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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Dev Dyn. Author manuscript; available in PMC Dec 5, 2008.
Published in final edited form as:
PMCID: PMC2596717
NIHMSID: NIHMS69729

Reciprocal Expression of lin-41 and the microRNAs let-7 and mir-125 During Mouse Embryogenesis

Abstract

In C. elegans, heterochronic genes control the timing of cell fate determination during development. Two heterochronic genes, let-7 and lin-4, encode microRNAs (miRNAs) that down-regulate a third heterochronic gene lin-41 by binding to complementary sites in its 3’UTR. let-7 and lin-4 are conserved in mammals. Here we report the cloning and sequencing of mammalian lin-41 orthologs. We find that mouse and human lin-41 genes contain predicted conserved complementary sites for let-7 and the lin-4 ortholog, mir-125, in their 3’UTRs. Mouse lin-41 (Mlin-41) is temporally expressed in developing mouse embryos, most dramatically in the limb buds. Mlin-41 is down-regulated during mid-embryogenesis at the time when mouse let-7c and mir-125 RNA levels are up-regulated. Our results suggest that mammalian lin-41 is temporally regulated by miRNAs in order to direct key developmental events such as limb formation.

Keywords: microRNAs, lin-41, let-7, mir-125, developmental timing, mouse embryogenesis, expression pattern, limb development, heterochronic gene

INTRODUCTION

Development occurs across the dimensions of both space and time. Although the mechanisms of spatial patterning have been well characterized, only a few of the genes involved in temporal regulation of development have been identified. These temporal regulatory genes, referred to as heterochronic genes, were first isolated in the nematode, Caenorhabditis elegans, where they control the timing of cell fate determination (Ambros and Horvitz, 1984). These temporal regulators have been ordered into a heterochronic pathway that includes transcription factors, RNA binding proteins, and small regulatory RNAs known as microRNAs (miRNAs). Many of the C. elegans heterochronic genes are highly conserved in other organisms including mice and humans (Pasquinelli et al., 2000; Slack et al., 2000; Moss and Tang, 2003). While this pathway has been well studied in C. elegans, the role of their orthologs in mammalian development is not known. Given that many of the mechanisms controlling development have been conserved across animal phylogeny, it is possible that orthologs of C. elegans heterochronic genes may be involved in controlling developmental timing in mammals.

C. elegans heterochronic genes control developmental timing in a subset of hypodermal cells, known as seam cells during post-embryonic development. Seam cells divide with a stem-cell like pattern at each larval stage but exit the cell cycle and terminally differentiate at the adult stage. Heterochronic genes can be divided into two categories, precocious and retarded, based on their mutant phenotype. For example, loss of function mutations in the lin-28 (Ambros and Horvitz, 1984), hbl-1 (Lin et al., 2003), and lin-41 (Slack et al., 2000) genes cause precocious terminal differentiation of seam cells during the fourth larval stage (L4) stage rather than at the adult stage. On the other hand, mutations in lin-4 and let-7 cause failure of seam cells to terminally differentiate appropriately and cells instead reiterate earlier larval fates at later developmental stages (Chalfie et al., 1981; Reinhart et al., 2000).

The heterochronic genes lin-4 and let-7 are temporally expressed during C. elegans development. lin-4 RNA is first detected late in the L1 stage (Feinbaum and Ambros, 1999) and this expression pattern correlates with its role in directing the progression of cell fates at the L1 to L2 transition. Similarly, let-7 expression is first detected in the L3 stage and functions to direct larval to adult cell fate transitions (Reinhart et al., 2000; Johnson et al., 2003). lin-4 and let-7 are the founding members of the miRNA family of non-coding regulatory RNAs (Lee et al., 1993; Reinhart et al., 2000). Hundreds of miRNAs have now been identified in many organisms, including mice and humans, but the functions of only a few of them have been determined (Bartel, 2004). Primary miRNA transcripts, known as pri-miRNA (Lee et al., 2002), are large sequences processed by Drosha RNase III endonuclease in the nucleus (Lee et al., 2003) into ~70 nucleotide precursors referred to as pre-miRNAs (Lee et al., 2002). Pre-miRNAs are then transported to the cytoplasm (Yi et al., 2003; Lund et al., 2004) where they are subsequently processed by the RNAse III enzyme DICER into mature ~22 nucleotide miRNAs (Lee et al., 2002). In general these small RNAs base pair with imperfect complementarity to target mRNA sequences in their 3’ untranslated region (3’UTR), and regulate gene expression at the translational level (Olsen and Ambros, 1999). In humans, however, miR-196 regulates its target, a HOX gene, by cleaving the mRNA transcripts (Yekta et al., 2004).

