receptor signaling and target gene expression at this stage suggests that the telencephalic primordium is the source of Fgf8 and acts as novel signaling center for non-autonomous axial patterning of the prospective neural retina.     

Evolution of axial patterning in elongate fishes

Within the ray-finned fishes, eel-like (extremely elongate) body forms have evolved multiple times from deeper-bodied forms. Previous studies have shown that elongation of the vertebral column may be associated with an increase in the number of vertebrae, an increase in the length of the vertebral centra, or a combination of both. Because the vertebral column of fishes has at least two anatomically distinct regions (i.e. abdominal and caudal), an increase in the number and relative length of the vertebrae could be region-specific or occur globally across the length of the vertebral column. In the present study, we recorded vertebral counts and measurements of vertebral aspect ratio (vertebral length/width) from museum specimens for 54 species representing seven groups of actinopterygian fishes. We also collected, from published literature, vertebral counts for 813 species from 14 orders of actinopterygian and elasmobranch fishes. We found that the number of vertebrae can increase independently in the abdominal and caudal regions of the vertebral column, but changes in aspect ratio occur similarly in both regions. These findings suggest that abdominal vertebral number, caudal vertebral number, and vertebral aspect ratio are controlled by separate developmental modules. Based on these findings, we suggest some candidate developmental mechanisms that may contribute to vertebral column patterning in fishes. Our study is an example of how comparative anatomical studies of adults can generate testable hypotheses of evolutionary changes in developmental mechanisms.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 97–116.     

The establishment of axial patterning in the maize leaf

The maize leaf consists of four distinct tissues along its proximodistal axis: sheath, ligule, auricle and blade. liguleless1 (lg1) functions cell autonomously to specify ligule and auricle, and may propagate a signal that correctly positions the blade-sheath boundary. The dominant Wavy auricle in blade (Wab1) mutation disrupts both the mediolateral and proximodistal axes of the maize leaf. Wab1 leaf blades are narrow and ectopic auricle and sheath extend into the blade. The recessive lg1-R mutation exacerbates the Wab1 phenotype; in the double mutants, most of the proximal blade is deleted and sheath tissue extends along the residual blade. We show that lg1 is misexpressed in Wab1 leaves. Our results suggest that the Wab1 defect is partially compensated for by lg1 expression. A mosaic analysis of Wab1 was conducted in Lg1+ and lg1-R backgrounds to determine if Wab1 affects leaf development in a cell-autonomous manner. Normal tissue identity was restored in all wab1+/- sectors in a lg1-R mutant background, and in three quarters of sectors in a Lg1+ background. These results suggest that lg1 can influence the autonomy of Wab1. In both genotypes, leaf-halves with wab1+/- sectors were significantly wider than non-sectored leaf-halves, suggesting that Wab1 acts cell-autonomously to affect lateral growth. The mosaic analysis, lg1 expression data and comparison of mutant leaf shapes reveal previously unreported functions of lg1 in both normal leaf development and in the dominant Wab1 mutant.     

Flexibility in the patterning and control of axial locomotor networks in lamprey

In lower vertebrates, locomotor burst generators for axial muscles generally produce unitary bursts that alternate between the two sides of the body. In lamprey, a lower vertebrate, locomotor activity in the axial ventral roots of the isolated spinal cord can exhibit flexibility in the timings of bursts to dorsally-located myotomal muscle fibers versus ventrally-located myotomal muscle fibers. These episodes of decreased synchrony can occur spontaneously, especially in the rostral spinal cord where the propagating body waves of swimming originate. Application of serotonin, an endogenous spinal neurotransmitter known to presynaptically inhibit excitatory synapses in lamprey, can promote decreased synchrony of dorsal-ventral bursting. These observations suggest the possible existence of dorsal and ventral locomotor networks with modifiable coupling strength between them. Intracellular recordings of motoneurons during locomotor activity provide some support for this model. Pairs of motoneurons innervating myotomal muscle fibers of similar ipsilateral dorsoventral location tend to have higher correlations of fast synaptic activity during fictive locomotion than do pairs of motoneurons innervating myotomes of different ipsilateral dorsoventral locations, suggesting their control by different populations of premotor interneurons. Further, these different motoneuron pools receive different patterns of excitatory and inhibitory inputs from individual reticulospinal neurons, conveyed in part by different sets of premotor interneurons. Perhaps, then, the locomotor network of the lamprey is not simply a unitary burst generator on each side of the spinal cord that activates all ipsilateral body muscles simultaneously. Instead, the burst generator on each side may comprise at least two coupled burst generators, one controlling motoneurons innervating dorsal body muscles and one controlling motoneurons innervating ventral body muscles. The coupling strength between these two ipsilateral burst generators may be modifiable and weakening when greater swimming maneuverability is required. Variable coupling of intrasegmental burst generators in the lamprey may be a precursor to the variable coupling of burst generators observed in the control of locomotion in the joints of limbed vertebrates.     

