Phosphorylation of Grainy Head by ERK Is Essential for Wound-Dependent Regeneration but Not for Development of an Epidermal Barrier

Phosphorylation of Grainy head by ERK is essential for wound-dependent regeneration but not for development of an epidermal barrier

Myungjin Kima and William McGinnisa,1

aSection of Cell and Developmental Biology, University of California at San Diego, La Jolla, CA 92093-0349

Edited* by Michael Levine, University of California, Berkeley, CA, and approved December 9, 2010 (received for review November 2, 2010) Grainy head (GRH) is a key transcription factor responsible for that cross-link the specialized apical extracellular matrix mole- epidermal barrier formation and repair, whose function is highly cules that make up the cuticle (4, 6). GRH also is required for conserved across diverse animal species. However, it is not known normal expression levels of Fasciclin 3 and Coracle proteins, how GRH function is reactivated to repair differentiated epidermal which are directly involved in mediating epidermal cell adhesion barriers after wounding. Here, we show that GRH is directly re- (4, 6, 14). A mutation of human Grhl2 results in progressive gulated by extracellular signal-regulated kinase (ERK) phosphory- hereditary deafness, which is presumably due to defective epi- lation, which is required for wound-dependent expression of GRH thelia in the cochlea, where Grhl2 is abundantly expressed (15). target genes in epidermal cells. Serine 91 is the principal residue in Drosophila GRH is a transcription factor that can bind to DNA GRH that is phosphorylated by ERK. Although mutations of the regulatory elements of Ddc and Ultrabithorax (Ubx) (16) and is ERK phosphorylation sites in GRH do not impair its DNA binding encoded by the grainy head (grh) gene, which was originally de- function, the ERK sites in GRH are required to activate Dopa decar- ﬁned by mutations that result in weak larval cuticle (6). The GRH boxylase (Ddc) and misshapen (msn) epidermal wound enhancers protein has a transactivation domain in the N terminus and DNA as well as functional regeneration of an epidermal barrier upon binding and dimerization domains in the C terminus of the pro- wounding. This result indicates that the phosphorylation sites are ﬁ

tein (17, 18) (Fig. 1A). High-af nity DNA binding of GRH BIOLOGY essential for damaged epidermal barrier repair. However, GRH requires homodimerization and induces activation or repression with mutant ERK phosphorylation sites can still promote barrier of GRH-dependent target gene transcription (17, 19, 20). How- DEVELOPMENTAL formation during embryonic epidermal development, suggesting ever, we still have very limited information on how the tran- that ERK sites are dispensable for the GRH function in establishing scriptional activity of GRH is regulated in cells and organisms. epidermal barrier integrity. These results provide mechanistic in- During Drosophila development, alternative splicing of grh sight into how tissue repair can be initiated by posttranslational transcripts generates two major protein isoforms, GRH-O and modification of a key transcription factor that normally mediates GRH-N (21). GRH-O is expressed in regions of the central the developmental generation of that tissue. nervous system (CNS), whereas GRH-N is expressed in barrier epithelia of the epidermis, foregut, hindgut, and tracheal system. embryo | Drosophila | cuticle Zygotic grh mutants die at the embryonic/larval transition with weak epidermal cuticle and discontinuous grainy sclerites in the nimals produce a protective epidermal barrier against head skeleton due to, in part, a lack of Ddc gene activation, a key Aphysical, chemical, and thermal damage, as well as dehy- enzyme involved in cuticular sclerotization (6). Clones of grh dration and pathogen infection. When the barrier is damaged, it mutant cells in the adult epidermis have defects in pigmentation is essential for animals to repair and regenerate the wounded and planar polarity (22). In addition, the mutation of grh leads to barrier structure to survive in a hostile environment. Biological a tortuous morphology of tracheal tubes (23). These abnormal- surface barrier function is conferred by epidermal cells, which in ities suggest that GRH plays diverse roles in establishing and mammals and arthropods produce the stratum corneum and maintaining epithelial barriers in normal development. cuticle, respectively. The stratum corneum consists of layers of We have shown that grh mutant embryos have abnormal repair a highly cross-linked matrix of dead keratinocytes, proteins, and of the cuticle barrier after epidermal wounding. The barrier re- lipids (1). In contrast, the cuticle barrier of Drosophila and other pair defects presumably results from a reduction of transcription insects consists of cross-linked lipids, proteins, and chitin (2). from a variety of GRH-target genes, one of which is Ddc (4). The In both Drosophila and mouse, transcription factors of the MAP kinase ERK is strongly activated in epidermal cells sur- Grainy head (GRH) family have been shown to be essential for rounding wounded sites, and ERK inhibition silences GRH- the development of epithelial barriers as well as the repair of mediated Ddc expression around epidermal wounds (4). From barriers after wounding (3–8). There is also evidence from this result, we inferred that ERK is in the signaling pathway from studies in Caenorhabditis elegans, Xenopus laevis, and Danio rerio injury to activation of GRH-dependent wound enhancers. It was indicating that GRH proteins have an evolutionarily conserved shown that GRH activity is controlled by the Breathless Re- role in the development and maintenance of epidermal barrier ceptor Tyrosine Kinase in the embryonic trachea (23) and that structure (9–11). All three murine homologs of GRH, Grainy GRH can be phosphorylated by ERK in vitro (20). However, head-like (Grhl) 1–3, are highly expressed in developing and there has been a lack of evidence supporting a direct relationship differentiated mouse epidermis (12). Although the mutation of murine Grhl1 results in mild defects in epidermal development and differentiation (13), mutations in Grhl3 gene results in se- Author contributions: M.K. and W.M. designed research; M.K. performed research; M.K. vere epidermal defects including inadequate skin barrier and contributed new reagents/analytic tools; M.K. and W.M. analyzed data; and M.K. and deficient wound repair, ultimately causing lethality at birth. This W.M. wrote the paper. phenotype is due in part to reduced expression of epidermal The authors declare no conflict of interest. protein cross-linking enzymes and cell adhesion proteins (3, 7). *This Direct Submission article had a prearranged editor. Analogous to the role of Grhl1 and Grhl3 proteins in mouse, 1To whom correspondence should be addressed. E-mail: [email protected]. GRH protein in Drosophila activates genes such as Dopa This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. decarboxylase (Ddc). Ddc is required to produce the quinones 1073/pnas.1016386108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1016386108 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 A S88PDS91P TAD DBD DD SST