The heterochronic miRNAs, let-7 and lin-4 regulate their targets by binding to complementary sites in their 3’UTRs. lin-4 represses the translation of the heterochronic genes lin-14 (Wightman et al., 1993; Ha et al., 1996; Olsen and Ambros, 1999) and lin-28 (Moss et al., 1997) at the L2 stage leading to decreased levels of LIN-14 and LIN-28 proteins upon lin-4 expression. The heterochronic gene, lin-41 is one known target of let-7. The retarded lethal phenotype of let-7 mutants is suppressed by lin-41 loss of function mutations and lin-41 is down-regulated in seam cells at the same time that let-7 is up-regulated (Reinhart et al., 2000; Slack et al., 2000). let-7 down-regulates lin-41 by binding to several complementary sequences in the lin-41 3’UTR (Vella et al., 2004a; Vella et al., 2004b). Although the mechanism of this repression is poorly understood (Reinhart et al., 2000; Slack et al., 2000), it involves inhibition of translation initiation (Pillai et al., 2005) and subsequent lin-41 mRNA degradation (Bagga et al., 2005).

The let-7 miRNA is highly conserved in several organisms including Drosophila and humans, and is temporally expressed in Drosophila and zebrafish, as well as in C. elegans (Pasquinelli et al., 2000). lin-4 is also conserved in mammals and has known mammalian orthologs, mir-125a and mir-125b (Lagos-Quintana et al., 2002; Lim et al., 2003). miR-125a and miR-125b differ from each other by only four bases, including a central diuridine insertion and a U to C change (Lagos-Quintana et al., 2002), and miR125b contains an extra A at its 3’ end. Not only are the heterochronic miRNAs themselves conserved, but mammalian lin-28 contains let-7 and miR-125a/b complementary sites in its 3’UTR (Moss and Tang, 2003), suggesting that the regulation of target genes by binding of miRNAs to their 3’UTRs is also likely conserved in mammals.

lin-41 is the first C. elegans heterochronic gene found to have mammalian homologs (Slack et al., 2000). The C. elegans LIN-41 protein is a member of the Ring finger B box Coiled Coil (RBCC) family of proteins (Slack et al., 2000). This family of zinc finger proteins (Saurin et al., 1996; Avela et al., 2000) includes proteins associated with human diseases such as the proteins mutated in most cases of promyelocytic leukemia, PML (Borden et al., 1995) and Mulibrey (muscle-liver-brain-eye) nanism disorder, MUL (Avela et al., 2000). All RBCC family members, including lin-41, possess several conserved protein motifs. These include a ring finger and one or two B boxes, which are domains rich in histidine and cysteine residues (Freemont, 1993) that chelate zinc and form protein-nucleic acid and protein-protein interactions (Slack and Ruvkun, 1998). RBCC family members also possess a coiled coil domain with multiple leucine residues and this domain is predicted to bind to other proteins. LIN-41, along with a subset of RBCC family proteins, also contains a C-terminal NHL domain which derives its name from the three proteins first identified with this motif: NCL-1, HT2A, and LIN–41 (Slack and Ruvkun, 1998). The NHL domain contains multiple repeats of a 44 amino acid sequence. The NHL containing protein, Drosophila BRAT, has been shown to bind to Pumilio through its NHL domain to subsequently mediate hunchback translational repression (Sonoda and Wharton, 2001). It is therefore possible that LIN-41 may also control translation of pattern formation genes.

