The RNA-Binding Protein LIN28B Regulates Developmental Timing in the Mammalian Cochlea

The RNA-binding protein LIN28B regulates developmental timing in the mammalian cochlea

Erin J. Goldena,b, Ana Benito-Gonzaleza,b, and Angelika Doetzlhofera,b,1

aSolomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and bCenter for Sensory Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205

Edited by Matthew W. Kelley, National Institutes of Health, Bethesda, MD, and accepted by the Editorial Board June 5, 2015 (received for review January 19, 2015) Proper tissue development requires strict coordination of pro- Lin28 genes encode for evolutionarily highly conserved RNA liferation, growth, and differentiation. Strict coordination is binding proteins (9) known to regulate larval developmental particularly important for the auditory sensory epithelium, where timing (heterochrony) in Caenorhabditis elegans (10). In humans deviations from the normal spatial and temporal pattern of and mice, Lin28a and its homolog Lin28b are critical regulators auditory progenitor cell (prosensory cell) proliferation and differ- of stemness, organismal growth, metabolism, tumorigenesis, and entiation result in abnormal cellular organization and, thus, tissue repair (11). LIN28A and LIN28B proteins promote a stem auditory dysfunction. The molecular mechanisms involved in the cell/progenitor-like state through two distinct mechanisms. First, timing and coordination of auditory prosensory proliferation and LIN28 proteins bind to and stabilize mRNAs encoding for cell differentiation are poorly understood. Here we identify the RNA- cycle regulators and growth stimulating genes, leading to in- binding protein LIN28B as a critical regulator of developmental creases in their protein abundance (12–15). Second, LIN28 timing in the murine cochlea. We show that Lin28b and its opposing proteins block let-7 microRNA (miRNA) biogenesis (16–19). let-7 miRNAs are differentially expressed in the auditory sensory Mature miRNAs are small noncoding RNAs that interact with lineage, with Lin28b being highly expressed in undifferentiated their targets by partial base pairing with complementary se- prosensory cells and let-7 miRNAs being highly expressed in their quences commonly found within the 3′ untranslated region progeny—hair cells (HCs) and supporting cells (SCs). Using recently (3′ UTR) of the target mRNA. In the majority of cases, miRNA developed transgenic mouse models for LIN28B and let-7g,we binding inhibits translation and/or destabilizes the target mRNA demonstrate that prolonged LIN28B expression delays prosensory (20). Similar to lin-28, let-7 was initially identified in C. elegans as cell cycle withdrawal and differentiation, resulting in HC and SC a heterochronic gene (10, 21). Let-7 miRNAs inhibit stem cell/ patterning and maturation defects. Surprisingly, let-7g overexpres- progenitor cell proliferation and promote differentiation by sion, although capable of inducing premature prosensory cell cycle targeting cell cycle and growth-associated genes (22–24). The exit, failed to induce premature HC differentiation, suggesting that Lin28 genes possess multiple let-7 binding sites in their 3′ UTR LIN28B’s functional role in the timing of differentiation uses let-7 and are subject to negative regulation by let-7 miRNAs, estab- independent mechanisms. Finally, we demonstrate that overexpres- lishing a double negative feedback loop (19). There is emerging sion of LIN28B or let-7g can significantly alter the postnatal pro- evidence for a critical role of the Lin28/let-7 axis in controlling duction of HCs in response to Notch inhibition; LIN28B has a self-renewal, lineage commitment, and differentiation during positive effect on HC production, whereas let-7 antagonizes this pro- neurogenesis (25). For instance, experiments in retinal explants cess. Together, these results implicate a key role for the LIN28B/let-7 provide compelling evidence that Lin28b and its opposing axis in regulating postnatal SC plasticity. miRNAs, let-7, mir-9, and mir-125, regulate the developmental

Lin28b | Let-7 | hair cell | cochlea | regeneration Significance

he auditory sensory epithelium, housed in the inner ear cochlea, The stereotyped cellular organization found within the Tis critical for our ability to perceive sound. This bilayered struc- mammalian auditory epithelium is key to its proper function. ture is composed of mechano-sensory hair cells (HCs), which lie Differentiation of this structure occurs under strict spatial atop a layer of glial-like supporting cells (SCs). Stereotyped organi- and temporal regulation to ensure that proper patterning is zation of these cells is essential for proper functioning of the mature achieved. Unlike other neuronal structures (e.g. the retina and cochlea. HCs and SCs arise from a common pool of progenitor cells cortex), where terminal mitosis and differentiation are linked, (prosensory cells), which in mammals withdraw from the cell cycle in these processes remain distinctly separated within the de- a highly synchronized apical-to-basal wave (1) that is closely followed veloping auditory epithelium. How coordination is achieved by an inverse basal-to-apical wave of differentiation (2). This unique remains largely unknown. Here we show that the RNA-binding spatial and temporal pattern of cell cycle withdrawal and differen- protein LIN28B times auditory prosensory cell cycle withdrawal tiation holds postmitotic prosensory cells in an undifferentiated state and differentiation through both let-7–dependent and let-7– for varying lengths of time, depending on their basal-to-apical loca- independent mechanisms. Additionally, we show that manipu- tion, and is thought to ensure the proper patterning of HCs and SCs. lation of the LIN28B/let-7 axis alters the capacity for postnatal Over the past several years, key regulators of prosensory cell pro- production of sensory hair cells (HC) in the absence of Notch liferation and differentiation have been identified (3, 4). P27/Kip1 signaling, revealing this axis as a potential candidate for future (CDKN1B), a cyclin-dependent kinase inhibitor, controls prosensory HC regeneration therapies. cell cycle withdrawal (5), whereas ATOH1, a basic helix-loop-helix Author contributions: E.J.G. and A.D. designed research; E.J.G., A.B.-G., and A.D. performed transcriptional activator, controls HC and SC differentiation (6, 7). research; E.J.G., A.B.-G., and A.D. analyzed data; and E.J.G. and A.D. wrote the paper. Atoh1 and p27/Kip1 loss-of-function studies indicate that prosensory The authors declare no conflict of interest. cell cycle exit and differentiation occur independently from each This article is a PNAS Direct Submission. M.W.K. is a guest editor invited by the Editorial other (5, 8); however, the molecular mechanisms coordinating the Board. timing of these processes remain unknown. 1To whom correspondence should be addressed. Email: [email protected]. Using microarray-based transcriptional profiling, we recently This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. identified Lin28b to be highly expressed in prosensory cells. 1073/pnas.1501077112/-/DCSupplemental.