WT 1063 AA 2A 1063 AAA AAA AAAAAAAAAAA Fig. 1. ERK phosphorylation sites in GRH protein. (A) Dia- PanA 1063 grams of wild-type and deletion mutant GRH proteins: N1 603 TAD, transcriptional activation domain; DBD, DNA binding N2 136 domain; DD, dimerization domain. Numbers indicate the N3 74 C-terminal residue of each protein. (B) Puriﬁed ERK was incubated with no substrates (lane C), fusion proteins con- CMN1WTN2 N3 BPCMN1WTN2 N3 BP B (kDa) C 10 taining wild-type (WT) GRH, deletion mutants GRH-N1, GRH-N2, and GRH-N3, or a positive control substrate (My- 190 8 γ 32 120 elin Basic Protein, MBP) in the presence of [ - P] ATP. Molecular size markers (kilodaltons) are indicated. Left 85 6 60 shows Coomassie blue-stained gel, and red dotted circles 50 4 indicate individual recombinant GRH proteins or the positive control substrate. Right shows 32P-autoradiography, 40 2 and blue dotted circles indicate the positions of the GST- GRH and MBP proteins. (C) Relative phosphorylation of GRH

Relative Phosphorylation Relative 0 25 by ERK quantiﬁed by densitometry. The bar graph indicates WT N1 N2 N3 MBP 20 Protein 32P the mean of three independent experiments, and error bars indicate SDs. (D) GRH S91A mutant displays a strong re-

A A A duction in the in vitro phosphorylation by ERK compared D 8 1 E n T 8 9 T A a with GRH WT. (E) GRH Pan Ala (PanA) mutation abolishes C W S S C W 2 P ERK-dependent phosphorylation compared with GRH WT 32P-GRH 32P-GRH and GRH S88A/S91A (2A) mutant. The data presented in this ﬁgure are representative of three independent experi- 32 GRH GRH ments. In D and E, Upper ( P-GRH) show autoradiographs and Lower (GRH) show Coomassie blue-stained gels.