Mammalian developmental timing is poorly understood even though many, if not all tissues are under temporal control during development. Since mammals contain orthologs of several of the C. elegans heterochronic genes, it is possible that a heterochronic pathway also exists in mammals to control developmental timing. To help elucidate the mechanisms of mammalian developmental timing, we cloned the human and mouse lin-41 genes and established that they closely resemble the C. elegans lin-41 gene. We also found that the mammalian lin-41 genes possess predicted complementary sites for both let-7 and lin-4 miRNA family members in their 3’UTRs. Our results show that lin-41 is temporally expressed during embryonic development in several tissues including the limb. Our expression analysis revealed that lin-41 is down-regulated at the same time that let-7c and miR-125a/b expression are up-regulated during mouse development. We speculate that lin-41 may be temporally controlled by the miRNAs let-7c and miR-125a/b, in the mouse to direct a variety of developmental processes, including limb development.

RESULTS

Identification of vertebrate lin-41 Orthologs

In order to identify mammalian orthologs for the C. elegans heterochronic gene, lin-41, we conducted a BLAST search (Altschul et al., 1997) using the C. elegans lin-41 sequence to probe the NCBI database. EST clones for human (Acc# AA169781), mouse (Acc# AW553438) and rat (Acc# BF408236) lin-41 orthologs were obtained from the IMAGE consortium and sequenced. The human EST clone was approximately 3 Kb in length, contains a start codon and a stop codon, and is therefore likely to be full length. Human lin-41 (HLIN-41) corresponded to human locus LOC131405 (Acc # XM_067369) however we noticed various sequence differences between our cDNA sequence and the predicted gene, including a mis-predicted intron and N terminus. Mouse lin-41 (Mlin-41) corresponded to mouse locus LOC382112 (Acc # XM_356199) while rat lin-41 (Rlin-41) corresponded to rat locus LOC301042 (Acc# XM_236676). We also identified lin-41 orthologs from several other species including chicken, dog, chimpanzee, cow, and zebrafish through BLAST or sequencing of cDNAs (Fig. 1 and Suppl. Fig. 1). All of the lin-41 orthologs posses the structural motifs present in members of the RBCC-NHL family, including the Ring finger, B-boxes, Coiled coil and NHL domains (Fig. 1A). We found that the RING fingers of the mammalian LIN-41 orthologs are highly homologous to one another, even though they differ in sequence from the C. elegans RING finger sequence. Comparison of amino acid sequences for vertebrate LIN-41 proteins shows extremely high identity (Suppl. Fig. 1). Amino acid sequence alignment between C. elegans lin-41 and HLIN-41 shows the highest degree of homology in the NHL domain (Fig. 1B). We have previously noted that the majority of known loss of function mutations for the C. elegans lin-41 gene are in its NHL domain (Slack et al., 2000) and these mutated residues are conserved in the human lin-41 gene. The NHL domains of vertebrates are almost identical (Suppl. Fig. 1). We therefore believe that the NHL domain is crucial for the function of the LIN-41 protein.

Fig. 1
LIN-41 structure and sequence

We also determined the degree of homology between the LIN-41 proteins, the most closely related C. elegans homologue, NHL-1, and the most closely related human RBCC-NHL homologues, BERP (El-Husseini and Vincent, 1999) and TRIM2 (Ohkawa et al., 2001; Reymond et al., 2001). The phylogenetic tree for the LIN-41 homologs (Fig. 1C) shows that the human and C. elegans LIN-41 sequences are more similar to one another than to BERP, TRIM2 or NHL-1 sequences. This reveals that HLIN-41 is likely to be the ortholog for C. elegans lin-41. Since the sequence of the lin-41 gene has been conserved across phylogeny, we speculate that the function of this gene in controlling developmental timing is likely to be conserved in mammals.

Mammalian lin-41 Orthologs Contain Predicted let-7 and miR-125a/b Complementary Sites in their 3’UTRs

