1. Clock Regulatory Elements Control Cyclic Expression of Lunatic fringe during Somitogenesis 
Susan E. Cole, John M. Levorse, Shirley M. Tilghman, Thomas F. Vogt 
Developmental Cell 
Volume 3, Issue 1, Pages (July 2002)
DOI: /S (02)

2. Figure 1
Characterization of Lfng Regulatory Regions in Transgenic Embryos
(A) A map of a genomic fragment including the first two exons (shaded boxes with the number of the exon) of Lfng is shown at the top, with positions of restriction sites used in isolating transgenes (X, XhoI; H, HindIII; A, AvrII; S, StuI; N, NotI). Comparison of expression patterns from constructs 1–5 allowed mapping of regions required for expression of LacZ at specific sites shown as heavy lines below the map. Dashed regions of the lines representing regulatory elements for the dermomyotome and hindbrain/neural tube represent the maximal extent of those regions if the data from construct 5 are excluded. The likely localization of the regulatory elements directing expression in the anterior PSM is shown as a solid line, although we can not formally exclude a role for regions in the first intron (dashed line). Numbers indicate distance from initiator methionine.
(B) A LacZ reporter gene was inserted at the NotI site 16 bp upstream from the translation start site, and constructs containing various amounts of flanking DNA were tested for reporter activity in 9.5–11.5 dpc founder animals. The number of transgenic lines that expressed LacZ in an Lfng-like pattern over the total number of independent transgene insertions is shown. Some sites of expression were only seen in later stage embryos; the numbers in those columns reflect the number of embryos of that age or older. N.V.: specific expression in the rostral compartment is not visible as it is obscured by perdurance of LacZ from the posterior PSM. However, the greater persistence of LacZ in the rostral half of the mature somites in constructs 1–3 and the examination of LacZ RNA in construct 3 supports the localization of these regulatory elements to 1.3 kb of the 5′ flank. Numbers for construct 3 include a single stable line.
Developmental Cell 2002 3, 75-84DOI: ( /S (02) )

3. Figure 2 Expression of β-Galactosidase in Transgenic Embryos
Construct 1 (A–D) is expressed throughout the PSM (B), the dermomyotomes of the mature somites (C), and the hindbrain and ventral neural tube (D). Due to perdurance of the LacZ protein, β-galactosidase staining is also seen in the most recently formed somites, although Lfng RNA is not expressed at these sites. Construct 2 (E–G) is expressed throughout the PSM (F) as well as in the dermomyotome of the mature somites (G). Construct 3 (H–J) is expressed throughout the PSM (I) and in the dermomyotome of the mature somites (J). Construct 4 (K–N) is expressed in the anterior, but not the posterior PSM (L), and β-galactosidase activity is also detected in the rostral regions of the most recently formed somites as well as in the dermomyotome of the mature somites (M) and the hindbrain and ventral neural tube (N). The forelimb bud is designated with an asterisk (C, G, J, and M).
Developmental Cell 2002 3, 75-84DOI: ( /S (02) )

4. Figure 3
Lfng RNA Overlaps the Mesp2 Expression Domain in the Rostral Compartment of the Forming Somite
Double-label RNA in situ hybridization was performed on 11.5 dpc embryos. Lfng RNA is orange and Mesp2 RNA is purple. Three major classes of expression patterns are shown ([A–C]; the arrow points to the most recently formed morphological somite boundary). In all cases, the band of Mesp2 expression is overlapped by a band of Lfng expression (indicated by an asterisk). Immediately anterior to this overlap is a band of cells that express neither gene. In some embryos (C), there is a narrow band of Lfng-expressing cells anterior to the band of Mesp2/Lfng-expressing cells. This probably represents Lfng RNA persisting in the future rostral half of the somite after the downregulation of Mesp2 as the somite boundary begins to form between S0 and S−I. A closer view of this region (D) confirms that Lfng RNA is not expressed in the region immediately rostral to the Mesp2 expression domain (marked by arrowhead). The region that is magnified in (D) is indicated by the white bar in (C). A schematic of the expression pattern is seen in (E). Somites are numbered to the right with dashed lines representing the boundary between the rostral (R) and caudal (C) compartments. Mesp2 RNA is shown in purple and Lfng RNA is shown in orange, with the area of overlap indicated as a hatched box. Cyclic Lfng expression (orange circle) is stabilized in the anterior PSM where it overlaps the domain of Mesp2 expression. At its most refined state, Lfng RNA is only expressed in the rostral compartment. The black arrow represents a somite border forming between S0 and S−I as the anterior-most band of Lfng expression is downregulated.
Developmental Cell 2002 3, 75-84DOI: ( /S (02) )