between ERK and GRH in late embryonic epidermal tissue and lation of the GRH 2A mutant suggested the existence of addi- the physiological importance of GRH phosphorylation in epi- tional minor phosphorylation sites. Therefore, we replaced all S dermal barrier generation and regeneration. To explore these or T residues in putative ERK phosphorylation sites (S-P or T- relationships, we first mapped ERK phosphorylation sites in the P), with A (GRH PanA mutant; Fig. 1A). The GRH PanA GRH-N protein. The identified sites were mutated to generate mutant protein is not detectably phosphorylated by ERK in vitro versions of GRH-N that had little or no phosphorylation by (Fig. 1E and Fig. S1B). Taken together, these data indicate that ERK. These wild-type and mutant forms of GRH-N were ex- ERK efficiently phosphorylates GRH on S-91, and phosphor- pressed in the epidermis of transgenic embryos that had no en- ylates other S-P and T-P sites such as S-88 with lower efficiency. dogenous GRH. To our surprise, we found that ERK phosphorylation sites in GRH-N are dispensable for developmental Mutations of ERK Phosphorylation Sites in GRH Do Not Impair Its DNA establishment of epidermally derived cuticular barriers and ex- Binding Function in Vitro. S-91 in GRH is located in a domain that pression of GRH-regulated epidermal cell adhesion proteins. inhibited in vitro DNA-binding affinity of bacterially overex- However, ERK sites are critical for GRH-dependent activation pressed GRH (18). Because ERK is necessary for wound- of epidermal barrier repair genes, which lead to regeneration of dependent GRH target gene expression, we hypothesized that an impermeable cuticle after wounding. ERK phosphorylation might influence the affinity of GRH to target DNA sequences such as those in the Ddc wound response Results enhancer. To test this hypothesis, we performed electrophoretic Identification of ERK Phosphorylation Sites in GRH. To determine mobility shift assays (EMSA) by using wild-type (GRH WT), which residues of GRH transcription factor are specifically nonphosphorylatable (GRH 2A and GRH PanA), and phos- phosphorylated by ERK, we carried out in vitro ERK kinase phomimetic (GRH S88E/S91E; hereafter, GRH 2E) GRH assays by using purified ERK enzyme and deletion mutants of proteins. GRH WT, 2A, PanA, or 2E variant proteins all bound GRH (Fig. 1A). The deletion mutant GRH-N3 is very weakly to Ddc enhancer sequences with indistinguishable affinities and phosphorylated by ERK compared with mutants N1 and N2, or specificities (Fig. S2). These results suggest that mutations of full-length (WT) GRH, which indicates that the major ERK ERK phosphorylation sites in GRH do not impair its DNA- phosphorylation sites are located between amino acids 74 and binding properties. However, we cannot completely exclude the 136 (Fig. 1 B and C). This region contains three potential ERK possibility that subtle quantitative difference in DNA-binding phosphorylation sites, which consist of serine (S) or threonine affinity between WT and mutant GRH proteins may influence its (T) followed by proline residues. These sites are S-88, S-91, and activity as a transcription factor. T-125. Interestingly, recent proteomic analysis has shown that phosphorylation of S-88 and S-91 in GRH can be detected in Substitution of Endogenous GRH with Transgenic GRH Mutants in Drosophila embryonic cell extracts (24). We substituted these Embryonic Epidermis. GRH is abundantly expressed in late em- potential ERK phosphorylation sites in GRH with alanines (A), bryonic epidermal cells (Fig. 2A), where it regulates genes that individually or in combination (Fig. 1A), and tested the in vitro produce a normal protective cuticular barrier (6), as well as genes phosphorylation of these GRH mutants by ERK. When com- that provide normal cell adhesion in the epidermis (14). To ex- pared with GRH WT, the S88A mutant shows a slight decrease amine the contribution of ERK-mediated GRH phosphorylation in ERK phosphorylation, whereas the S91A mutant shows to developmental epidermal structure formation, we generated a strong decrease (Fig. 1D and Fig. S1A). The GRH S88A/S91A flies with UAS-GRH WT, 2A, PanA, and 2E transgenes inserted double mutant (hereafter, GRH 2A) is phosphorylated by ERK in an identical genomic site using the phiC31-integrase system. to ≈20% of WT (Fig. 1E and Fig. S1B). The weak phosphory- We tested a variety of GAL4 protein expression driver lines for