Early onset of phenotype and cell patterning in the embryonic zebrafish retina

The regular arrangement of retinal cone cells in a mosaic pattern is a common feature of teleosts. In the zebrafish, Brachydanio rerio, the retinal cone mosaic comprises parallel rows consisting of a repeating motif of four cone types. In order to elucidate the temporal and spatial aspects of the genesis of the cone mosaic in the developing retina, we generated a monoclonal antibody that specifically binds to the double cone photoreceptor of the adult. We first saw staining in the developing retina with this antibody, FRet 43, at 48 hours postfertilization, the time at which the first photoreceptor cells undergo their final mitotic division. We then injected embryonic fish with the thymidine analog, 5-bromo-2'-deoxyuridine (BrdU), confirming with a double-labeling experiment that the onset of FRet 43 antigenicity occurs within three hours of the cellular division that generates the double cone photoreceptors. Then we stained tangential sections of the 54-hour embryonic retina with FRet 43, further showing that cells devoid of staining alternate with stained pairs of cells in a pattern that is consistent with the arrangement of photoreceptors in the adult cone mosaic. These results indicate that a marker of the double cone phenotype is expressed at approximately the same time as cellular birthday and that the mosaic patterning is present within 6 hours of this expression.     

The morphology and topographic distribution of AII amacrine cells in the cat retina

When cat retina is incubated in vitro with the fluorescent dye, 4',6-diamidino-2-phenyl-indole (DAPI), a uniform population of neurons is brightly labelled at the inner border of the inner nuclear layer. The dendritic morphology of the DAPI-labelled cells was defined by iontophoretic injection of Lucifer yellow under direct microscopic control: all the filled cells had the narrow-field bistratified morphology that is distinctive of the AII amacrine cells previously described from Golgi-stained retinae. Although the AII amacrines are principal interneurons in the rod-signal pathway, their density distribution does not follow the topography of the rod receptors, but peaks in the central area like the cone receptors and the ganglion cells. There are some 512 000 AII amacrines in the cat retina and their density ranges from 500 cells per square millimetre at the superior margin to 5300 cells per square millimetre in the centre (retinal area is 450 mm2). The isodensity contours are kite-shaped, particularly at intermediate densities, with a horizontal elongation towards nasal retina. The cell body size and the dendritic dimensions of AII amacrines increase with decreasing cell density. The lobular dendrites in sublamina a of the inner plexiform layer span a restricted field of 16-45 microns diameter, while the arboreal dendrites in sublamina b form a varicose tree of 18-95 microns diameter. The dendritic field coverage of the lobular appendages is close to 1.0 (+/- 0.2) at all eccentricities whereas the coverage of the arboreal dendrites doubles within the first 1.5 mm and then remains constant at 3.8 (+/- 0.7) throughout the periphery.     

Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo

Gastrulation in higher vertebrate species classically commences with the generation of mesoderm cells in the primitive streak by epithelio-mesenchymal transformation of epiblast cells. However, the primitive streak also marks, with its longitudinal orientation in the posterior part of the conceptus, the anterior-posterior (or head-tail) axis of the embryo. Results obtained in chick and mouse suggest that signals secreted by the hypoblast (or visceral endoderm), the extraembryonic tissue covering the epiblast ventrally, antagonise the mesoderm induction cascade in the anterior part of the epiblast and thereby restrict streak development to the posterior pole (and possibly initiate head development anteriorly). In this paper we took advantage of the disc-shape morphology of the rabbit gastrula for defining the expression compartments of the signalling molecules Cerberus and Dickkopf at pre-gastrulation and early gastrulation stages in a mammal other than the mouse. The two molecules are expressed in novel expression compartments in a complementary fashion both in the hypoblast and in the emerging primitive streak. In loss-of-function experiments, carried out in a New-type culturing system, hypoblast was removed prior to culture at defined stages before and at the beginning of gastrulation. The epiblast shows a stage-dependent and topographically restricted susceptibility to express Brachyury, a T-box gene pivotal for mesoderm formation, and to transform into (histologically proven) mesoderm. These results confirm for the mammalian embryo that the anterior-posterior axis of the conceptus is formed first as a molecular prepattern in the hypoblast and then irrevocably fixed, under the control of signals secreted from the hypoblast, by epithelio-mesenchymal transformation (primitive streak formation) in the epiblast.Edited by D. Tautz     