In C. elegans, it has been shown that the lin-41 gene is regulated by the let-7 miRNA through let-7 complimentary sites (LCS) in its 3’UTR (Vella et al., 2004a; Vella et al., 2004b). The C. elegans lin-41 3’UTR also contains lin-4 complementary sites (Slack et al., 2000) suggesting that multiple miRNAs may control this single gene target. We therefore manually scanned the 3’UTRs of the mammalian lin-41 orthologs and found that they contained let-7 (LCS) and miR-125a/b miRNA complementary sites (MCS) in their 3’UTRs (Figs. 2A, 2B, 2C), with features resembling those of the sites in the C. elegans lin-41 3’UTR. The human lin-41 3’UTR contains 4 predicted LCSs, while the mouse lin-41 3’ UTR, which is longer than the human 3’UTR sequence, contains three additional LCSs with the potential to base pair with let-7 (Fig. 2A, 2C, 2D). While the human lin-41 3’UTR contains one miR-125 site (MCS), the mouse lin-41 3’UTR contains four additional MCS sites (Fig. 2B). The presence of these sites implies that in mammals lin-41 may also be regulated by the let-7 and lin-4 orthologs. An alignment of the human and mouse 3’UTR sequences and the putative let-7 and miR-125a/b binding sites shows that the sites are almost completely conserved (Fig. 2D). This evolutionary conservation indicates that the sites are likely to be functionally relevant, perhaps in the negative regulation of mammalian lin-41 by let-7 and miR-125a/b.

Fig. 2Fig. 2
let-7 complementary sites (LCS) and miR-125a/b complementary sites (MCS) in the 3’UTR of mammalian lin-41

Expression Analysis of lin-41, let-7, and miR-125a/b in the Mouse Embryo

Previous data has shown that the miRNAs let-7 and lin-4 and their homologues are temporally regulated during development in organisms such as C. elegans, Drosophila, and zebrafish (Feinbaum and Ambros, 1999; Pasquinelli et al., 2000; Reinhart et al., 2000; Aravin et al., 2003; Bashirullah et al., 2003). The proposed target of these miRNAs, lin-41, has also been shown to be temporally expressed during C. elegans development (Slack et al., 2000; Bagga et al., 2005). In order to investigate whether these heterochronic genes are likewise temporally expressed in mammals, we analyzed the expression patterns of the lin-41, let-7 and mir-125a/b genes during mouse development by northern blot. Mouse embryos were collected at various stages of development from embryonic day 8 (E8.0) through newborn and total RNA blots were probed for let-7c, mir-125a, mir-125b, and Mlin-41. We found that Mlin-41, let-7c, and mir-125a/b displayed reciprocal expression patterns in the developing mouse embryo (Fig. 3A, 3B). Robust expression of a Mlin-41 band was detected at E8.5 and then expression gradually decreased until E11.0. At later developmental times, Mlin-41 expression was undetectable (Fig. 3A). In a reciprocal fashion, let-7c expression was first detected at about E8.5 and increased to the highest levels around E14.5 and remained elevated through the newborn stage (Fig. 3B). The expression of miR-125a was first detected at E8.0, and peaked around E12.5 and then gradually decreased (Fig. 3B). Interestingly, miR-125b was detected two stages later, at E10.5, peaked at E12–E14.5, and then decreased (Fig. 3B). We noted that the 70-nucleotide precursor (pre-miRNA) for let-7c, miR-125a and miR-125b were readily detected by northern blot (Fig. 3B) and were also temporally expressed during mouse development. Pre-miRNA levels varied for the three miRNAs, and pre-mir-125b was more abundant than the mature mir-125b, suggesting that pre-mir-125b is poorly processed at stages E10.5–E12.5. However, the developmental relevance of this observation is not known. Our data show that the expression of the miRNAs is up-regulated at the time when expression of Mlin-41, a proposed target of these miRNAs, decreases. The reciprocal expression patterns exhibited by the miRNAs are consistent with a model whereby the miRNAs let-7c and miR-125a/b may regulate Mlin-41.

Fig. 3
Northern blots of mouse embryo RNA probed for Mlin-41(A), miR-125a miR-125b and let-7c (B). The β-Actin control and ethidium bromide stained gel are shown as a loading control in (A), U6 is shown as the loading control in (B).