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1016386108 Kim and McGinnis Downloaded by guest on September 28, 2021 grhIM +/ grhIM: e22c hrg IM: e22c>GRHWT zygotes (Fig. 2 A and C–G). These results suggest that grh-null ABC embryos with e22c-driven GRH expression could be used to test the importance of GRH phosphorylation in its two roles, ﬁrst in epidermal development, and second in epidermal wound repair. GRH

25µm GRH Phosphorylation Is Not Required for the Expression of GRH A’ B’ C’ Target Cell Adhesion Molecules During Epidermal Development. To understand a role of GRH phosphorylation in late embryonic

Fas3 epidermal cells, we monitored the expression levels of embryonic GRH targets, Fasciclin 3 (Fas3) or Coracle (14). The levels of Fas3 (Fig. 2 B′ and G) and Coracle (Fig. S3B′ and Fig. S5A) were ﬁ IM IM 2A IM PanA IM 2E signi cantly reduced in epidermal cells of grh homozygotes. grh : e22c>GRH grh : e22c>GRH grh : e22c>GRH IM DEF However, grh homozygotes did not have reduced levels of DE- cadherin, which we used as a control for antibody penetration of late-stage embryos that have begun cuticle synthesis (Fig. S4 and GRH Fig. S5B). The reduced expression level of Fas3 or Coracle by grh null mutation was rescued by expression of GRH WT (Fig. 2C′ D’ E’ F’ and Fig. S3C′), ERK site mutants of GRH (Fig. 2 D′ and E′ and Fig. S3 D′ and E′), or phosphomimetic GRH (Fig. 2F′ and Fig. S3F′)ingrhIM mutant background, at comparable level to those Fas3 of grhIM/+ heterozygote controls (Fig. 2G and Fig. S5A). These data show that ERK phosphorylation sites in GRH are not re- G quired for the developmental expression of Fas3 and Coracle in late embryonic epidermis. 1.4 GRH Fas3 1.2 * 1 GRH Phosphorylation Is Not Necessary for Epidermal Barrier Integrity.

0.8 BIOLOGY 0.6 The larval cuticle that develops in grh mutants is weak and easily 0.4 ruptured, and the head skeleton is grainy and discontinuous (6), DEVELOPMENTAL 0.2 Relative Intensity 0 (Fig. 3B). To investigate the role of ERK phosphorylation of ABCDEF GRH in head skeleton structure and cuticle barrier formation, we analyzed the cuticular phenotypes of grh-null embryos with IM Fig. 2. Fas3 expression in grh mutants expressing various GRH forms in e22c-driven expression of GRH WT, GRH 2A, GRH PanA, and late embryonic epidermal cells. All images are dorsolateral views of stage 16 GRH 2E proteins. The severe cuticular defects in body cuticle embryos with genotypes indicated at the top of each pair of images. The fi embryos were simultaneously stained with anti-GRH (red, A–F) and anti- and head skeletal structures of grh-null embryos were signi - Fasciclin 3 (Fas3) antibodies (green, A’–F’). All images were taken with cantly, albeit not completely, rescued by e22c-driven GRH WT identical settings from experiments done in parallel on the same day. (Scale (Fig. 3C). To our surprise, we found that the embryos that bars: 25 μm.) (G) Quantification of GRH (filled bars) and Fas3 (open bars) expressed the ERK site mutants of GRH (GRH 2A and GRH expression levels shown in the stage 16 embryonic epidermal cell with fol- PanA), or the phosphomimetic GRH 2E, under e22c control, lowing labels: A, grhIM/+; B, grhIM, e22c-GAL4; C, grhIM, e22c > GRH WT; D, grhIM, e22c > GRH 2A; E, grhIM, e22c > GRH PanA; and F, grhIM, e22c > GRH 2E. Asterisk denotes statistical significance (P = 0.0037). There are no sig- IM nificant differences in GRH and Fas3 expression levels between grh het- grhIM/+ grhIM: e22c grhIM: e22c>GRHWT IM B erozygote and grh homozygote expressing GRH WT, ERK site mutant GRH A C (GRH 2A or GRH PanA), or phosphomimetic GRH (GRH 2E). The bar graph indicates the mean of values from more than three embryos, and error bars indicate SEs. 100µm D grhIM: e22c>GRH2A E grhIM: e22c>GRHPanA F grhIM: e22c>GRH2E their ability to produce GRH transgenic proteins at physiological levels in embryonic epidermis. The GAL4 driver lines under the control of the act5C, da,andarm enhancers strongly induced GRH ≈ expression in late embryonic epidermal cells ( 10-fold higher than grhIM/+ grhIM: e22c grhIM: e22c>GRHWT endogenous levels) and caused embryonic or larval lethality. How- G H I ever, the epidermal e22c-GAL4 driver in combination with GRH

