126 Nucleic Acids Research, 2009, Vol. 37, No. 1 Figure 5. DNM3os, DNM3, HIF-1 and Twist-1 are expressed together during development. The expression of DNM3os, DNM3 (A) and Hif1 and Twist-1 (B) was measured in the whole-cell embryo by RT-PCR at various stages of embryonic development. Western blot showing Twist-1 protein expression at the diﬀerent stages of development is also shown (C). in cells transfected with the (–640:–357) promoter region lacking the E-box (Figure 4B). We next sought to conﬁrm that Twist-1 expression during development coincided with that of DNM3os, miR-214 and miR-199a and we analyzed transcript expression at E10.5, E12.5, E14.5 and E16.5. Figure 5A shows that DNM3os mRNA levels were ﬁrst expressed at E10.5, were maximal at E12.5 and began to decrease at E14.5. The sense DNM3 mRNA transcript was also detected from E12.5 onward (Figure 5A). Western and QT-RTPCR showed that the expression of the Twist-1 transcription factor and mRNA, respectively, was also maximal at E12.5 and decreased at E14.5 (Figure 5B). HIF-1 has been shown to directly regulate Twist-1 expres- sion (18,19) and we also showed that HIF-1 mRNA expression corresponded closely with that of Twist-1 being expressed maximally at E12.5 (Figure 5B). Furthermore, Taqman analyses showed that the expres- sion of miR-199a-5p, miR-199a-3p and miR-214 levels were highest at E12.5 and this corresponded with peak DNM3os and Twist-1 expression (Figure 6A). Finally, we used in situ hybridization to observe the expression of the single-stranded DNM3os, miR-199a-3p, miR- 199a-5p and miR-214. The results showed that DNM3os was strongly expressed in the cerebellum, midbrain, nasal process and fore- and hindlimb buds and that expression of miR-214 mirrored that of DNM3os. However, miR- 199a-3p was most strongly expressed in the hindbrain, while miR-199a-5p was strongly expressed in the limb buds (Figure 6B). DISCUSSION It was observed previously that a noncoding 7.9-kb DNM3os fragment is expressed during development and that its expression may be under the control of Twist-1(6). Here we present promoter deletion studies to show that the bHLH transcription factor Twist-1 drives the expression of the 7.9-kb antisense transcript via an E-Box within the Dynamin-3 gene intron and that the transcript encodes the miR-199a and miR-214 genes. In addition, we show that inhibiting the expression of Twist- 1 resulted in decreased miR-199a and miR-214 levels, indi- cating that under normal physiological conditions Twist-1 can drive the expression of the miR-199a/214 cluster. We further show that mir-199a and mir-214 are developmen- tally regulated, with expression beginning at E11.5, being maximal at E12.5 and falling at E14.5. The expression of the transcription factor HIF1 (known to drive the expres- sion of Twist-1) Twist-1, and miR-199a and miR-214 were coincident during development. Together, these data strongly suggest that Twist-1 drives DNM3os, miR- 199a and miR-214 expression during development. Furthermore, in situ studies on E12.5 embryos showed that the miRNAs were expressed speciﬁcally in mouse cerebellum, midbrain, nasal process and fore and hind limb buds. Previous studies have examined the role of miRNAs in ES cells and embryonic development by knocking out the enzymes (Dicer, Drosha and DGCR8) that are involved in miRNA biogenesis. These studies have revealed a crucial role for miRNAs in ES cell diﬀerentiation, and the control of development (20). MiRNA-speciﬁc studies have shown that miR-214 regulates the speciﬁcation of zebra ﬁsh muscle cell types by targeting a negative regulator of Hedgehog signaling (21) and miR-199a has been shown to regulate COX-2 expression in mouse uterus during implantation (22). We have previously shown that during establishment of the brain coronal suture, Twist-1 knockout results in abnormal cell proliferation and osteoblast diﬀerentiation. Results that suggest