Dynamic decapentaplegic signaling regulates patterning and adhesion in the Drosophila pupal retina

The correct organization of cells within an epithelium is essential for proper tissue and organ morphogenesis. The role of Decapentaplegic/Bone morphogenetic protein (Dpp/BMP) signaling in cellular morphogenesis during epithelial development is poorly understood. In this paper, we used the developing Drosophila pupal retina--looking specifically at the reorganization of glial-like support cells that lie between the retinal ommatidia--to better understand the role of Dpp signaling during epithelial patterning. Our results indicate that Dpp pathway activity is tightly regulated across time in the pupal retina and that epithelial cells in this tissue require Dpp signaling to achieve their correct shape and position within the ommatidial hexagon. These results point to the Dpp pathway as a third component and functional link between two adhesion systems, Hibris-Roughest and DE-cadherin. A balanced interplay between these three systems is essential for epithelial patterning during morphogenesis of the pupal retina. Importantly, we identify a similar functional connection between Dpp activity and DE-cadherin and Rho1 during cell fate determination in the wing, suggesting a broader link between Dpp function and junctional integrity during epithelial development.     

miR-196 regulates axial patterning and pectoral appendage initiation

Vertebrate Hox clusters contain protein-coding genes that regulate body axis development and microRNA (miRNA) genes whose functions are not yet well understood. We overexpressed the Hox cluster microRNA miR-196 in zebrafish embryos and found four specific, viable phenotypes: failure of pectoral fin bud initiation, deletion of the 6th pharyngeal arch, homeotic aberration and loss of rostral vertebrae, and reduced number of ribs and somites. Reciprocally, miR-196 knockdown evoked an extra pharyngeal arch, extra ribs, and extra somites, confirming endogenous roles of miR-196. miR-196 injection altered expression of hox genes and the signaling of retinoic acid through the retinoic acid receptor gene rarab. Knocking down rarab mimicked the pectoral fin phenotype of miR-196 overexpression, and reporter constructs tested in tissue culture and in embryos showed that the rarab 3′UTR is a miR-196 target for pectoral fin bud initiation. These results show that a Hox cluster microRNA modulates development of axial patterning similar to nearby protein-coding Hox genes, and acts on appendicular patterning at least in part by modulating retinoic acid signaling.     

Polychaetoid controls patterning by modulating adhesion in the Drosophila pupal retina

Correct cellular patterning is central to tissue morphogenesis, but the role of epithelial junctions in this process is not well-understood. The Drosophila pupal eye provides a sensitive and accessible model for testing the role of junction-associated proteins in cells that undergo dynamic and coordinated movements during development. Mutations in polychaetoid (pyd), the Drosophila homologue of Zonula Occludens-1, are characterized by two phenotypes visible in the adult fly: increased sensory bristle number and the formation of a rough eye produced by poorly arranged ommatidia. We found that Pyd was localized to the adherens junction in cells of the developing pupal retina. Reducing Pyd function in the pupal eye resulted in mis-patterning of the interommatidial cells and a failure to consistently switch cone cell contacts from an anterior-posterior to an equatorial-polar orientation. Levels of Roughest, DE-Cadherin and several other adherens junction-associated proteins were increased at the membrane when Pyd protein was reduced. Further, both over-expression and mutations in several junction-associated proteins greatly enhanced the patterning defects caused by reduction of Pyd. Our results suggest that Pyd modulates adherens junction strength and Roughest-mediated preferential cell adhesion.     

Inductive patterning of the embryonic brain in Drosophila

In vertebrates (deuterostomes), brain patterning depends on signals from adjacent tissues. For example, holoprosencephaly, the most common brain anomaly in humans, results from defects in signaling between the embryonic prechordal plate (consisting of the dorsal foregut endoderm and mesoderm) and the brain. I have examined whether a similar mechanism of brain development occurs in the protostome Drosophila, and find that the foregut and mesoderm act to pattern the fly embryonic brain. When the foregut and mesoderm of Drosophila are ablated, brain patterning is disrupted. The loss of Hedgehog expressed in the foregut appears to mediate this effect, as it does in vertebrates. One mechanism whereby these defects occur is a disruption of normal apoptosis in the brain. These data argue that the last common ancestor of protostomes and deuterostomes had a prototype of the brains present in modern animals, and also suggest that the foregut and mesoderm contributed to the patterning of this 'proto-brain'. They also argue that the foreguts of protostomes and deuterostomes, which have traditionally been assigned to different germ layers, are actually homologous.     