E3864–E3873 | PNAS | Published online July 2, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1501077112 Downloaded by guest on September 28, 2021 timing of retinal neurogenesis (26). Here, using recently developed PNAS PLUS iLIN28B and ilet-7g transgenic mouse lines (27), we show that Lin28b functions as a developmental timer in the murine cochlea through both let-7–dependent and let-7–independent mechanisms. Furthermore, we demonstrate that reexpression of LIN28B enhances the ability of early postnatal SCs to switch cell fate and transdifferentiate into HCs in response to Notch inhibition and that let-7 overexpression antagonizes this process. Together, these results suggest an important role for the LIN28B/let-7 axis in regulating SC plasticity. Results Lin28b and let-7 miRNAs Are Differentially Expressed in the Auditory Sensory Lineage. To characterize the spatial and temporal expression pattern of the Lin28b/let-7 axis during cochlear differentiation, a series of RNA in situ hybridization (ISH) and quantitative PCR (qPCR) experiments were performed. In addition to Lin28b, we characterized the expression of the high mo- bility group transcription factor Hmga2, which has been shown to promote organismal growth and stemness in other systems (28, 29). Similar to Lin28b, both human and murine Hmga2 harbor let-7 binding sites in their 3′ UTRs and are negatively regulated by the let-7 miRNAs (23, 24, 30, 31). In the mammalian cochlea, prosensory cell differentiation follows a steep basal-to-apical gradient, whereby midbasally located HCs differentiate before more apically located HCs. Our analysis revealed that both Lin28b and

Hmga2 transcripts are expressed in prosensory cells but are rapidly BIOLOGY

down-regulated in a basal-to-apical fashion upon the onset of HC DEVELOPMENTAL differentiation (Fig. 1 A–I′). At embryonic day (E)13.0, Lin28b and Hmga2 were coexpressed with Sox2 in prosensory cells (Fig. 1 A–C) (32). At E14.5, following the onset of Atoh1 expression in nascent HCs, Lin28b and Hmga2 transcript expression was reduced in the differentiating cochlear base and mid turn (Fig. 1 D–F). At the peak of HC differentiation (E16.5), Lin28b and Hmga2 expression was only maintained in the most apical segment of the cochlear duct, which had not yet differentiated (Fig. 1 G–I′). Quantification by qPCR and Western blot revealed that the more than fourfold reduction in Lin28b transcript levels between E13.5 and E16.5 (Fig. 1J) was accompanied by a nearly fivefold decline in LIN28B protein levels (Fig. 1 K and L). This dramatic decline in LIN28B levels in the differentiating cochlea strongly correlated with a rise in let-7 miRNA expression Fig. 1. Members of the Lin28b/let-7 axis are differentially expressed in the (Fig. 1 M–S and Fig. S1). The murine let-7 family is comprised developing cochlea. (A–I′) ISH-based analysis of Lin28b and Hmga2 expres- of nine mature let-7 miRNA species (let-7a, let-7b, let-7c, let-7d, sion before (A–C) and during cochlear differentiation (D–I′). Sox2 (A) marks let-7e, let-7f, let-7g, let-7i, and mir-98) (33). Our qPCR analysis prosensory cells, and Atoh1 (D, G, and G′) marks HCs. Brackets (A–C) indicate revealed that eight of nine mature let-7 miRNA species were the prosensory domain. Arrows (E and H) indicate Lin28b expression within the developing spiral ganglion. High power images of the apical turn of G–I expressed at detectable levels in the developing cochlea and with ′– ′ the exception of let-7i, their expression steadily increased with are shown in G I .(J) RT-qPCR analysis of relative Lin28b, Hmga2,andAtoh1 mRNA expression within the cochlear epithelium before (E13.5), during the advancement of cochlear differentiation and maturation (E16.5), and following (P2) differentiation. Rpl19 was used as an endogenous (Fig. 1S). We also analyzed the expression of mir-125b, a ver- reference gene. Data are mean ± SEM. (K and L) LIN28B protein quantifi- tebrate homolog of the C. elegans heterochronic gene lin-4 (34), cation within the cochlea epithelium at stages acutely surrounding the onset known to negatively regulate Lin28a/b expression (35). Our of HC differentiation. (M–R) ISH-based analysis of let-7c and let-7f expression qPCR analysis revealed that similar to mature let-7 miRNAs, within the early postnatal (P3) cochlea (M–O) and vestibular crista (P–R). expression of mature mir-125b increased with the advancement of Mir-183 (M and P) marks cochlear and vestibular HCs. Arrowheads and lines cochlear differentiation (Fig. 1S). ISH-based analysis using locked (M–O) indicate cochlear IHCs and OHCs. (Scale bars: A–I′,100μm; M–R,50μm.) nucleic acid (LNA) probes revealed that in the terminally dif- (S) RT-qPCR analysis of mature let-7 miRNA expression within the cochlear epi- ferentiated cochlea postnatal day (P)3 mature let-7f and let-7c thelium before (E13), during (E18), and following (P2) differentiation. The snoRNA U6 was used as an endogenous control. Data are mean ± SEM. miRNAs were expressed in cochlear and vestibular HCs and cells of the cochlear and vestibular ganglion. In contrast to mir-183, whose expression was confined to HCs and cells of the cochlear and vestibular ganglion (36, 37), let-7f and let-7c were also being highly expressed in undifferentiated prosensory cells, and expressed in SCs and nonsensory epithelial cells of the greater mature let-7 miRNA species being highly expressed in terminally epithelial ridge (Fig. 1 M–R). In the differentiating cochlea differentiated HCs and SCs. (E16.0), let-7c and let-7f were expressed in nascent HCs, and in LIN28B the case of let-7c, in surrounding SCs, closely following the basal- Overexpression Delays Prosensory Cell Differentiation. Based to-apical wave of HC differentiation (Fig. S1). In summary, our on the opposing expression pattern of Lin28b and let-7 miRNAs expression analysis revealed reciprocal expression of Lin28b and in the differentiating murine cochlea, and the known hetero- mature let-7 miRNAs in the developing cochlea, with Lin28b chronic function of this axis in C. elegans larval development