transgenic proteins in a WT genetic background allowed survival to 50µm

adulthood, suggesting that the transgenic GRH expression by the grhIM: e22c>GRH2A grhIM: e22c>GRHPanA grhIM: e22c>GRH2E e22c-GAL4 driver could be used for gene rescue studies for GRH J K L epidermal function. We first tested whether UAS-GRH protein expression levels using the e22c-GAL4 driver were similar to the levels of endogenous GRH in late embryonic stage by immunostaining with Fig. 3. Expression of GRH transgenes rescues head skeleton morphology affinity-purified GRH-specific antibodies. GRH protein is and epidermal integrity in grh-null embryos. Cuticle preparations showing detected in epidermal nuclei of grhIM/+ heterozygotes, but not in head skeleton structure (A–F) and results of epidermal permeability assay – – μ grhIM homozygotes (Fig. 2 A and B). At late embryonic stages, (G L) in stage 17 embryos with indicated genotypes. (Scale bars: A F, 100 m; G–L,50μm.) All of the GRH protein variants largely rescued the head skeletal grh mutants with e22c-GAL4-driven expression of GRH WT, defects (B) and permeability defects (H) characteristic of grhIM homozygous GRH 2A, GRH PanA, and GRH 2E proteins showed GRH mutants. The rescued animals at the embryo/larval transition displayed protein expression specifically in epidermal cells, at levels that nearly normal denticle belts and cuticles that were resistant to the charac- IM/+ were indistinguishable to endogenous GRH in grh hetero- teristic stretching and bursting of grhIM mutant cuticles.

Kim and McGinnis PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 also rescued the head skeleton and body cuticle defects of grh grhIM/+ grhIM: e22c grhIM: e22c>GRHWT mutants (Fig. 3 D–F) to an extent comparable with GRH WT. ABC These results indicate that ERK phosphorylation of GRH is not crucial for the formation of cuticle structure during late em-

bryogenesis. .47-GFP

We next wanted to determine whether ERK phosphorylation Ddc 50µm of GRH is needed for establishment of epidermal barrier function. To test this possibility, we developed a whole-mount cuticle permeability assay based on a previous protocol (25). In this grhIM: e22c>GRH2A grhIM: e22c>GRHPanA grhIM: e22c>GRH2E assay system, the vitelline membrane is permeabilized by an or- D E F ganic solvent, and the epidermal/cuticular barrier integrity can be assayed by exclusion of Rhodamine B dye from the body cavity. grhIM/+ heterozygotes at stage 17 can exclude the dye, but .47-GFP IM grh homozygotes cannot (Fig. 3 G and H and Fig. S6), con- Ddc sistent with the idea that grh mutant embryos have permeable epidermal barriers, presumably due to incomplete cuticle for- 100% mation and/or cuticle breaks. Interestingly, the epidermal barrier G moderate integrity defect exhibited in the grh mutant embryos was rescued 80% strong

by e22c-driven expression of GRH 2A, GRH PanA, or GRH 2E 60% (Fig. 3 J–L), to an extent comparable with the rescue provided by -GFP) e22c-driven GRH WT (Fig. 3I and Fig. S6). These data support 40%