Mlin-41 Expression

To further elucidate the biological function of lin-41 during early mammalian development, whole mount in situ hybridization on wild-type (CD-1) mouse embryos was performed using labeled RNA probes spanning a 1 Kb region of the Mlin-41 gene (pBRM1) and temporal and spatial expression patterns were determined (Fig. 4). lin-41 expression was detected at E9.5 in early forelimb buds. By E10.5, expression was localized to the distal end of the forelimb with more intense staining in the posterior region of the limb bud (Fig. 4C & E). Mlin-41 was also weakly detected in the newly formed hindlimb bud. Expression was observed in the dorsal root ganglia, brachial arches, eyespot, and developing brain at this stage. By E11.0, when the limb buds are more prominent, expression in the forelimb buds appeared to diminish and strong expression was detected in the posterior distal region of the hindlimb bud (data not shown). Based on these dynamic expression patterns in mouse embryos, we propose that Mlin-41 plays a role in limb development as well as in nervous system and craniofacial development. let-7, the predicted regulator of Mlin-41, is expressed in the anterior region of the mouse embryo limbs at day E11.5 (Mansfield et al., 2004), consistent with let-7 being a negative regulator of lin-41 there. In Figure 4F we depict a model of the reciprocal expression pattern of Mlin-41 and let-7c in a forelimb bud based on our in situ expression data and published data (Mansfield et al., 2004). The chicken lin-41 (clin-41) gene has a similar expression pattern in developing limbs (J. Lancman and J. Fallon personal communication, see accompanying paper in this issue).

Fig. 4
lin-41 in situ hybridization of whole mount mouse embryos

DISCUSSION

In C. elegans, heterochronic genes control the timing of organ formation during development. We postulated that mammalian homologs of C. elegans heterochronic genes could be involved in regulating key temporally controlled events during mammalian development. In this report, we have identified several vertebrate orthologs of the C. elegans lin-41 heterochronic gene and found that they all contain protein motifs placing them in the RBCC-NHL family. An alignment of the sequence of the lin-41 gene products shows that they are highly homologous to each other, most notably in the NHL domain. This strong sequence conservation suggests that the function of LIN-41 may be conserved across animal phylogeny.

In addition we have shown through in situ and northern blot analyses that Mlin-41 is temporally expressed in early mouse development. Mlin-41 expression is most pronounced in the developing limb bud and craniofacial and nervous tissues of the mouse embryo, coinciding with temporally regulated developmental events in these regions. Expression in both the fore- and hindlimb buds was observed to be dynamic during stages E9.5–E11.5, with expression initially observed throughout each limb bud, but restricted to the posterior distal regions at later times. This is consistent with data from the Fallon lab showing lin-41 expression in chick and mouse limb buds (J. Lancman and J. Fallon personal communication). The formation of the mammalian limb is thought to be under temporal control (Summerbell et al., 1973), but the regulatory mechanisms governing timing of gene expression here are not well understood. The current model states that different parts of the limb are specified along the proximal-distal axis at the early limb bud stage and then these cells expand as the limb develops (Dudley et al., 2002; Sun et al., 2002; Tickle, 2003). During this expansion, the cells become irreversibly differentiated to their limb fates with proximal tissues differentiating earlier than distal tissues. However, little is known about how positional specification occurs or how it is temporally regulated. We hypothesize that Mlin-41 could regulate specific genes involved in limb formation and outgrowth.

We found that the miRNAs, let-7, mir-125a and mir-125b are also temporally expressed during mouse development. Although all of these miRNAs were first detected at different times (around E8.5–E10.5), they demonstrated peak expression during stages E12.5–E14.5. We note that the timing of increased expression of these miRNAs correlates roughly with the time when organogenesis is well underway. Given that these miRNAs control timing of organ formation in C. elegans, we propose that they may also regulate timing of organ development in mammals as well. This is consistent with a report of let-7 expression in limb buds (Mansfield et al., 2004) around this time.