Golden et al. PNAS | Published online July 2, 2015 | E3865 Downloaded by guest on September 28, 2021 (10), we hypothesized that Lin28b might be functioning as an intrinsic regulator of developmental timing in the mammalian cochlea. To address the function of the Lin28b/let-7 axis in the murine cochlea, we made use of the iLIN28B transgenic mouse line (27). In this mouse line, a flag-tagged human LIN28B transgene is under the control of a tetracycline-responsive promoter element (TRE). When combined with a ubiquitous (Rosa26)or inner ear-specific (Pax2) reverse tetracycline transactivator (rtTA) transgene, doxycycline (dox) administration (beginning at E8.5) resulted in robust LIN28B overexpression within the developing cochlear duct (Fig. S2 A–F). In addition, because of LIN28B’s inhibitory function on let-7 miRNA biogenesis, LIN28B overexpression resulted in more than an 80% reduction in mature let-7 miRNA expression within the developing cochleae of iLIN28B transgenic embryos (Fig. S2G). To determine whether LIN28B overexpression might inhibit or delay prosensory cell differentiation, dox was continuously administered starting at E8.5, and HC-specific Atoh1/nEGFP reporter expression was used to monitor HC differentiation in E13.0 Rosa26-iLIN28B and control cochlear explants over 3 d (Fig. 2 A–J). HC differentiation follows a steep basal-to-apical gradient and a less steep medial-to-lateral gradient, causing basally located HCs to differentiate before more apically located HCs and medial IHCs to differentiate before more lateral OHCs + (2, 8). After 16 h in vitro (HIV), a stripe of GFP IHCs was visible in the cochlear base of control explants (Fig. 2 B and J). In contrast, LIN28B overexpressing cochlear explants remained Fig. 2. LIN28B overexpression delays auditory HC differentiation. (A)Ex- undifferentiated after 16 HIV (Fig. 2 C and J). Sixteen hours perimental design: iLIN28B overexpression was induced beginning at E8.5 later (32 HIV), IHCs in LIN28B overexpressing cochlear cultures and Rosa26-iLIN28B (iLIN28B tg/+; Rosa26 M2-rtTA tg/+) and nontransgenic became apparent (Fig. 2 E and J) and over the next 32 h, HC littermate control (Rosa26 M2-rtTA tg/+) cochlear epithelia were cultured differentiation progressed at a similar rate in control and LIN28B beginning at E13.0. In vitro HC differentiation was monitored over the following 3 d by using the Atoh1/nEGFP reporter. (B–I) Atoh1/nEGFP reporter overexpressing cultures (Fig. 2J). Consistent with the observed expression (EGFP, green) marks nascent HCs. Asterisks indicate EGFP ex- + delay in HC differentiation in vitro, both IHC and OHC differ- pression within HCs of the vestibular sacculus. (J) Length of the EGFP HC entiation was less advanced in acutely isolated E15.5 Rosa26- stripe was used to quantify the extent of HC differentiation in control versus iLIN28B cochlear tissue compared with control (Fig. 2S). Rosa26-iLIN28B cochlear cultures. Data expressed as mean ± SEM (n = 5 To rule out the possibility that the observed delay in cochlear animals per group, *P < 0.05). (K and L) Overlays of low-power fluorescent HC differentiation was due to a more general developmental images of acutely isolated E15.5 control (Pax2-Cre tg/+; rtTA tg/+) and Pax2- delay, an inner ear-specific LIN28B overexpression approach iLIN28B (Pax2-Cre tg/+; rtTA tg/+; iLIN28B tg/+) cochlear ducts stained for the was used (Fig. S2 B, C, E, and F). Dox was continuously ad- hair cell specific protein MYO6 (white). Red arrowheads indicate the IHC domain, and yellow arrowheads indicate the OHC domain. (M–R) Cross- ministered starting at E8.5, and the basal-to-apical extent of HC sections through the basal, mid, and apical turns of control and Pax2-iLIN28B differentiation was analyzed in acutely isolated E15.5 Pax2- cochleae. MYO6 staining (red) marks HCs, and p27/Kip1 (green) staining iLIN28B and control cochlear tissue. Immunostaining for HC- marks the postmitotic sensory domain. (S and T) Analysis of acutely isolated specific protein myosin VI (MYO6) (38) revealed that similar to E15.5 cochlear ducts (as demonstrated in J and K) from both the global global overexpression (Rosa26-iLIN28B), selective, inner ear- Rosa26-iLIN28B line and inner ear-specific Pax2-iLIN28B line compared with specific LIN28B overexpression (Pax2-iLIN28B) delayed audi- their nontransgenic littermates (control). Data expressed as mean ± SEM (n = tory HC differentiation. In E15.5 nontransgenic control animals, 3–7 animals per group, *P < 0.05, n.s., not significant). (Scale bars: B–I, K,and + μ – μ MYO6 IHCs were present throughout nearly the entire length L, 200 m; M R,50 m.) + of the cochlear duct (Fig. 2 K, M, O, and T) and MYO6 OHCs were already evident in the cochlear base (Fig. 2 K, M, and T). In suggest that LIN28B overexpression just hours before HC differ- contrast, cochlear tissue from Pax2-iLIN28B transgenic litter- + entiation is sufficient to delay its onset. mates contained no MYO6 OHCs yet (Fig. 2 L, N, and T) and + MYO6 IHCs (Fig. 2 L, P, and T) were not found as far apically LIN28B Overexpression Results in a Delay in Prosensory Cell Cycle Exit. as in control cochlear tissue. We next assayed the effects of LIN28B overexpression on Next we wanted to determine whether more acute LIN28B prosensory cell proliferation. Lin28b functions as an oncogene in overexpression was sufficient to delay the onset of HC differ- many tumors (39–41); however, little is known about Lin28b’s entiation. Dox-mediated induction of LIN28B protein takes role in controlling cell proliferation during normal development. ∼24 h (Fig. S3B). To account for this 24-h delay in induction, In the mammalian cochlea, prosensory cells withdraw from the LIN28B expression was induced at E12.5 and cochlear explant cell cycle in a striking apical-to-basal gradient. Murine prosensory cultures were obtained 12 h later, at E13.0 (Fig. S3A). Analysis of cells exit the cell cycle within a 48-h time window, with apical HC-specific Atoh1/nEGFP expression in these cultures revealed prosensory cells exiting the cell cycle as early as E12.5 and the that HC differentiation in E13.0 LIN28B overexpressing cochlear most basal progenitors exiting as late as E14.5 (1, 42). To de- explants was significantly delayed compared with controls (Fig. S3 termine whether higher-than-normal LIN28B protein levels might C–K), suggesting that within hours of being expressed, LIN28B delay prosensory cell cycle exit, a series of EdU pulse–chase ex- exerts its negative effect on HC differentiation. A qualitatively periments were performed by using the Pax2-iLIN28B transgenic similar delay in the HC differentiation was also observed when line. Dox was induced starting at E8.5, and EdU incorporation in E13.5 cochlear explants were acutely infected with murine Lin28b- differentiated HCs and SCs of Pax2-iLIN28B transgenic and expressing lentivirus (Fig. S3 L–W). Taken together, these data nontransgenic cochleae was analyzed at E18.5. Cytoplasmic my-

E3866 | www.pnas.org/cgi/doi/10.1073/pnas.1501077112 Golden et al. Downloaded by guest on September 28, 2021 PNAS PLUS BIOLOGY DEVELOPMENTAL

Fig. 3. LIN28B overexpression delays progenitor cell cycle withdrawal and causes mispatterning of the auditory sensory epithelium. (A–L) EdU incorporation (red) in HCs (MYO7a, blue) and SCs (SOX2, green) was analyzed at E18.5 following a single EdU pulse at E13.5. Shown are whole-mount preparations of basal, mid, and apical cochlear segments from control and Pax2-iLIN28B inner ears. Yellow asterisks mark ectopic IHCs, and dashes indicate missing OHCs. (M–O) Quantification of HC-specific EdU incorporation in E18.5 control (gray) and Pax2-iLIN28B (red) cochlear tissue following a single EdU pulse at E12.5 (M), a single EdU pulse at E13.5 (N), or once daily EdU pulses from E14.5 through E17.5 (O). Data expressed as mean ± SEM (n = 2, *P < 0.05). (P and Q) Cross-sections of E18.5 control and Pax2-iLIN28B cochleae. EdU (red) was given once daily from E14.5 to E17.5. HCs are marked by MYO7A (white) and SC subtypes are marked by P27 (green) and SOX2 (blue) immunostaining. Asterisk (Q) marks an ectopic SC disrupting the bilayered patterning of the cochlear epithelia in an iLIN28B cochlea. (R) Quantification of HC and SC subtypes per cochlear cross-section in control (gray) and Pax2-iLIN28B (red) cochleae. Data expressed as mean ± SEM (n = 3 animals per group, 5 cross-sections per animal; *P < 0.05). (S) RT-qPCR expression analysis of let-7 target genes associated with growth and proliferation in E14.5 control (gray) and Pax2-iLIN28B (red) cochlear epithelia. Data expressed as mean ± SEM (n = 3, *P < 0.05). d, Deiter’s cell; h, Hensen’s cell; i, phalangeal cell; p, pillar cell. (Scale bars: 50 μm.)