our conclusion that the developmental function of GRH in ( Ddc .47 20% epidermal development and cuticle barrier formation does not Wound Response 0% A require ERK-dependent phosphorylation. T n c : : A : a : E 2 IM W IM 2 IM P IM 2 2 h H h H h H h H e R R R R /+ : gr gr gr gr M M G G G G I I > > > > ERK Phosphorylation of GRH Is Required for Epidermal Wound h h c c c c 2 2 2 2 gr gr 2 2 2 2 Enhancer Activation. GRH target genes such as Ddc and mis- e e e e shapen (msn) are rapidly transcribed around epidermal wounds where ERK is activated (4, 26). Because ERK is also activated in Fig. 4. GRH phosphorylation is required for wound-dependent activation of the Ddc 0.47-GFP reporter. The expression pattern of the reporter (A–F)sur- cells surrounding wound sites (4), we hypothesized that phos- rounding epidermal wounds is shown in stage 17 embryos with indicated phorylation of GRH by ERK might be required for activation of genotypes. Arrows indicate sites of injury. Dotted lines indicate the embryonic epidermal wound response genes. To explore this possibility, we boundaries. (G)Quantification of wound responsiveness is represented in first tested an epidermal wound enhancer from the Ddc gene that a bar graph. Wound responses were classified as strong (black, GFP signal >6 IM was fused to a GFP reporter gene (4). In grh homozygous cells distal from the wound site) or moderate (gray, 3–6 cells distal from the mutants, the Ddc-GFP wound enhancer is only weakly activated wound site). The number of analyzed embryos was as follows: grhIM/+ (n = 103); in a few embryos around epidermal wounds (Fig. 4 B and G), grhIM, e22c-GAL4 (n =164);grhIM, e22c > GRH WT (n =112);grhIM, e22c > GRH IM IM whereas 73% of the grh mutants with e22c-driven GRH WT 2A (n = 140); grh , e22c > GRH PanA (n = 120); and grh , e22c > GRH 2E μ displayed moderate-to-strong activation of the enhancer around (n = 138). (Scale bars: 50 m.) epidermal wound sites (Fig. 4 C and G). In contrast, the wound- dependent activation of the Ddc wound enhancer was dramati- ERK Phosphorylation of GRH Is Critical for Functional Epidermal cally reduced in grh mutants with the e22c-driven GRH 2A Barrier Restoration After Epidermal Injury. To explore roles of protein (Fig. 4D). GRH PanA expression in the background of GRH phosphorylation in repair of damaged epidermal barrier, grh null mutant was indistinguishable from the Ddc reporter we performed an epidermal barrier dye permeability assay to activation seen in grh mutants alone (Fig. 4 E and G). These assess the functionality of the restored cuticle barrier after results suggest that ERK phosphorylation of S-88 and S-91 res- wounding at late embryonic stages. In the assay, whereas grhIM/+ idues in GRH is crucial, but that other ERK phosphorylation heterozygous controls showed efficient regeneration of barrier sites contribute to the activity of GRH on the Ddc epidermal ′ IM wound enhancer after wounding. The phosphomimetic form integrity after wounding (Fig. 5 A and A ), as expected, grh GRH 2E (Fig. 4F) is comparable with GRH WT in its ability to homozygous embryos were highly permeable to dye with or without wounding (Fig. 5 B and B′). The e22c-driven GRH WT rescue Ddc wound enhancer activation after wounding in a grh ′ ′ mutant background (Fig. 4G). However, GRH 2E does not ac- (Fig. 5 C and C ), as well as GRH 2E (Fig. 5 F and F ), dra- tivate Ddc-GFP in unwounded embryos, suggesting that ERK matically rescued the ability of grh-null epidermal cells to re- fi generate an impermeable barrier after wounding, at the similar phosphorylation of GRH alone is not suf cient for wound- IM/+ induced gene activation, and that other wound-induced signaling level to grh heterozygotes (Fig. 5G). However, intriguingly, the grh-null embryos expressing GRH 2A (Fig. 5 D and D′)or input is needed for the wound-responsive gene activation. IM We have shown that GRH induces expression of another GRH PanA (Fig. 5 E and E′), which were able to rescue grh wound responsive gene msn (26). To monitor GRH activity in defects in developmental barrier generation (Fig. 3 G–L), still wounded epidermal cells, we used a Msn in vivo wound response showed very significant defects in epidermal barrier regeneration reporter after epidermal wounding. The msn wound enhancer is after wounding (Fig. 5G). These data suggest that GRH phos- strongly activated around epidermal wounds in grhIM/+ hetero- phorylation by ERK is important for the functional regeneration zygotes, but not in grhIM homozygous mutant embryos (26), (Fig. of a damaged epidermal barrier after wounding. S7 A, B, and F). Although e22c-driven GRH WT can moderately rescue activation of the msn wound response enhancer in epi- Discussion dermal cells of grhIM mutants, GRH 2A and GRH PanA showed We have discovered that posttranslational modification of GRH no such rescue function (Fig. S7 C–F). In summary, our results by ERK phosphorylation is an important molecular signaling suggest that ERK phosphorylation sites are required for GRH to event activating its function so it can regenerate impermeable activate downstream genes that mediate repair after epidermal epidermal barriers after wounding. Our evidence indicates that wounds in late embryos. ERK phosphorylation of GRH is not critical for its role in the