We have shown that the timing of let-7c and miR-125a/b RNA expression occurs just before Mlin-41 expression begins to decrease. In addition, each of the vertebrate lin-41 cDNAs that we examined possess multiple predicted let-7 and miR-125a/b complementary sites in their 3’UTRs. Due to their reciprocal expression patterns and the presence of miRNA complementary sites in the lin-41 3’UTRs, we propose that miR-125a/b and let-7 are likely to regulate lin-41. We therefore hypothesize that the regulation of lin-41 by the miRNAs let-7 and mir-125a/b/lin-4, may be conserved from C. elegans to vertebrates. Based on the reciprocal northern blot expression data, we believe that miR-125a/b and let-7 negatively regulate lin-41 at the level of gene transcription, mRNA stability or post-transcriptional processing similar to the mechanisms proposed for C. elegans lin-41 (Bagga et al., 2005). Similar to Mlin-41, mammalian lin-28 also contains let-7 and miR-125a/b complementary sites in its 3’UTR (Moss and Tang, 2003), and in mammalian neuronal cell lines lin-28 RNA levels are down-regulated as let-7 and mir-125a/b RNA is up-regulated (Sempere et al., 2004). It has been shown that miRNAs could regulate their targets at the transcriptional level in plants (Bao et al., 2004), and at the RNA degradation level in plants and animals (Llave et al., 2002; Yekta et al., 2004; Jing et al., 2005). The few miRNAs identified in C. elegans and Drosophila, such as let-7 and lin-4, appear to regulate their targets by blocking protein accumulation or translation from their target mRNAs resulting in decreased target mRNA levels (Bagga et al., 2005). The phenomenon of miRNAs regulating target genes through complementary sites in their 3’UTRs is likely to be conserved from C. elegans through mammals and the mechanism by which this regulation occurs may be similar. Notwithstanding, based on its homology and expression pattern, mammalian lin-41 and the miRNAs studied here may be involved in regulating specific genes directing key developmental events, including limb formation.

EXPERIMENTAL PROCEDURES

EST clones and sequencing

We use BLAST to search the NCBI database with the C. elegans lin-41 cDNA as a probe. Human and mouse EST clones with high homology were ordered from Invitrogen and ATCC. These EST cDNAs were sequenced and analyzed using the DNAstar Lasergene Seqman program. Structural domains were determined as compared to the known C. elegans LIN-41 protein using MegAlign. EST clones: human lin-41 EST Acc # AA169781 (pBRM18), mouse lin-41 EST Acc # AW553438 (pBRM12), zebrafish EST Acc # AI584314 (pBRM31).

Sequencing primers for pBRM18/AA169781 included T3 and T7, ORHEST5 (5’-gccgacgagccgccgcccaagaac-3’), OHEST5 (5’-tgctggttgcacagtgtc-3’), OHEST6 (5’-cacagcagctcacactcg-3’), OHEST7 (5’-cgagtgtgagctgctgtg-3’), OHEST8 (5’-gcagcggcgcagcagctc-3’), OHEST16 (5’-gccctttcaaggtggtggtcaac-3’), OHEST18 (5’-cggcagcttcctgtgcaagtttgg-3’), OHEST19 (5’-gtggccaccaccgcgtcgagcagg-3’), OHEST20 (5’-cacacccaaaacacagaaacctagg-3’), OHEST21 (5’-gcccagaaagcgtgccgactggcag-3’), OHEST22 (5’-gtcagccaccacgatcctgcgtg-3’).

Sequencing primers for pBRM12/AW553438 included T7/M13–20, BM121 (5’-caacaaaaacacagacac-3’), BM122 5’-(gccaagaggcttgccagag-3’) BM123 (5’-gccctcctgaacagtccctgatg-3’), BM124 (5’-cgccccctgaccaggccctgtacc-3’). Sequencing primers for pBRM31/AI584314 included OZEST1 (5’ctgcacgccttctgcagg-3’) OZEST2 (5’-ggcgatccatttgcccg-3’) OZEST3 (5’-gatcttcaagccttgtgg-3’) OZEST4 (5’-gacttttgccagcaccatg-3’) OZEST5 (5’cccatcatgatcgtatcc-3’) OZEST6 (5’-gtgagtcggaccctctgg-3’) OZEST7 (5’-gccctccttatccacgcag-3’) OZEST8 (5’-aaggagattttgcacatc-3’)

We deposited the results of our cDNA sequencing in Genbank: Hlin-41 #, Mlin-41 #, Zlin-41 #