osin VII a (MYO7A) staining was used to identify HCs, and nu- conducted using the Rosa26-LIN28B transgenic line revealed clear p27/Kip1 and SOX2 staining were used to identify SCs that at E13.5 LIN28B overexpressing HC and SC precursors (5, 43). In the first set of experiments timed mated dams received expressed P27/Kip1 protein at much lower levels than in control a single injection of EdU at E13.5, the peak of cell cycle with- cochlea (Fig. S4 C–D′). To determine whether the altered pat- drawal. These experiments revealed that at stage E13.5, basally tern of HC and SC precursor proliferation was due to a delay in located HC and SC precursors in both control and LIN28B the initial onset of cell cycle withdrawal, a single injection of overexpressing cochlea were actively dividing and incorporated EdU was administered at E12.5 and EdU incorporation in api- EdU at a similar rate (Fig. 3 A–D and N). However, whereas in cally located HCs was analyzed at E18.5. Our analysis revealed control cochlear tissue HC and SC precursors located further that at E12.5 the majority of apical prosensory cells was actively apically had largely withdrawn from the cell cycle, in the LIN28B, cycling in LIN28B overexpressing cochleae, whereas in control overexpressing cochleae HC and SC precursors continued to cochleae, prosensory cells had started to withdraw from the cell proliferate and incorporate EdU at a significantly higher rate cycle (Fig. 3M). Consistent with a shift in timing rather than a than controls (Fig. 3 E–L and N). Moreover, experiments permanent derailment of prosensory cell cycle withdrawal, EdU

Golden et al. PNAS | Published online July 2, 2015 | E3867 Downloaded by guest on September 28, 2021 injections for three consecutive days beginning at E14.5, only labeled basally located HCs in LIN28B overexpressing cochleae. No EdU-labeled cells were observed within the auditory sensory epithelium further apically, indicating that once postmitotic, HC and SC precursors permanently withdrew from the cell cycle in the LIN28B overexpressing cochlea (Fig. 3 O–Q). Further- more, no proliferation was detected in either control or iLIN28B sensory epithelia at E15.5 (Fig. S4 A and B). At E18.5, HC differentiation and patterning is largely complete in the murine cochlea. In nontransgenic littermate control cochleae, the typical stereotyped pattern of one row of IHCs and three rows of OHCs (Fig. 3 A, E, and I) resting on a single layer of SCs (Fig. 3 C, G, K, and P) was observed throughout the length of the cochlear duct. In contrast, the stereotyped organization of HCs and SCs was disrupted in the cochlear base of E18.5 Pax2-iLIN28B transgenic embryos. Ectopic IHCs were frequently observed in the basal segment of the LIN28B overexpressing cochleae (Fig. 3B and Fig. S4H). In addition, OHCs were frequently missing, resulting in patches of sensory epithelium containing only two rows of OHCs (Fig. 3B and Fig. S4I). These HC patterning defects were mostly confined to the cochlear base; cellular patterning of the auditory sensory epithelium in more apical segments of the LIN28B overexpressing cochleae was largely normal (Fig. 3 F and J and Fig. S4 H and I). Interestingly, despite prolonged proliferation of both HC and SC precursors, only the number of SCs was increased in the iLIN28B cochleae (Fig. 3R and Fig. S4 F and G). SCs were more densely packed, and in contrast to the single SC layer in control cochleae (Fig. 3 C, G, K, and P), SCs were frequently found stacked on top of each other within LIN28B overexpressing cochleae (Fig. 3 D, H, L, and Q). In addition to the observed cellular patterning defects, maturation of HCs and SCs was delayed in iLIN28B cochleae as evidenced by delayed down-regulation of the prosensory marker SOX2 in HCs (Fig. 3 P and Q) and delayed up- regulation of the SC-specific marker S100 (Fig. S4 J and K). What is the molecular basis of the LIN28B-mediated delay in prosensory cell cycle withdrawal and differentiation? One possible mechanism is that the up-regulation of let-7 target genes due to lower than normal let-7 levels alters the timing of prosensory proliferation and differentiation. Recent studies have revealed a critical role for the let-7 targets Igf2bp1 (Imp1), Hmga2, and Lin41 (Trim71) in neuronal stem cell/progenitor cell main- tenance (29, 31, 44). Our analysis of let-7 target gene expression revealed that LIN28B overexpression significantly increased transcript levels of Igf2bp1, Lin41, and Hmga2 (Fig. 3S). Fur- thermore, LIN28B overexpression significantly increased the expression of the let-7 targets N-Myc (Mycn) and Cyclin D1 (ccnd1) (45, 46), which have been shown to positively regulate prosensory cell proliferation in the murine cochleae and vestibular organs (47–49) (Fig. 3S).

Let-7 Overexpression Results in Premature Progenitor Cell Cycle Exit but Does Not Cause Precocious Differentiation. To test whether Fig. 4. Let-7 overexpression accelerates progenitor cell cycle withdrawal – ′ LIN28B regulates prosensory proliferation and differentiation but not differentiation. (A B ) Cross-sections of E14.5 control and Pax2 iLet-7 cochleae. A single EdU pulse was given at E13.5 and EdU incorporation via a let-7–dependent mechanism, we made use of transgenic + (green) within SOX2 (red) prosensory progenitor cells was analyzed after mice that express a Lin28a/b-resistant form of let-7g under the 24 h. High-power images of the basal turn are shown in A′ and B′. control of a tetracycline response element (Fig. S5A) (27). When (C) Quantification of EdU incorporation shown in A–B′. Data expressed as combined with either a ubiquitous (Rosa26 M2-rtTA) or inner mean ± SEM (n = 2, *P < 0.05). (D and E) Bright field and green fluorescence ear-specific (Pax2-rtTA) rtTA, dox administration induced a more image overlays of acutely isolated E14.5 control and Rosa26 iLet-7 cochlear than 15-fold increase in let-7g miRNA expression within the ducts. Atoh1/nEGFP reporter (EGFP, green) marks nascent HCs. Asterisks de- developing cochlear duct (Fig. S5 B–D). Let-7g overexpression note EGFP expression within HCs of the vestibular sacculus. (F and G)Analysis significantly reduced Hmga2 mRNA (Fig. 4 H and I and Fig. of the extent of HC differentiation in E14.5 control and Rosa26-iLet-7 cochlear ducts shown in D and E. Data expressed as mean ± SEM (n = 4, *P < 0.05). S5G) and LIN28B protein levels (Fig. S5 E and F). To determine – let-7 (H K) ISH-based analysis of Hmga2 (H and I) and Atoh1 (J and K) expression whether higher than normal levels might cause precocious in E14.5 Pax2-iLet-7 and control cochlear tissue. (L and M) HC phenotype in prosensory cell cycle withdrawal, dox-fed timed-mated dams E18.5 control and Pax2-iLet-7 cochlear whole mounts. HCs are marked by received a single injection of EdU at E13.5 and EdU incor- phalloidin (green). Shown is the basal turn. Arrowheads indicate IHC do- poration within Pax2-iLet-7 transgenic and control embryos was main, brackets indicate OHC domain, and yellow dashes indicate missing analyzed 24 h later. Our EdU incorporation analysis revealed a OHCs. (Scale bars: A–B′ and H–K,50μm; D and E, 200 μm.)

