Delta1 expression during avian hair cell regeneration

Jennifer S. Stone ) 0 1 Edwin W. Rubel 0 1 0 Key words: Notch, Delta, Hair cell , Differentiation, Regeneration, Chick 1 Virginia Merrill Bloedel Hearing Research Center, Department of Otolaryngology and Head and Neck Surgery, University of Washington , Seattle, WA 98195-7923 , USA Portions of this study were reported at the 1998 Association for Research in Otolaryngology Midwinter Conference Postembryonic production of hair cells, the highly specialized receptors for hearing, balance and motion detection, occurs in a precisely controlled manner in select species, including avians. Notch1, Delta1 and Serrate1 mediate cell specification in several tissues and species. We examined expression of the chicken homologs of these genes in the normal and drug-damaged chick inner ear to determine if signaling through this pathway changes during hair cell regeneration. In untreated post-hatch chicks, Delta1 mRNA is abundant in a subpopulation of cells in the utricle, which undergoes continual postembryonic hair cell production, but it is absent from all cells in the basilar papilla, which is mitotically quiescent. By 3 days after drug-induced hair cell injury, Delta1 expression is highly upregulated in areas of cell proliferation in both the utricle and basilar papilla. Delta1 mRNA levels are elevated in progenitor cells during DNA synthesis and/or gap 2 phases of the cell cycle and expression is maintained in both daughter cells SUMMARY The otocyst, the precursor to the specialized epithelia of the inner ear, arises from a thickened invagination of the ectoderm. In birds, 8 distinct patches of sensory epithelium differentiate within the otocyst. Three cristae (located in the ampullae of the semicircular canals) and four macculae (the utricle, saccule, lagena and maccula neglecta) serve vestibular functions. The elongated basilar papilla is auditory in function. A subset of cells that form within these patches hair cells serves to transduce the energy of motion and sound into interpretable neural signals. The other primary cell type support cells provides mechanical and physiological support to hair cells and the epithelium. Hair cells and support cells are organized in a precise array of alternating cell types; numerous support cells surround each hair cell and, as a result, hair cells do not contact each other. Mammals are born with their full compliment of hair cells, but permanent sensory deficits ensue after injury to mature inner ear epithelia, because production of new hair cells does not occur. In contrast, in the vestibular epithelia of postembryonic birds, hair cells are removed from the epithelium via apoptosis immediately after mitosis. Delta1 expression remains upregulated in cells that differentiate into hair cells and is downregulated in cells that do not acquire the hair cell fate. Delta1 mRNA levels return to normal by 10 days after hair cell injury. Serrate1 is expressed in both hair cells and support cells in the utricle and basilar papilla, and its expression does not change during the course of druginduced hair cell regeneration. In contrast, Notch1 expression, which is limited to support cells in the quiescent epithelium, is increased in post-M-phase cell pairs during hair cell regeneration. This study provides initial evidence that Delta-Notch signaling may be involved in maintaining the correct cell types and patterns during postembryonic replacement of sensory epithelial cells in the chick inner ear. and replaced by mitotic regeneration (Jorgensen and Mathiessen, 1988; Roberson et al., 1992; Weisleder and Rubel, 1992; Kil et al., 1997). Further, after hair cells are experimentally damaged in the vestibular epithelia of post-hatch chicks, basal levels of cell proliferation are increased and new hair cells and support cells are produced (reviewed in Oesterle and Rubel, 1996). There is no ongoing mitotic activity or cell production in the mature avian basilar papilla (Oesterle and Rubel, 1993). However, regeneration of new hair cells and support cells is stimulated there following experimental damage (reviewed in Rubel, 1992; Cotanche et al., 1994). During both spontaneous and damage-induced hair cell regeneration in the inner ear, the cellular organization of the sensory epithelium is precisely maintained. Very little is know about the molecules that direct the formation of the correct numbers, types and patterns of cells during hair cell regeneration. During development, several molecules, such as bone morphogenic proteins, retinoic acid-associated proteins and various transcription factors are expressed differentially across the axis of the otocyst, suggesting they play a role is specifying different regions within the inner ear (reviewed in Corey and Breakefield, 1994; Fekete, 1996; Whitfield et al., 1997; Fritzsch et al., 1998). It has been proposed that the formation of the precise array of hair cells and support cells is regulated by lateral inhibition, as emerging hair cells prevent the surrounding uncommitted cells from differentiating into hair cells (Lewis, 1991; Goodyear et al., 1995), and that rearrangement of cells after their differentiation works to perfect the pattern (Goodyear and Richardson, 1997). The initial process lateral inhibition (Wigglesworth, 1940) is mediated in several biological systems by the lin-12/Notch family of extracellular receptors (reviewed in Lewis, 1996; Kimble and Simpson, 1997; Weinmaster, 1997). These receptors are activated by members of the Delta-Serrate-Lag2 family of ligands. The ultimate effects of ligand-mediated activation of Notch are to inhibit expression of proneural genes that activate neural differentiation (reviewed in Lee, 1997), and of Delta (Haenlin et al., 1994; Hinz et al., 1994; Kunisch et al., 1994; Chitnis and Kintner, 1996; Heitzler et al., 1996; Ma et al., 1996; de la Pompa et al., 1997). Lateral inhibition through Notch signaling drives cell fate decisions during development in a wide variety of tissues across many species, ranging from the central nervous systems of mammals (Bao and Cepko, 1997; de la Pompa et al., 1997), chicks (Austin et al., 1995; Henrique et al., 1997a) and frogs (Coffman et al., 1993; Chitnis et al., 1995; Dorsky et al., 1995, 1997) to feather development in chicks (Crowe et al., 1998). A similar mechanism has been proposed to occur in the otocyst (Lewis, 1991), throughout which patches of precursor cells with the potential to form hair cells and support cells are scattered (Knowlton, 1967). Notch and the genes for its ligands, Delta and Serrate, are expressed in the developing otocyst of chicks (Myat et al., 1996; Adam et al., 1998), mammals (Lindsell et al., 1996) and zebrafish (Dornseifer et al., 1997; Appel and Eisen, 1998; Haddon et al., 1998a), and Notch signaling appears to be necessary for normal development of hair cells in the inner ear (Haddon et al., 1998b). Notch signaling is also important for cell specification in some adult vertebrate tiss (...truncated)