Northern Blot Analysis

Mating of CD-1 (wt) mice was performed by housing a male mouse with one or more female mice. The presence of a vaginal plug was designated as E0.5. CD-1 mouse embryos from stage E8.0 to newborn were dissected into cold PBS, flash frozen in liquid nitrogen and stored at −80°C. Total RNA for Mlin-41 analysis was prepared by homogenizing embryos in Trizol, purifying with chloroform, and precipitating the RNA with isopropanol. RNA was then washed with 75% ethanol and dissolved in RNase free water or RNA Storage Solution from Ambion. Approximately 20mg of RNA was loaded in each lane of a 1% agarose TAE gel. The gel was transferred to positively charged nitrocellulose membrane (Brightstar-Plus from Ambion) and then UV crosslinked. The probe was a 1 Kb DNA sequence encoding the C-terminus of Mlin-41, called pBRM1, labeled using random decamers and [α-32P]dATP with Exonuclease free Klenow. The probe was applied to the blot overnight followed by high and low stringency washes. The blot was probed with β-Actin as a loading control as well. The β-Actin probe was a 1.1 Kb fragment of the mouse β-Actin gene accession # X03672, the β-Actin mRNA is 1892 bp in length.

Total RNA for miRNA analysis was extracted from homogenized mouse embryos using the guanidinium thiocyanate single-step method (Current Protocols in Molecular Biology 4.2.1). Approximately 20 µg of total RNA was run on a Criterion urea-10% polyacrylamide gel (Bio-Rad) and electrophoretically transferred to a Zeta-Probe membrane (Bio-Rad) using a Criterion Blotter (Bio-Rad). The blot was then UV-crosslinked (Stratagene UV Stratalinker) and baked at 80°C for 1 hr. Hybridization and washing steps were performed as previously described by Reinhart et al., 2000. Radioactive probes used to detect let-7c (5’-aaccatacaacctactacctca-3’), mir-125a (5’-tcacaagttagggtctcaggga-3’), and mir-125b (5’-cacaggttaaagggtctcaggga-3’) were made using the StarFire Oligonucleotide Labeling System (IDT). Given that our Northern blots showed different patterns for mir-125a and mir-125b, we deduce that our probes were specific for each miRNA, and did not cross-hybridize. We cannot rule out the possibility that let-7c is not detecting other let-7 isoforms. This northern blot was then stripped of all probe and subsequently reprobed with U6 to normalize lanes for loading. Probe pU6 (5’-GCAGGGGCCATGCTAATCTTCTCTGTATTG-3’) was 5’-end labeled with γ-32P ATP by methods described previously (Reinhart et al., 2000).

in situ Hybridization

Antisense and sense RNA probes were generated from the Mlin-41 cDNA sequence in pBRM1. For labeling, 5–10 µg of DNA was digested with XhoI (antisense) or EcoRI (sense) and was purified by phenol:chloroform:IAA extraction and ethanol precipitation. Probes were labeled with digoxigenin using the Ambion MAXIscript™ in vitro transcription kit containing T3 polymerase for antisense probe and T7 polymerase for sense probe. Since the pBRM1 probes were approximately 1 KB in length they were reduced to 200–300 bp fragements for better hybridization (W. Zhong lab protocol).

Embryos from E9.5 and E10.5 were dissected from pregnant females into PBS, fixed in 4% paraformaldehyde, dehydrated in methanol series, bleached in hydrogen peroxide, digested with proteinase K and then fixed in a paraformaldehyde/glutaraldehyde mix. Embryos were hybridized with the pBRM1 probe overnight at 65°C. Embryos were subsequently washed and incubated overnight with antibody for digoxigenin. Color was detected using BM Purple AP Substrate (Roche) and then the embryos were photographed.

Supplementary Material

1

Suppl. Fig. 1. Alignment of C-terminal regions of vertebrate LIN-41 proteins.

ACKNOWLEDGEMENTS

Grant Information: Yale Center for Musculoskeletal Disorders (to F.J.S.); NIH:1RO1GM64701 (to F.J.S.); NIH/NSRA:F32GM071157 (to A.E-K.).

We would like to thank H. Grosshans and K. Olsson Carter for critical reading of this manuscript, members of the Slack laboratory for helpful comments, the Fallon lab for sharing unpublished materials, the Ruddle and Zhong lab for protocols, reagents and equipment, and Petur Petersen for help with mouse techniques and procedures. This work was supported by a grant from the Yale Center for Musculoskeletal Disorders and A. E-K. was supported by NIH/NSRA Grant F32GM071157.

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