E3868 | www.pnas.org/cgi/doi/10.1073/pnas.1501077112 Golden et al. Downloaded by guest on September 28, 2021 + PNAS PLUS dramatic decrease in the number of proliferating SOX2 prosensory cells upon let-7g overexpression (Fig. 4 A–C). In let-7g overexpressing cochleae, less than 5% of basally located prosensory cells incorporated EdU compared with more than 30% in control (Fig. 4C). This reduced rate of prosensory cell proliferation is likely due to the premature up-regulation of p27/ Kip1. At E12.5, broad P27/Kip1 protein expression was seen in the apical segment of Pax2-iLet-7 cochlear ducts, whereas control cochlear sensory epithelia did not yet express p27/Kip1 protein (Fig. S5 L and M). Unsurprisingly, global (Rosa26-iLet-7) as well as inner ear-specific (Pax2-iLet-7) let-7 overexpression caused defects in cochlear outgrowth (Fig. 4F and Fig. S5H), and at E18.5, Pax2-iLet-7 cochleae were 40% shorter than controls (Fig. S5H). Likely due to premature prosensory cell cycle withdrawal, both IHC and OHC density was significantly reduced (Fig. S5 I and J) and the third row of OHCs was frequently missing in the Pax2-iLet-7 cochleae (Fig. 4 L and M and Fig. S5K). Surprisingly, the timing of HC differentiation appeared to be unaffected by let-7 overexpression. At E14.5, both Rosa26-iLet-7 cochleae and nontransgenic littermate control cochleae had a similar extent of HC differentiation along the length of the cochlear duct. Atoh1/nEGFP-positive IHCs were observed only within the basal half of the cochlear duct; the apical half remained undifferentiated (Fig. 4 D–G). Similarly, no difference in HC-specific Atoh1 mRNA expression was observed in E14.5 Pax2-iLet-7 transgenic cochlear tissue compared with nontransgenic littermate controls (Fig. 4 J

and K). In summary, our analysis suggests that the Lin28b/let-7 BIOLOGY

circuit controls the timing of prosensory cell cycle withdrawal DEVELOPMENTAL in the developing cochlea. However, in contrast to LIN28B overexpression, let-7g overexpression had little effect on the timing of HC differentiation, suggesting that Lin28b uses a distinct, let-7–independent mechanism in regulating the timing of HC differentiation.

The LIN28B/let-7 Axis Modulates the Capacity for SC Conversion in the Absence of Notch Signaling. In the mammalian cochlea, HC production is restricted to embryonic development. Loss of HCs is permanent and is a leading cause of human deafness. However, recent studies suggest that in a permissive environment, murine SCs can function as HC progenitors and generate/regenerate HCs (50). One of the key pathways limiting SC-to-HC conversion is the Notch signaling pathway. During embryonic development, activation of Notch signaling within a subset of prosensory cells inhibits the activation of a HC-specific program, limiting these cells to a SC fate (51–53). Inhibition of Notch signaling using γ-secretase inhibitors results in the generation of new HCs within the embryonic and neonatal murine cochlea (54–56); although the ability of SCs to dedifferentiate and convert to a HC fate declines rapidly within the first postnatal week of life. In the adult animal, only SCs located in the cochlear apex respond to Notch inhibition and infrequently convert to a HC fate (57). Fig. 5. The LIN28B/let-7 axis modulates the capacity for SC conversion in the Recently, two studies have suggested that Lin28a/b reexpression absence of Notch signaling. (A–G) LIN28B reexpression promotes SC-to-HC can counteract the developmental decline in cell regeneration conversion in the absence of Notch signaling. (A) Schematic of HC re- and tissue repair. In both cases, let-7 repression was necessary to generation assay. LIN28B overexpression was induced starting at E15.5, and mediate this effect, although let-7 repression alone was not suf- cochlear explants from P2 Rosa26-iLIN28B pups and nontransgenic littermates (control) were cultured in the presence of DAPT or DMSO. SC-to-HC ficient to enhance tissue repair (58, 59). To determine whether conversion was quantified after 3 DIV. (B–E) Shown are midbasal segments LIN28B reexpression might enhance SC plasticity, LIN28B was of P2 control and iLIN28B cochlear explants after 3 d of DMSO (B and C)or reexpressed in the late embryonic (E15.5) cochlea and the ability DAPT (D and E) treatment. HCs are marked by Atoh1/nEGFP (green) and of SCs to generate new HCs in response to γ-secretase inhibitor MYO7A (red). (F and G) Quantification of B–E. Data expressed as mean ± treatment was analyzed in early postnatal (P2) cochlear explant SEM (n = 3, *P < 0.03). (H–N) Let-7 overexpression antagonizes SC conversion cultures (Fig. 5A). We limited our analysis to the cochlear in the absence of Notch signaling. (H) Schematic of HC regeneration assay. midbase where early postnatal SCs only infrequently convert Let-7 overexpression was induced starting at E17.5 and cochlear explants to HCs in response to Notch inhibition (54). In contrast to early from P2 Rosa26-iLet-7 pups and nontransgenic littermates (control) were LIN28B LIN28B cultured in the presence of DAPT or DMSO. SC-to-HC conversion was embryonic (E8.5) induction, late embryonic quantified after 3 DIV. (I–L) Shown are midapical segments of P2 control and induction starting at E15.5 did not alter the number or organi- – iLet-7 cochlear explants after 3 d of DMSO (I and J) or DAPT (K and L) zation of HCs and SCs (Fig. S6 A E), and no difference in the treatment. HCs are marked by Atoh1/nEGFP (green) and MYO7A (red). number of HCs was observed in untreated P2 control and (M and N) Quantification of I–L. Data expressed as mean ± SEM (n = 4–5 per Rosa26-iLIN28B cochlear explants after 3 d in culture (Fig. 5 B, group, *P ≤ 0.02). (Scale bars: 50 μm.)