The morphology and topographic distribution of substance-P-like immunoreactive amacrine cells in the cat retina

In cat retinal wholemounts, substance-P-like immunoreactivity (SP-IR) was localized in a distinct population of amacrines whose cell bodies were normally placed in the ganglion cell layer. Although displaced amacrines accounted for 80-95% of the SP-IR amacrines in peripheral retina, this proportion decreased considerably within the area centralis, accounting for 50-80% of the labelled cells at maximum density. The SP-IR cells in both the inner nuclear and ganglion cell layers gave rise to well-defined varicose dendrites of uniform appearance that stratified around 60% depth (S3/S4) of the inner plexiform layer. In addition, sparse fine dendrites in stratum 1 (S1) could sometimes be traced to inner nuclear cells and occasionally to displaced amacrines. The combined SP-IR cell density ranged from less than 50 cells mm-2 in the far periphery to more than 500 cells mm-2 in the area centralis; the maximum density showed little individual variation despite wide differences in the proportion of displaced cells. The 39,000 SP-IR amacrines in a mapped retina had a triangular topographic distribution, with intermediate isodensity lines extending vertically in superior retina and horizontally along both arms of the visual streak. Colocalization experiments established that all SP-IR cells in cat retina showed GABA-like immunoreactivity, and that the SP-IR amacrines were quite distinct from the cholinergic amacrines identified by choline acetyltransferase immunohistochemistry.     

Nanos plays a conserved role in axial patterning outside of the Diptera

Axial patterning is a fundamental event in early development, and molecules involved in determining the body axes provide a coordinate system for subsequent patterning. While orthologs of Drosophila bicoid and nanos play a conserved role in anteroposterior (AP) patterning within at least a subset of Diptera, conservation of this process has not yet been demonstrated outside of the flies. Indeed, it has been argued that bicoid, an instrumental "anterior" factor in Drosophila melanogaster, acquired this role during the evolution of more-derived dipterans. Interestingly, the interaction of Drosophila maternal nanos and maternal hunchback provides a system for patterning the AP axis that is partially redundant to the anterior system. Previous studies in grasshoppers suggest that hunchback may play a conserved role in axial patterning in this insect, but this function may be supplied solely by the zygotic component of hunchback expression. Here we provide evidence that the early pattern of zygotic grasshopper Hunchback expression is achieved through translational repression that may be mediated through the action of grasshopper nanos. This is consistent with the notion that an anterior gradient system is not necessary in all insects and that the posterior pole "probably conveys longitudinal polarity on the ensuing germ anlage".     

A role for MKP3 in axial patterning of the zebrafish embryo

Fibroblast growth factors (FGFs) are secreted molecules that can activate the RAS/mitogen-activated protein kinase (MAPK) pathway to serve crucial functions during embryogenesis. Through an in situ hybridization screen for genes with restricted expression patterns during early zebrafish development, we identified a group of genes that exhibit similar expression patterns to FGF genes. We report the characterization of zebrafish MAP kinase phosphatase 3 (MKP3; DUSP6 - Zebrafish Information Network), a member of the FGF synexpression group, showing that it has a crucial role in the specification of axial polarity in the early zebrafish embryo. MKP3 dephosphorylates the activated form of MAPK, inhibiting the RAS/MAPK arm of the FGF signaling pathway. Gain- and loss-of-function studies reveal that MKP3 is required to limit the extent of FGF/RAS/MAPK signaling in the early embryo, and that disturbing this inhibitory pathway disrupts dorsoventral patterning at the onset of gastrulation. The earliest mkp3 expression is restricted to the future dorsal region of the embryo where it is initiated by a maternal beta-catenin signal, but soon after its initiation, mkp3 expression comes under the control of FGF signaling. Thus, mkp3 encodes a feedback attenuator of the FGF pathway, the expression of which is initiated at an early stage so as to ensure correct FGF signaling levels at the time of axial patterning.     

Semi-automated topographic mapping of fracture surfaces through stereo-photogrammetry

By equipping a standard Hilger-Watts stereo-photogrammetry viewer with transducers, sets of (X, Y, Z) coordinates corresponding to features within fracture surfaces can be obtained. Transducers mounted so as to monitor the the X and Y translations of the viewing tray, when incorporated with a third transducer to measure the rise and fall of the light spot used to measure apparent height changes, supply three voltage signals corresponding to the coordinates of any feature within the fracture surface. These signals are then processed through an analog/digital converter and the digitized signals stored on diskette on an ATT 6300 computer. The voltage files can then be converted to calibrated, normalized files of (X, Y, Z) coordinates using a menu-driven program. Inexpensive commercially available software is then incorporated to use this data to generate calibrated topographic maps. In this way, detailed contour maps, as well as carpet plots and line profiles, can be generated from any fracture surface.