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1016386108 Kim and McGinnis Downloaded by guest on September 28, 2021 grhIM/+ grhIM : e22c grhIM: e22c>GRHWT developmental transcriptional activation function of GRH was AB C not significantly altered by the mutation of ERK phosphorylation sites (Fig. S8). However, ERK phosphorylation of GRH-binding affinity might enhance its binding to a coactivator or prevent its fi 50 m binding to a corepressor (29) that is speci c during wound response. Because GRH 2E expression did not constitutively ac- A’ B’ C’ tivate transcription of GRH target genes in both developmental and wound response contexts, the phosphorylation of GRH alone is not likely to be sufficient for triggering activation of the wound response genes. Given that both GRH and FOS-D are required for the induction of Ddc and msn (26), and that FOS grhIM: e22c>GRH2A grhIM: e22c>GRHPanA grhIM: e22c>GRH2E function can be also activated by ERK phosphorylation (30), we D E F believe that wound-induced signaling input through FOS or other transcription factors is also necessary for the transcription of these wound response genes along with the phosphorylation of GRH. Therefore, the ERK-dependent phosphorylation of both GRH and FOS upon injury may activate both transcription D’ E’ F’ factors to synergistically induce many target genes that facilitate epidermal barrier regeneration. Although GRH function in Drosophila embryonic epidermis is critical for both developmental generation and wound-triggered regeneration of epidermal barriers, it was not known whether it G 100% is required for the process of reepithelialization (epidermal

80% p = 0.0004 wound closure) after wounding (31). Mammalian Grhl3 has been 60% p = 0.008 shown to be required for the keratinocyte wound closure in tissue

40% culture, mainly through its activation of RhoGEF19 (8). How- BIOLOGY 20% ever, in Drosophila, we found that the wound closure phenotypes

IM DEVELOPMENTAL Barrier Permeability Barrier Permeability After Wound Repair 0% between wild-type and grh homozygous embryos were in- ABCDEF distinguishable (Fig. S9), indicating that Drosophila GRH is not critical for reepithelialization after wounding. Fig. 5. ERK phosphorylation sites in GRH are required for functional epi- Given that we have demonstrated the importance of the ERK- dermal barrier regeneration after wounding. Fluorescence images show GRH axis in transcriptional activation of epidermal wound re- results of epidermal barrier permeability assays performed after wounding and wound repair in stage 17 embryos. Genotypes are indicated at the top sponse genes, the signals and receptors upstream of ERK are of of each pair of images. Magnified images of wounded areas (A–F) and great interest. Mammalian cell culture studies suggest that re- whole embryo images (A’–F’) are shown. Dotted lines indicate the embryonic ceptor tyrosine kinases (RTK) are responsible for the wound- boundaries. (G) Quantification of epidermal barrier integrity phenotypes dependent activation of ERK (32). In Drosophila, stitcher (stit) after wound repair. Bar graph indicates percentile of permeable embryos in encodes a Ret-family tyrosine kinase that contributes to trans- IM/+ indicated groups at stage 17 after epidermal wound repair: A (grh ; n = mission of an epidermal wound signal, because null mutations in IM IM > IM 95); B (grh , e22c-GAL4; n = 99); C (grh , e22c GRH WT; n =67);D (grh , stit result in a partial inhibition of wound-induced ERK phos- e22c > GRH 2A; n = 90); E (grhIM, e22c > GRH PanA; n = 114); and F (grhIM, e22c > GRH 2E; n = 68). P values indicating the significance of the barrier phorylation, and reduced activation of wound enhancers in em- repair defects are indicated in Fig. 5G. bryonic epidermal cells (33). It is very probable that another