Golden et al. PNAS | Published online July 2, 2015 | E3869 Downloaded by guest on September 28, 2021 C, and F). Treatment with the γ-secretase inhibitor DAPT for Lin28b Acts Through a let-7–Dependent Mechanism To Time Prosensory 3 d significantly increased the number of MYO7A and Atoh1/ Cell Cycle Withdrawal but Not Differentiation. The regulatory nEGFP-positive HCs in both P2 control and Rosa26-iLIN28B functions of the RNA binding protein LIN28B and its opposing cochlear explants (Fig. 5 D–G); however, LIN28B overexpressing let-7 miRNAs have been extensively studied in stem cells, tumor cochlear explants generated significantly more new HCs than models, and adult tissues; however, little is known about their controls (Fig. 5 F and G). Interestingly, these newly generated function during embryonic development. Here we provide evi- HCs largely lacked EdU incorporation, suggesting that they dence that the LIN28B/let-7 circuit is critical for the proper originated from postmitotic cells. Taken together, our finding timing of prosensory cell cycle exit in the murine cochlea. We suggests that LIN28B reexpression increased the frequency of show that higher-than-normal LIN28B protein levels delayed direct SC-to-HC conversion in response to Notch inhibition. the onset of prosensory cell cycle exit, whereas increased let-7 To determine whether let-7 overexpression would have the levels resulted in premature cell cycle exit. The opposing effects opposite effect and limit SC plasticity, let-7g overexpression was of LIN28B and let-7g overexpression suggest that relative induced in the late embryonic cochlea (E17.5) and HC genera- LIN28B/let-7 levels set the timing of prosensory cell cycle with- tion in the absence of γ-secretase activity was analyzed in P2 drawal in the murine cochlea. Furthermore, our finding that let-7g cochlear explant cultures (Fig. 5H). For these experiments, we is sufficient to induce premature cell cycle withdrawal suggests limited our analysis to the cochlear midapex. In this more apical that LIN28B times prosensory cell cycle exit, at least in part, by segment, early postnatal SCs readily convert to HCs when Notch repressing let-7 biogenesis. Interestingly, although both global and signaling is inhibited (54). Let-7 overexpression in the late em- cochlear-specific LIN28B overexpression was sufficient to delay bryonic (E17.5) cochlea did not alter the number or organization the onset of HC differentiation, similar let-7g overexpression strat- of HCs and SCs (Fig. S6 F–J), and no difference in the number of egies failed to induce premature HC differentiation. The failure to HCs was observed in untreated P2 control and Rosa26-iLet-7 induce earlier HC differentiation is in stark contrast to let-7’sdif- cochlear explants after 3 d in culture (Fig. 5 I, J,andM). Strik- ferentiation-promoting function in other tissues. For example, ingly, we found that let-7 overexpression significantly decreased overexpression of let-7 in neuronal progenitors not only inhibits the number of MYO7A and Atoh1/nEGFP-positive HCs that proliferation, but induces premature neuronal differentiation as were generated following DAPT treatment (Fig. 5 K–N). Whereas well (29, 44, 45, 61, 62). How can this be explained? In many tis- P2 control explant cultures had more than a 95% increase in sues, including the developing brain, timing of differentiation is HCs following DAPT treatment, P2 Rosa26-iLet-7 cultures had tightly linked to timing of progenitor cell cycle exit. However, in the only a 30% increase in HCs (Fig. 5N). Thus, our findings suggest murine cochlea the developmental timing of these processes is that let-7 down-regulation is necessary for SC-to-HC conversion largely uncoupled (5, 8). This difference is illustrated by the diver- in the absence of Notch signaling and that let-7 overexpression gent behavior of the let-7 target Mycn within different tissue models. antagonizes this process. Similar to let-7 overexpression, loss of Mycn causes premature prosensory cell cycle withdrawal without altering the timing of HC Discussion differentiation within the developing cochlea (48, 63). In contrast, The differentiating inner ear cochlea expresses hundreds of brain-specific deletion of Mycn results in both premature progenitor miRNA species and RNA binding proteins, indicating that post- cell cycle exit and premature neuronal differentiation (64). Alter- transcriptional regulation is likely to play an important role in natively, it could be reasoned that the failure of let-7 to induce controlling the dynamics of cochlear development. Here we have premature HC differentiation is due to a lack of inductive cues that demonstrated that the RNA-binding protein LIN28B is a critical would allow for an earlier onset of HC differentiation. The lack of component of a timing mechanism that regulates both prosensory inductive cues being the reason is rather unlikely, however, because cell cycle withdrawal and differentiation in the murine cochlea. loss of inhibitory Sonic Hedgehog (Shh) signaling is sufficient to Furthermore, our data suggests a role for the LIN28B/let-7axisin trigger premature HC differentiation (65–67). regulating HC regeneration and SC plasticity, identifying a novel candidate pathway for future gene-based HC regeneration therapies. Let-7–Independent Functions of LIN28B. It is becoming increasingly clear that LIN28 proteins can function in a let-7–independent Regulation of Lin28b/let-7 Expression in the Developing Cochlea. We manner (68, 69). Several recent genomewide studies have have found that the evolutionary conserved Lin28b/let-7 circuit revealed that LIN28A and LIN28B proteins directly alter the is active in the auditory sensory lineage. Lin28b is specifically protein abundance of hundreds of their mRNA targets through expressed in prosensory progenitors and is rapidly down-regulated transcript stabilization and/or modulation of translation effi- upon differentiation, whereas levels of mature let-7 miRNAs rise ciency (14, 15, 70–72). Consistent with an important role in gene during prosensory differentiation and are highly expressed in regulation, among the top LIN28B targets are RNA binding early postnatal HCs and SCs. LIN28B inhibits mature let-7 proteins, transcriptional regulators, and RNA splicing factors miRNA processing and likely serves a critical role in let-7’s (15, 70). How might LIN28B alter the timing of HC differenti- posttranscriptional regulation within the developing cochlea. ation? To date two pathways, the Insulin-like growth factor Despite the mutually antagonistic relationship between Lin28b (Igfr1)-phosphatidylinositol 3-kinase (PI3K)/Akt pathway and and the let-7s, however, it is unlikely that the initial down- the Shh pathway, have been shown to influence timing of HC regulation of Lin28b is mediated by let-7 miRNAs. Instead, it is differentiation within the murine cochlea (65–67, 73). LIN28B more plausible that the heterochronic miRNA mir-125b medi- has been shown to positively regulate Igf1r-PI3K/Akt pathway ates Lin28b’s down-regulation. Similar to the let-7 miRNAs, mir- (27); however, loss of Igfr1 within the developing cochlea does 125b targets the expression of Lin28b; however, unlike the let-7 not mirror LIN28B’s effects on HC differentiation. In the Igfr1 miRNAs, Lin28b and mir-125b do not share a mutually antag- mutant cochlea, prosensory cell cycle exit occurs prematurely onistic relationship, and LIN28B is unable to regulate mir-125b’s while HC differentiation is delayed (73). Functional connections expression (19, 35, 60). Alternatively, it is possible that in the between the Shh signaling pathway and LIN28B have not yet developing cochlea, Lin28b’s expression is mainly regulated on a been established; however, the tools described here will allow transcriptional level. In stark contrast to the wealth of knowledge future examination of this link. related to Lin28b’s regulation by miRNAs, relatively little is known about the transcription factors controlling Lin28b’s The LIN28B/let-7 Axis Influences SC Plasticity. LIN28 proteins have expression during mammalian development, and further investi- been shown to promote cell reprogramming, tissue repair, and gations are warranted. regeneration (58, 59, 74, 75). HC loss is among the leading

