Caltech Biological Imaging Center

Teaching at the Woods Hole Marine Biological Laboratory

Embryology

Zebrafish Development and Genetics

Sensorineural hearing loss (SNHL) is one of the more common birth defects and approximately 20% of these patients have inner ear malformations that are readily visible using radiological examination. What causes these inner ear malformations and why they should lead to SNHL, as well as balance disorders, is often unknown. Our research on frog inner ear development is addressing this question.

Most patients with congenital SNHL do not have such obvious inner ear malformations but may have more subtle defects such as loss or abnormal differentiation of specific inner ear cell types such as the mechanosensory hair cells. The developmental processes leading to the differentiation of mechanosensory hair cells and statoacoustic ganglion (SAG) neurons (which wire the hair cells into the central nervous system) from the early otic epithelium remain unclear. Members of the Pax-Six-Eya-Dach gene regulatory network, involved in the development of numerous organs and tissues in both the fly Drosophila and vertebrates have been proposed to play an important role in inner ear development. Loss of Eya1 function in mice results in major ear, craniofacial and kidney defects while mutations in human EYA1 result in the phenotypically similar branchio-oto-renal (BOR) or branchio-otic (BO) syndromes, the latter lacking kidney abnormalities. Interestingly, at least 3 mutations in human SIX1 can also result in BOR/BO syndrome.

Mirror duplications of the developing inner ear

The main goal of the frog research is to determine which molecules and regions of the otic placode are required for the normal patterning of the developing inner ear with the hope of understanding and treating human inner ear malformations. We discovered that ablating half of the placode or otocyst along the anterior-posterior (A-P) axis, in the frog Xenopus, results in a high percentage of mirror image duplicated inner ears. The regenerated mirror half is derived from the remaining placode and not surrounding tissues. In zebrafish, the only gene mutations that result in mirror duplicated inner ears are those belonging to the Hedgehog (Hh) signaling pathway. In contrast, mice use Hh and another secreted signaling molecule, Wnt (an amalgam of wingless and int-1, the first mammalian Wnt identified), for dorsal-ventral (D-V) patterning. Blocking Hh signaling in Xenopus, as seen in zebrafish, results in 2 mirror image anterior halves and suggests that Hh signaling is necessary for posterior patterning. The ability to generate mirror duplicated inner ears provides an assay for studying the molecules and regions of the developing inner ear that are required for normal patterning. These studies are being done in collaboration with Dr. Caryl Forristall of the University of Redlands.

The transcription factor six1 inhibits neuronal and promotes hair cell fate in the developing inner ear

The main goal of the zebrafish research is to understand at the molecular level how placodal cells choose between neuronal, nonsensory or sensory cell fates. To address this, we manipulate gene expression in zebrafish embryos, and monitor these cell types. Recently, genes belonging to the Pax-Six-Eya-Dach regulatory network, well characterized in fly eye development, have been implicated in the development of the inner ear and its ganglion. We have found that the zebrafish homeodomain containing transcription factor six1 (homologous to fly sine oculis) differentially affects hair cell versus neuronal fate in developing ears and is the first gene identified to do so. Six1 gene function is knocked down using antisense morpholino oligonucleotides or increased by injecting mRNA at the single cell stage. Six1 loss of function results in fewer hair cells and more statoacoustic ganglion (SAG) neurons while six1 gain of function results in more hair cells and fewer neurons. The affects of six1 on hair cells and neurons is already apparent when these cells first differentiate. This and the fact that six1 is not downstream of the early neurogenic gene neurogenin1 suggest an early role in the development of these two inner ear cell lineages.

Time-lapse Quicktime movie of figure 5 of Developmental Biology publication.

Two dorsal and ventral regions of the otic placode of stage 23-25 embryos were labeled simultaneously with two vital dye solutions: DiD (green) and DiI-CM(red). A z-series stack of images (10 planes of focus) were collected every 5 minutes over a 10 hour period at two different fluorescent wavelengths, cy3 and cy5 (Chroma filters), sequentially.

Within the first 200 minutes, green cells move ventrally and co-localize with the red cells that are splitting into two groups. This group of cells remains together for 1-2 hours and disappears. In the more dorsal region, green and red cells co-localized and stay together for almost 4 hours. They flip around clockwise just before they separate and then become co-localized again.
Anterior is to the left, posterior to the right; dorsal is up and ventral is down.

Andres Collazo, PhD, Section Chief

Olivier Bricaud, PhD, Post-doctoral Scientist

Aldo Castillo, Research Assistant

Aicha Castillo, Confocal Technician

Sung-Hee Kil, Ph.D., Post-doctoral Scientist

Erik Waldman, Research Fellow

Kalpana Desai, Research Assistant

Caryl Forristall, Ph. D., Professor
University of Redlands

Shuo Lin, Ph. D., Professor
University of California at Los Angeles

Gerry Weinmaster, Ph. D., Professor
University of California at Los Angeles