Research Projects

Our laboratory uses genetics and molecular biology to study pattern formation in the zebrafish embryo. The rapid development and simple anatomy of this teleost embryo, together with recently developed techniques for reverse genetics and a nearly complete genome sequence, make zebrafish a powerful molecular genetic system for studying the mechanisms of development. We are interested in how gene functions translate into cell behaviours and the formation of tissues and organs. We focus on two main areas:

1) Neural crest cell fate specification and formation of the craniofacial skeleton

2) Cell interactions and formation of the anterior-posterior axis of the nervous system

More recently we have begun to take an evolutionary approach using cichlid fish from Africa to map genes underlying natural craniofacial variation in the wild.


1) Craniofacial Development and Genetics

How do bones acquire their distinct sizes and shapes during development? Most bones of the skull are derived from cranial neural crest (NC) cells in the embryo, which migrate into the pharyngeal arches to form the facial skeleton. Defects in NC cells cause some of the most common birth defects in humans such as cleft palate, jaw and tooth defects and certain kinds of deafness. These cells have to find the appropriate segment (arch 1 versus 2) and acquire the proper dorsal-ventral (D-V) identity (lower versus upper jaw) within each arch. Over the years our work has focused on characterizing zebrafish mutants with craniofacial defects, which has led to the identification of novel molecular mechanisms underlying both the segmentation and D-V development of the facial skeleton. Our recent work has shown how Endothelin-1 (Edn1), Bone morphogenetic protein (Bmp) and Wnt signaling are integrated to determine craniofacial patterning (Alexander et al., 2011; Zuniga et al., 2011; Alexander et al., 2014).

Bmp and Edn1 Signaling in Dorsal-Ventral Patterning of the Craniofacial Skeleton

Bmp and Edn1 Signaling in Dorsal-Ventral Patterning of the Craniofacial Skeleton

Planar Cell Polarity and Skeletal Development

Stacks of chondrocytes are the basic building blocks of endochondral bones. We recently found that a mutant in REREa disrupts both the stacking and polarity of craniofacial cartilage. It appears to do so by interfering with planar cell polarity (PCP) signaling through the atypical cadherins Fat3 and Dachsous2 (Dchs2) (Le Pabic et al., 2014). This is the first evidence implicating Fat/Dchs signaling in skeletal cell polarity in vertebrates.


Fat3 and Dchs2 Signaling in Planer Cell Polarity of Craniofacial Cartilage

2) Cell Migration and Fate Specification

Neural Crest (NC) Cell Adhesion During Migration

In addition to craniofacial cartilage and bone, NC cells also form peripheral neurons and glia, all of the body’s pigment cells, and many other derivatives. An over-arching goal of the lab is to understand how such migratory cells acquire their fates. Are they specified prior to migration or totally reliant on their migratory environments? Understanding this could help in the diagnosis and treatment of diseases that disrupt NC (e.g. cleft palate, neurofibromatosis, melanoma), including strategies for production of NC-like stem cells.

How are migratory cell behaviors controlled by cell adhesion? NC cells rapidly alter distributions of adhesion molecules called cadherins at their surfaces during migration. Cadherins of the adherens junctions that hold epithelial cell together are downregulated in order for NC cells to undergo an epithelial-to-mesenchymal transition (EMT). Secreted Wnts induce some of these changes in NC adhesion and also promote specification of NC-derived pigment cells. We showed that the transcription factor Ovo1, which is a direct Wnt target, controls migration of pigment precursors by regulating the intracellular movements of N-cadherin (Ncad). Ovo1 represses expression of several Rab GTPases known to modulate cellular localization of cadherins, and many Rab GTPases are strongly expressed in NC (Piloto and Schilling, 2010).

Regulation of N-cadherin (Ncad) trafficking in NC cells by Ovo1

Regulation of N-cadherin (Ncad) trafficking in NC cells by Wnt signaling and Ovo1

More recently we found that Rabconnectin 3a (Rbc3a), which regulates the vATPase responsible for endocytic vesicle acidification, is required for Wnt signaling, Ncad trafficking, NC cell EMT and pigment cell development. Rbc3a controls migration of pigment precursors by regulating the intracellular movements of the Wnt receptor, Fz7. Rbc3a regulates the v0a1 subunit of vATPase, and knockdown of v0a1 disrupts NC development similar to Ovo1 and Rbc3a (Tuttle et al., 2014).