E3870 | www.pnas.org/cgi/doi/10.1073/pnas.1501077112 Golden et al. Downloaded by guest on September 28, 2021 causes of human deafness, because in contrast to nonmammalian was harvested. Both Rosa26-iLIN28B and Pax2-iLIN28B overexpression were PNAS PLUS vertebrates, mammals are unable to regenerate damaged HCs. induced at E8.5, except for the Rosa26-iLIN28B litters used in the HC re- Thus, uncovering mechanisms that promote HC regeneration/ generation experiments, which were induced at E15.5. To induce Rosa26- repair within the mammalian inner ear remains a major focus iLet-7 expression, dox treatment was begun at E11.5. To induce Pax26-iLet-7 expression, dox treatment was begun at E9.5. For the iLet-7 hair cell re- within the auditory research community. For nonmammalian generation experiments, Rosa26-iLet-7 was induced at E17.5. For the Rosa26- vertebrates, SCs are the main source of postembryonic HC iLIN28B and iLet-7 lines, nontransgenic littermate controls expressed only production and HC replenishment after trauma (76). Strikingly, the Rosa26 M2-rtTA transgene (M2-rtTA tg/+). For the Pax2-iLIN28B and iLet-7 mammalian SCs can generate HCs under certain permissive lines nontransgenic littermate controls expressed only the rtTA-EGFP and Pax2- conditions, such as Notch inhibition or Wnt overstimulation, but Cre transgenes (rtTA-EGFP tg/+; Pax2-Cre tg/+). this ability rapidly declines during the first week of life (57, 77–79). Here, we show that LIN28B reactivation in the early postnatal Tissue Isolation. Embryos were removed from timed-mated females and murine cochlea enhances the generation of new HCs in response staged by using the EMAP eMouse Atlas Project Theiler staging criteria (www. to Notch inhibition. The vast majority of newly generated HCs in emouseatlas.org/emap/ema/home.html). Inner ears were collected and dis- sected in HBSS (Life Technologies). To free the cochlear epithelium (E13– our experimental paradigm were generated by a nonmitotic pro- E16), the vestibular portion of the inner ear was removed and the remaining cess, suggesting that this increase in newly generated HCs was due cochlear portion was incubated in calcium-magnesium-free PBS (Life Tech- to an increased number of SCs that converted into HCs in re- nologies) containing dispase (1 mg/mL; Life Technologies) and collagenase sponse to Notch inhibition. How does LIN28B reexpression alter (1 mg/mL; Worthington) for 8 min then placed in DMEM-F12 (Life Tech- the response of SCs? A recent study showed that LIN28A reac- nologies) containing 10% (vol/vol) FBS for 30 min, the cochlear duct was tivation in adult mice boosts tissue repair by reprogramming cel- then freed from surrounding mesenchyme by manual dissection with lular metabolism (58), and it is possible that enhancement of 30-gauge needles. To obtain cochlear sensory epithelia of stages E18 and oxidative SC metabolism had a positive effect on the ability of SCs older, the cochlear capsule and the spiral ganglion were removed before dispase/collagenase treatment. to switch cell fate and convert into HCs. Alternatively, it is possible that LIN28B reexpression altered the differentiation state of RNA Extraction and qPCR. Total RNA including mature miRNAs was extracted SCs by down-regulating let-7 miRNA expression. For instance, in from cochlea epithelia samples by using the miRNeasy Micro Kit (QIAGEN). zebrafish, Lin28 becomes reactivated in Müller glia after damage, For mRNA expression analysis, mRNA was reverse transcribed into cDNA by and promotes Müller glia dedifferentiation and subsequent retina using the iScript cDNA synthesis kit (Bio-Rad). SYBR Green-based qPCR

regeneration, at least in part by decreasing let-7 miRNA levels was performed by using Fast SYBR Green Master Mix reagent (Applied BIOLOGY

(80). Indeed, let-7 down-regulationwasfoundtobenecessaryto Biosystems, Life Technologies no. 4385612) and gene-specific primers. For DEVELOPMENTAL promote cellular regeneration in multiple models of murine tissue miRNA expression analysis, predesigned TaqMan Assays (Applied Biosystems, ’ repair (58). Similarly, we found that let-7 overexpression signifi- Life Technologies) were used according to manufacturer s instructions for two-step RT-qPCR. qPCRs were carried out in triplicate by using a StepOne cantly antagonized SC-to-HC conversion in the absence of Notch Plus Real-Time PCR System (Applied Biosystems, Life Technologies). Relative signaling, suggesting that let-7 down-regulation is necessary for gene expression was analyzed by using the ΔΔCT method (83). The ribosomal SC dedifferentiation in the murine cochlea. Could let-7 miRNAs gene Rpl19 was used as an endogenous reference gene for SYBR Green-based contribute to the developmental decline in SC’s regenerative ca- assays, and the snoRNA U6 was used as an endogenous reference gene for pacity? Let-7 miRNAs are highly expressed in early postnatal HCs TaqMan-based miRNA measurements. qPCR primer sequences are listed in and SCs, and there is tentative evidence that cochlear let-7 ex- Table S2. pression further increases during postnatal maturation (36). Fu- ture experiments are needed to link the rise in let-7 expression to Immunoblotting. Cochlear epithelia were placed in lysis buffer (50 mM Hepes, an age-related decline in regenerative capacity. 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 0.2% SDS, pH 7.9) In summary, we have demonstrated that the Lin28b/let-7 supplemented with fresh Complete Protease Inhibitor (Roche). Equal amounts of cochlear lysate were resolved on NuPAGE 4–12% Bis-Tris Gels (Novex, Life miRNA axis is active within the developing cochlea and plays Technologies) and transferred to PVDF membrane by electrophoresis. Mem- a key role in coordinating the timing of progenitor cell cycle branes were blocked in 10% BSA in TBST and immunoblotted: rabbit anti- withdrawal and the onset of differentiation. Interestingly, LIN28B Lin28b 1:1,000 (Cell Signaling no. 5422), mouse anti-β-actin 1:3,000 appears to act through both let-7–dependent and let-7- (Ambion no. AM4302), rabbit anti-Flag 1:1,000 (Cell Signaling no. 2368). HRP- independent mechanisms in coordinating these processes and conjugated secondary antibodies from Jackson ImmunoResearch were used at further studies investigating these let-7–independent pathways a concentration of 1:7,500 (goat anti-rabbit IgG no. 111–035-003; sheep anti- – are needed. Lastly, our findings indicate that the LIN28B/let-7 mouse IgG no. 515 035-003). Signal was revealed by using a Western Light- ’ axis regulates the capacity for SC-to-HC conversion in the ab- ening-ECL kit (Perkin-Elmer) according to manufacturer s instructions. sence of Notch signaling, making it an interesting candidate for ISH and Immunostaining. Embryo heads (E11–E16) or inner ears (E18 and future HC regeneration therapies. older) were fixed in 4% (vol/vol) paraformaldehyde in PBS overnight at 4 °C then cryoprotected through sucrose gradient: 10% (wt/vol) sucrose for 30 Materials and Methods min at room temperature (RT), 15% sucrose for 1 h at room temperature, Mouse Breeding and Genotyping. All experiments and procedures were ap- 50:50 30% sucrose:OCT overnight at 4 °C. Before cryosectioning, samples proved by the Johns Hopkins University Institutional Animal Care and Use were submerged in OCT (Tissue-Tek, Sakura) and flash frozen. Fourteen Committees protocol, and all experiments and procedures adhered to Na- micrometer sections were collected on SuperFrost Plus slides (Fisher). tional Institutes of Health-approved standards. The Atoh1/nEGFP transgenic pBluescript II (Stratagene) and pGem-T easy (Promega) vectors containing line was obtained from Jane Johnson (University of Texas Southwestern full-length mouse Atoh1, Sox2, Lin28b,andHmga2 cDNA were used as Medical Center, Dallas, TX) (81). The iLIN28B and ilet-7g transgenic lines templates to synthesize digoxigenin-labeled antisense RNA probes accord- were obtained from George Q. Daley (Children’s Hospital, Boston, MA) (27). ing to the manufacturer’s specifications (Roche). Custom-made 5′ digox- The Pax2-Cre line was obtained from Andrew Groves (Baylor College, ygenin-labeled locked nucleic acid probes (miRCURY LNA) from Exiqon were Houston, TX) (82). The M2-rtTA (stock no. 006965) and rtTA-EGFP (stock no. used for let-7f and let-7c ISH. Probes were detected with the anti–DIG-AP 005572) lines were purchased from Jackson Laboratories. Mice were geno- (alkaline phosphatase) conjugated antibody (Roche), and the color re- typed by PCR and genotyping primers are listed in Table S1. Mice of both actions were developed by using BM Purple AP Substrate (Roche). Detailed sexes were used in this study. All mouse lines were maintained on a mixed protocol is available upon request. background of C57BL/6 and CD-1. Embryonic development was considered Immunostaining was performed according to the manufacturer’sspecifi- as E0.5 on the day a mating plug was observed. cations. Primary antibodies: rabbit anti-myosin VI (1:1,000, Proteus no. 25– To induce iLIN28B and iLet-7 expression, dox was delivered to time-mated 6791), rabbit anti-myosin VIIa (1:500, Proteus no. 25–6790), goat anti-SOX2 females via ad libitum access to feed containing 2 g of doxycycline per kg (1:500, Santa Cruz no. sc-17320), mouse anti-p27/Kip1 (1:200, NeoMarkers/ of feed (Bioserv no. F3893), which was continued until the stage the tissue Thermo no. MS-256-P1), rabbit anti-S100 (1:500, Abcam no. ab868), and rabbit

