Academic journal article Genetics

Genetic Screen in Drosophila Melanogaster Uncovers a Novel Set of Genes Required for Embryonic Epithelial Repair

Academic journal article Genetics

Genetic Screen in Drosophila Melanogaster Uncovers a Novel Set of Genes Required for Embryonic Epithelial Repair

Article excerpt

ABSTRACT

The wound healing response is an essential mechanism to maintain the integrity of epithelia and protect all organisms from the surrounding milieu. In the "purse-string" mechanism of wound closure, an injured epithelial sheet cinches its hole closed via an intercellular contractile actomyosin cable. This process is conserved across species and utilized by both embryonic as well as adult tissues, but remains poorly understood at the cellular level. In an effort to identify new players involved in purse-string wound closure we developed a wounding strategy suitable for screening large numbers of Drosophila embryos. Using this methodology, we observe wound healing defects in Jun-related antigen (encoding DJUN) and scab (encoding Drosophila αPS3 integrin) mutants and performed a forward genetics screen on the basis of insertional mutagenesis by transposons that led to the identification of 30 lethal insertional mutants with defects in embryonic epithelia repair. One of the mutants identified is an insertion in the karst locus, which encodes Drosophila β^sub Heavy^-spectrin. We show β^sub Heavy^-spectrin (β^sub H^) localization to the wound edges where it presumably exerts an essential function to bring the wound to normal closure.

WOUND healing is essential to organisms throughout the animal kingdom. It must occur for restoring tissue integrity after injury both during embryonic and adult life. Epithelia, in particular, act as a physical barrier protecting living organisms and their organs from the surrounding environment and have evolved robust mechanisms to ensure their integrity. Simple embryonic epithelial tissues have an extraordinary capacity to reseal small discontinuities very rapidly and efficiently through an epithelial resealing mechanism. This was initially described in the chick embryo (Martin and Lewis 1992), but seems to be conserved across species as it was shown to also occur in mouse, frog, and fly embryos (McCluskey et al. 1993; Davidson et al. 2002; Wood et al. 2002). In all these systems, small epithelial wounds close via the cooperation of three distinct mechanisms: the assembly of an actomyosin purse string in the epithelial cells at the wound margin, the protrusive activity of epithelial cells at the margin, and the contraction and ingression of deep cells when those are exposed (see Jacinto et al. 2001;Martin and Parkhurst 2004; Garcia-Fernandez et al. 2009 for review).

Advances in live imaging of Drosophila embryos expressing fluorescent proteins made time-lapse microscopy of the epithelial healing process possible and the exact sequence of cell movements to be determined (Wood et al. 2002). The cells at the wound margin constrict their apical edges through the action of an actomyosin cable that assembles just minutes after wounding and is linked intercellularly through adherens junctions. Concomitant with the formation of the purse string, cells at the wound margin begin to extend actin-rich protrusions. When opposing wound margins come into close proximity, filopodia and lamellipodia from opposing flanks make contact and they appear to pull the wound margins toward one another. For laserinduced oval wounds of ~10 by 20 µm, the entire healing process can be completed in just over 2 hours (Wood et al. 2002).

The signaling cascades that regulate the epithelial resealing process are just beginning to be unraveled, but the known molecular mechanisms appear to be conserved in both vertebrates and invertebrates, namely the involvement of Grainy-head (GRH) transcription factors or the JNK signaling cascade, transduced by JUN/FOS transcriptional complexes (Ramet et al. 2002; Li et al. 2003; Ting et al. 2003, 2005a,b; Galko and Krasnow 2004;Mace et al. 2005). In the fly, the expression of some genes at the wound site is dependent on functional GRH and JUN/FOS dimers (AP1) binding sites in their promoter region (Mace et al. 2005; Pearson et al. 2009). These observations are consistent with abnormal wound healing in grh or basket/DJNK mutants' larval cuticle and the activation of JNK signaling pathway at wild-type larval wound sites (Galko and Krasnow 2004; Mace et al. …

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