Andres Collazo

Fringe differentially modulates Jagged1 and Delta1 signalling throughNotch1 and Notch2

Proteins encoded by the fringe family of genes are required to modulate Notch signalling in a wide range of developmental contexts. Using a cell co-culture assay, we find that mammalian Lunatic fringe (Lfng) inhibits Jagged1-mediated signalling and potentiates Delta1-mediated signalling through Notch1. Lfng localizes to the Golgi, and Lfng-dependent modulation of Notch signalling requires both expression of Lfng in the Notch-responsive cell and the Notch extracellular domain. Lfng does not prevent binding of soluble Jagged1 or Delta1 to Notch1-expressing cells. Lfng potentiates both Jagged1- and Delta1-mediated signalling via Notch2, in contrast to its actions with Notch1. Our data suggest that Fringe-dependent differential modulation of the interaction of Delta/Serrate/Lag2 (DSL) ligands with their Notch receptors is likely to have a significant role in the combinatorial repertoire of Notch signalling in mammals.

Regulation of polarized extension and planar cell polarity in the cochlea by the vertebrate PCP pathway.

The mammalian auditory sensory organ, the organ of Corti, consists of sensory hair cells with uniformly oriented stereocilia on the apical surfaces and has a distinct planar cell polarity (PCP) parallel to the sensory epithelium. It is not certain how this polarity is achieved during differentiation. Here we show that the organ of Corti is formed from a thicker and shorter postmitotic primordium through unidirectional extension, characteristic of cellular intercalation known as convergent extension. Mutations in the PCP pathway interfere with this extension, resulting a shorter and wider cochlea as well as misorientation of stereocilia. Furthermore, parallel to the homologous pathway in Drosophila melanogaster, a mammalian PCP component Dishevelled2 shows PCP-dependent polarized subcellular localization across the organ of Corti. Taken together, these data suggest that there is a conserved molecular mechanism for PCP pathways in invertebrates and vertebrates and indicate that the mammalian PCP pathway might directly couple cellular intercalations to PCP establishment in the cochlea.

The transcription factor six1 inhibits neuronal and promotes hair cell fate in the developing zebrafish (Danio rerio) inner ear.

The developmental processes leading to the differentiation of mechanosensory hair cells and statoacoustic ganglion neurons from the early otic epithelium remain unclear. Possible candidates include members of the Pax–Six–Eya–Dach (paired box–sine oculis homeobox–eyes absent–dachshund) gene regulatory network. We cloned zebrafish six1 and studied its function in inner ear development. Gain- and loss-of-function experiments show that six1 has opposing roles in hair cell and neuronal lineages. It promotes hair cell fate and, conversely, inhibits neuronal fate by differentially affecting cell proliferation and cell death in these lineages. By independently targeting hair cells with atoh1a (atonal homolog 1a) knockdown or neurons with neurog1 (neurogenin 1) knockdown, we showed that the remaining cell population, neurons or hair cells, respectively, is still affected by gain or loss of six1 function. six1 interacts with other members of the Pax–Six–Eya–Dach regulatory network, in particular dacha and dachb in the hair cell but not neuronal lineage. Unlike in mouse, six1 does not appear to be dependent on eya1, although it seems to be important for the regulation of eya1 and pax2b expression in the ventral otic epithelium. Furthermore, six1 expression appears to be regulated by pax2b and also by foxi1 (forkhead box I1) as expected for an early inducer of the otic placode. Our results are the first to demonstrate a dual role for a member of the Pax–Six–Eya–Dach regulatory network in inner ear development.

Balancing cell numbers during organogenesis: Six1a differentially affects neurons and sensory hair cells in the inner ear

While genes involved in the differentiation of the mechanosensory hair cells and the neurons innervating them have been identified, genes involved in balancing their relative numbers remain unknown. Six1a plays a dual role by promoting hair cell fate while inhibiting neuronal fate in these two lineages. Genes homologous to six1a act as either transcriptional activators or repressors, depending on the partners with which they interact. By assaying the in vivo and in vitro effects of mutations in presumptive protein-protein interacting and DNA-binding domains of Six1a, we show that, in the developing zebrafish inner ear, Six1a promotes hair cell fate by acting as a transcriptional activator and inhibits neuronal fate by acting as a transcriptional repressor. We also identify several potential partners for Six1a that differ between these two lineages. The dual role of Six1a in the developing otocyst provides a mechanism for balancing the relative number of hair cells and neurons during organogenesis of the inner ear.