Golden et al. PNAS | Published online July 2, 2015 | E3871 Downloaded by guest on September 28, 2021 anti-dsRed (1:500 Clontech no. 632496). Alex Fluor (488, 546, and 633) la- Hair Cell Generation Assay. P2 cochlear surface preparations were cultured on beled secondary antibodies were used to visualize staining (1:1,000, Molec- SPI black membranes (SPI Supplies, Structure Probe) in DMEM-F12 (Life ular Probes/Life Technologies). Stereocilia were visualized with fluorescently Technologies) containing 1x N2 supplement (Life Technologies), 5 ng/mL EGF labeled phalloidin (1:500, Molecular Probes/ Life Technologies). For anti-p27/ (Sigma), 2.5 ng/mL FGF (Sigma), 100 U/mL penicillin (Sigma), and 10 μg/mL dox Kip1 immunostaining, sections underwent antigen retrieval by boiling in 1x (Sigma). Experimental cultures were treated with 10 μM DAPT (γ-secretase Dako Ready-to-Use Target Retrieval Solution (Dako no. S1700) in a 95 °C inhibitor IX, Calbiochem-EMD Biosciences) to block Notch signaling. Control water bath for 20 min, followed by a 45-min cooling incubation at RT. cultures received DMSO (0.04%). Media was refreshed once at 24 h, and cultures were fixed at 72 h. To monitor proliferation, explants were grown in μ Hair Cell Differentiation Assay. E13.5 undifferentiated Atoh1/nEGFP cochlear the presence of 3 M EdU. For HC quantification, cochlear explant cultures – μ – μ epithelia, spiral ganglion, and surrounding mesenchyme were cultured on SPI were subdivided into four parts: apex (0 1,500 M); mid apex (1,500 3,000 M); – μ – black membranes (SPI Supplies, Structure Probe) in DMEM-F12 (Life Technol- mid base (3,000 4,500 M); base (4,500 5,500). A sampling (four per region) ogies) containing 1x N2 supplement (Life Technologies), 5 ng/mL EGF (Sigma), of high-power confocal z-stack images were taken through the HC and SC 2.5 ng/mL FGF (Sigma), 100 U/mL penicillin/streptomycin (Sigma), and 10 μg/mL layers within these regions, and HC density and proliferation were manually quantified by using Imaris software (Bitplane) and ImageJ. HCs were iden- dox (Sigma). Cultures were grown in a 5% CO /20% O humidified incubator. 2 2 tified by MYO7A and Atoh1/nEGFP coexpression. To monitor HC differentiation, native nuclear GFP expression (Atoh1/nEGFP) was imaged every 8–12 h for 3 d by using fluorescent stereomicroscopy (Leica). + Lentiviral Expression System. Control and experimental constructs (mCherry Length of the GFP HC stripe was quantified by using ImageJ64 software (NIH). vs. Lin28bF2AmCherry) were maintained in FUW lentiviral-vector backbone. Multistep PCR was used to link full-length murine Lin28b ORF to a mCherry Proliferation Assay. EdU (5-ethynyl-2′-deoxyuridine, Life Technologies) was reporter via a F2A linker sequence (84). High titer lentiviral stocks were reconstituted in PBS and administered at 50 μg per gram of body weight prepared as described (85). Undifferentiated Atoh1/nEGFP cochlear epithe- to time-mated females by i.p. injection. Click-iT Alexa Fluor 546 Kit (Life lial explants were incubated in a 1:1 mix of lentivirus and culture media at Technologies) was used to detect incorporated EdU according to the man- room temperature for 2-h before plating. Cultures were maintained as de- ’ ufacturer s specifications. MYO7A and SOX2 immunostaining labeled HCs scribed above. Detailed protocols are available upon request. and SCs. Cochleae were divided into three equal portions (base, mid, apex) and high-power confocal (Zeiss) z-stack images (4 images per region, rep- Statistical Analysis. Values are presented as mean ± SE (SEM), n = animals per resenting ∼700 microns) were taken through the HC and SC layers within group. All results were confirmed by at least two separate experiments. Two- each of these regions. The percentage of HCs and SCs that had incorporated tailed Student’s t tests were used to determine confidence interval. P values ≤0.05 EdU was manually quantified within Photoshop and ImageJ. were considered significant. P values >0.05 were considered not significant.

Cochlear Length and Cell Patterning Analysis. Cochlear surface whole mounts ACKNOWLEDGMENTS. We thank the members of the A.D. Laboratory and were prepared from E18.5 iLIN28B and nontransgenic littermate embryos and Center for Sensory Biology for the help and advice provided throughout immunostained for MYO7A and SOX2. Low-power fluorescent images of the the course of this study; Randy Reed, Mollie Meffert, Shan Sockanathan, HC layer were assembled in Photoshop CS5 (Adobe), and the length of the and Seth Blackshaw for fruitful discussions regarding this project; Neil M. entire cochlear sensory epithelium was measured in ImageJ64 (NIH). For cell Neumann for training and patience with the Imaris software; Jane density and patterning analysis, cochleae were divided into three equal Johnson for the Atoh1/nEGFP mice; Andrew Groves for the Pax2-Cre mice; and George Q. Daley for iLIN28B and iLet-7 mice. This work was sup- portions (base, mid, apex) and high-power confocal (Zeiss) z-stack images ported by March of Dimes Basil O’Connor Grant FY11-84 (to A.D.) and ∼ (4 images per region, representing 700 microns) were taken through the HC National Institutes of Health Grants DC012972 (to E.J.G.), DC013477 (to A.B.-G.), and SC layers within each of these regions. Images then were manually DC011571 (to A.D.), and DC005211 (Sensory Mechanisms Research quantified within Photoshop and ImageJ. Core Center).

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