developmental establishment of epidermal cell adhesion and cuticular barriers in late embryogenesis. Interestingly, putative Epidermal Injury ERK phosphorylation sites are also found in the N-terminal ? domain of Grhl3, a mammalian homolog of GRH. Given that Stit grhl3 mutant mice display defects in both developmental skin Phosphorylation by ERK ERK Phosphorylation sites barrier formation and wound-induced repair (3), and that ERK is NOT required are REQUIRED P P is required for mammalian wound repair (27), it is plausible that P PP P ERK phosphorylation of GRH might be an evolutionarily con- GRH GRH served event in animal epidermal wound repair. Drosophila GRH and mammalian Grhl proteins do not share extended blocks of amino acid sequence homology that include S-P or T-P motifs that are characteristic of ERK consensus sites (18), but Ddc msn functional phosphorylation sites can show rapid sequence drift Developmental Generation during evolution (28). of Epidermal Barrier Mutations of ERK phosphorylation site residues in GRH do Wound-dependent not detectably influence its affinity to DNA binding sites in vitro, Regeneration of Epidermal Barrier but the phosphorylation sites are required for GRH functional activity on epidermal wound enhancers in late embryonic epidermal cells. The wound-specific activity of GRH does not ap- Fig. 6. A model showing GRH regulation by phosphorylation in un- pear to involve phosphorylation-dependent nuclear localization, wounded and wounded late embryonic epidermal cells. In normal developmental conditions, GRH activates genes required for formation of the because the GRH protein is constitutively nuclear, either when cuticle and epidermal integrity in a manner that is independent of ERK expressed by endogenous promoters or epidermal GAL4 drivers phosphorylation. However, ERK phosphorylation of GRH is crucial for its (21). In addition, as measured by the strength of overexpressed activation of epidermal wound response genes and restoration of epidermal ERK-site mutants of GRH in activating Ddc expression, the barrier function.

Kim and McGinnis PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 RTK(s) are responsible for the activation of ERK after epider- riers (Fig. 6). This model may also apply to c-Jun in mammals, mal wounding observed in stit null mutant embryos. The PVR which does not require JNK-dependent phosphorylation sites for RTK is a good candidate because its function has been shown to developmental eyelid and neural tube closure, but does require be required for wound healing in larval epidermal tissue (34). those sites for closure of epidermal wounds (36, 37). Thus, some The RTK-mediated activation of ERK-GRH axis appears to regeneration processes in tissues or organs of diverse animal mediate other biological roles in addition to its role in embryonic species after injury may be initiated through a similar molecular epidermal barrier repair. GRH has been reported to mediate mechanism—posttranslational reactivation of essential tran- Torso RTK-dependent repression of the tailless gene in early scription factors that are normally involved in developmental embryogenesis (20). Therefore, although the ERK-GRH axis is morphogenesis. dispensable for late embryonic epidermal barrier development, it appears to function in other developmental contexts. These dis- Materials and Methods tinct functions presumably depend on the context of different Details on plasmid constructs and biochemical analyses, Drosophila genetics transcriptional enhancers with different transcription factor codes. and cuticular preparation, generation of GRH antisera, epidermal wound- More importantly, the current findings suggest an important ing, epidermal barrier permeability assay, and epidermal wound closure mechanism that may underlie injury-induced tissue regeneration. assay are described in SI Materials and Methods. Immunostaining was done After wounding or amputation, developmental programming as described (4). Images of wounded embryos were obtained with a Leica must be reinitiated to recover the original structure of the SP2 laser-scanning upright confocal microscope, choosing representative damaged tissue (35). In the context of the epidermis, GRH is embryos as described (26). a key transcription factor for generating a normal epidermal barrier during development in a manner independent of ERK ACKNOWLEDGMENTS. We thank J. C. Pearson for fly lines, constructs, and experimental advice; members of the W.M. laboratory for helpful sugges- phosphorylation. However, GRH is also persistently expressed in tions; S. Jeong for sharing EMSA protocol; R. Aroian, E. Bier, and M. Karin for terminally differentiated epidermal cells of the embryo, larvae sharing reagents and equipment; B. Moussian, C. Samakovlis and A. Jacinto (21), and adult, and its function in barrier repair must be rapidly for personal communications; the Bloomington Stock Center, T. H. Millard, and robustly reactivated after wounding. In our model, a semi- and H. J. Bellen for fly lines; and the Developmental Studies Hybridoma Bank for antibodies. We also are grateful to J. H. Lee for valuable comments. This dormant state of GRH can be overcome by ERK phosphoryla- work was supported by National Institutes of Health Grant R01GM077197 tion to regain its ability to transcriptionally activate target genes (to W.M.) and Korea Research Foundation Grant KRF-2008-357-C00126 like Ddc, msn, and many others that regenerate epidermal bar- (to M.K.).

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