Ablation Studies on the Developing Inner Ear Reveal a Propensity for Mirror Duplications

The inner ear develops from a simple ectodermal thickening known as the otic placode. Classic embryological manipulations rotating the prospective placode tissue found that the anteroposterior axis was determined before the dorsoventral axis. A small percentage of such rotations also resulted in the formation of mirror duplicated ears, or enantiomorphs. We demonstrate a different embryological manipulation in the frog Xenopus: the physical removal or ablation of either the anterior or posterior half of the placode, which results in an even higher percentage of mirror image ears. Removal of the posterior half results in mirror anterior duplications, whereas removal of the anterior half results in mirror posterior duplications. In contrast, complete extirpation results in more variable phenotypes but never mirror duplications. By the time the otocyst separates from the surface ectoderm, complete extirpation results in no regeneration. To test for a dosage response, differing amounts of the placode or otocyst were ablated. Removal of one third of the placode resulted in normal ears, whereas two‐thirds ablations resulted in abnormal ears, including mirror duplications. Recent studies in zebrafish have demonstrated a role for the hedgehog (Hh) signaling pathway in anteroposterior patterning of the developing ear. We have used overexpression of Hedgehog interacting protein (Hip) to block Hh signaling and find that this strategy resulted in mirror duplications of anterior structures, consistent with the results in zebrafish. Developmental Dynamics 236:1237–1248, 2007.

Developmental Variation, Homology, and the Pharyngula Stage

Understanding how development varies both inter- and intraspecifically can be important for systematic and evolutionary studies. This review will explore three different ways such understanding can be applied to evolutionary analyses. First, developmental data can be useful for homology determination. Interspecific variation in development has been thought to make developmental data poor candidates for determining homology. However, an updated developmental criterion that is more broadly comparative and mechanistic augments the available criteria used in homology determination. Second, modern cell and molecular biology are providing a better understanding of the many developmental processes involved in a structures formation and will augment the number of characters available for phylogenetic analyses. Recent work has revealed that what had been thought to be a highly conserved developmental stage, the pharyngula (the phylotypic and zootypic stage of craniates) is highly variable. This variation can be seen in the development of such tissues as neural crest and placodes. These tissues are particularly interesting from a phylogenetic standpoint because they and the structures they form contribute to key synapomorphies of craniates. Finally, understanding developmental processes and how they form the variety of morphologies seen in nature will help in constructing the transformations that occurred during evolution. One such example involves descriptions of how lateral line development is affected in different mutant lines of zebrafish. The many species of teleost fishes express great variation in the patterns of their lateral lines, and this is often an important systematic character. Understanding the genetic basis of lateral line development would help not only in hypothesizing possible transformational series but also in determining how many genes may have been required for these transformations.

Use of Confocal Microscopy in Comparative Studies of Vertebrate Morphology

Laser scanning confocal microscopy provides a means to acquire and analyze images of complex morphological structures and to help place molecules or cells of interest in their proper morphological context. Confocal microscopy is a form of fluorescence microscopy that sharpens the images collected by visualizing the light from only one plane of focus. This allows for the collection of multiple focal planes in what is called a z-stack, which provides three-dimensional data. Five steps that any investigator using a confocal microscope should follow are described: (1) labeling and (2) mounting of specimens for viewing, (3) optimizing the image on the confocal, and (4) collecting and (5) analyzing of confocal image data. We describe three specific protocols incorporating these steps from our work on vertebrate inner ear development. The first two describe a collection of z-stacks in living, fluorescently labeled, and intact embryos. The second protocol is for time-lapse imaging of multiple focal planes at each time point. The third protocol describes confocal imaging of preserved material double labeled with antibodies and by retrograde labeling of neurons via axonal uptake. Finally, three alternative or complementary approaches to standard confocal microscopy are described and discussed.