Rbc3a Controls Endocytic Trafficking Pathways Critical for Wnt Signaling in NC Migration


ECM Assembly and Muscle-Tendon Interactions at Myotendinous Junctions

Migrating cells also interact with ECM as they differentiate, a dramatic example of which occurs in myotendinous junctions (MTJs) that link muscles to bones. Cranial neural crest cells form the tendons of the head and these cells are thought to specify sites of muscle attachments to form a functional musculoskeletal system. We recently showed that a major component of the tendon ECM called Thrombospondin-4b (Tsp4b) maintains MTJ integrity in zebrafish and acts as a scaffold for other ECM components (Subramanian and Schilling, 2014). Overexpression of Tsp4, either fish or human, rescues muscle detachment and strengthens normal tendons, suggesting that it may be a potential therapeutic for tendon injuries. We are currently investigating the functions of the tendon determinant, Scleraxis, and several ECM proteins in the early development and maintenance of MTJs in zebrafish.


Thrombospondins Controls ECM Assembly Required for Tendon Maintenance at Myotendinous Junctions


Anterior-Posterior Patterning and Retinoic Acid Signaling

Another major area of research in the lab for many years focuses on the establishment of signaling gradients and their roles in cell fate decisions. The vitamin A derivative, retinoic acid (RA) is one such signal that is thought to act as a classic morphogen – specifying fates in a concentration-dependent manner – to pattern segments called rhombomeres along the anterior-posterior axis of the hindbrain. Our earlier work focused on the role of RA in determining segment identities and neuronal cell fates, while our more recent work focuses on RA degradation and the dynamics and robustness of the RA gradient itself.


Systems Biology: Spatial Dynamics in Retinoid Signaling

(White et al., 2007; White and Schilling, 2008; Cai et al., 2012; Zhang et al., 2012)

An integrated signaling network shapes the RA gradient to pattern the hindbrain

An integrated signaling network shapes the RA gradient to pattern the hindbrain


Zebrafish Models for Complex Birth Defects

Cornelia de Lange Syndrome (CdLS) is a multi-organ system birth defect syndrome caused by a defect in cohesin, which mediates sister chromatid cohesion. Most cases of CdLS are due to haploinsufficiency for Nipped-B-like (Nipbl), which facilitates cohesin loading. There is growing evidence that cohesin and Nipbl are also involved in transcriptional regulation. We developed a model of nipbl-deficiency in zebrafish, and showed that morpholino knockdown produces a spectrum of specific heart and gut/visceral organ defects with similarities to those in CdLS. Analysis of nipbl morphants further revealed that, as early as gastrulation, expression of genes involved in endodermal differentiation (sox32, sox17, foxa2, and gata5) and left-right patterning (spaw, lefty2, and dnah9) is altered. Experimental manipulation of the levels of several such genes—using RNA injection or morpholino knockdown—implicated both additive and synergistic interactions in causing observed developmental defects. These findings support the view that birth defects in CdLS arise from collective effects of quantitative changes in gene expression. Interestingly, both the phenotypes and gene expression changes in nipbl morphants differed from those in mutants or morphants for genes encoding cohesin subunits, suggesting that the transcriptional functions of Nipbl cannot be ascribed simply to its role in cohesin loading.

(Muto et al., 2011)

Multiple roles for Nipbl in embryonic heart and gut development

Multiple roles for Nipbl in embryonic heart and gut development

Zebrafish as a Model for Studying Lens Physiology and Cataract Formation

Cataracts are the leading cause of blindness, due to the lens becoming opaque. One critical process for maintaining lens transparency is water permeability through channels consisting of Aquaporin-0 (Aqp0) proteins, but how Aqp0 is regulated and whether or not it has other functions remain unclear. In collaboration with Jim Hall’s lab in Physiology and Biophysics at UCI we recently found that zebrafish have two Aqp0s, only one of which acts as a water channel in permeability assays (Froger et al., 2010; Clemens et al., 2013). Interestingly, loss of function of either one causes cataracts in larval fish, and we are currently investigating other possible functions for Aqp0 (e.g. adhesion) that might be necessary for lens cell transparency.

An Antibody Library Against Zebrafish Proteins

The zebrafish field has relied for far too long on in situ hybridization to detect mRNA, and very few antibodies that recognize mammalian proteins cross-react with their fish relatives. Our lab has recently begun a collaboration with a local company, GeneTex Inc., located in Irvine, with the goal of generating a large library of new antibodies and validating them by staining of zebrafish tissue.