5. Figure 4
Dynamic LacZ RNA Expression Recapitulates Endogenous Lfng Expression
In situ hybridization analysis of LacZ RNA in embryos transgenic for construct 3 reveals dynamic expression within the PSM (A and B). Double labeling reveals that the expression of LacZ RNA largely overlaps the expression pattern of Lfng (C and D). This overlap is seen regardless of the moiety used to label each probe and the substrate and order of revelation. In (C), Lfng RNA (purple) was developed before the LacZ signal (orange). In panel D, the probes were reversed, with LacZ developed first (purple) and Lfng developed second (orange). In all cases, the expression patterns overlap, indicating cyclic expression of the LacZ RNA.
Developmental Cell 2002 3, 75-84DOI: ( /S (02) )

6. Figure 5
Comparative Sequence and Transgene Analysis of Lfng Reveals an Evolutionarily Conserved Sequence Necessary and Sufficient for Expression in the Posterior PSM
(A) A map of the mouse genomic region containing exon 1 of Lfng is shown at the top. Directly beneath are indicated regulatory regions mapped using transgenic analysis: pPSM, posterior PSM; aPSM, anterior PSM; dm, dermomyotome; hb/nt, hindbrain/ventral neural tube. Dashed lines indicate the maximal extent of those regions. The human genomic sequence surrounding exon 1 of Lfng is depicted horizontally at the left. Regions of high conservation between the two species appear as diagonal lines within the dot plot. The four colored boxes demarcate conserved regions that overlap with the regulatory elements identified by transgene analysis. Some of these regions were also independently identified by Morales et al. (2002), presented in this issue, who identified our conserved region 2 as block A, our conserved region 3 as block B, and the region of lower conservation in the proximal promoter as block C.
(B) A summary of transgene expression patterns for constructs 6–10. The numbers of the transgenic lines that expressed LacZ at each site over the total number of independent transgene insertions are shown. Numbers for construct 7 include two stable lines. NV: not specifically visible due to perdurance of LacZ from the caudal PSM; -: no expression expected.
Developmental Cell 2002 3, 75-84DOI: ( /S (02) )

7. Figure 6 Expression Patterns Observed in Constructs 6–10
Construct 6 contains conserved region 1 flanking the sequences from construct 4, and is expressed in the anterior PSM but not in the posterior PSM (A). Construct 7 contains conserved region 2 flanking the sequences contained in construct 4, and directs expression in the posterior PSM (B). If conserved region 2 sequences are deleted (construct 8), expression is again observed in the anterior, but not in the posterior PSM (C). In construct 9, conserved region 2 is placed in a construct containing the hsp68 promoter. Strong LacZ activity is seen throughout the PSM (D). Construct 7 drives cyclic expression in the PSM based on the expression pattern of LacZ RNA (E and F) and the overlap of LacZ RNA (purple) and Lfng RNA (orange) (G and H). In construct 10, the Hoxc8 enhancer was used in place of FCE1 from construct 7. Some of the signal derives from Hoxc8-driven neural tube expression, but it is clear that a constant level of LacZ RNA is seen in the caudal PSM, with strong expression always visible in the tailbud (n = 4). The domain of LacZ RNA expression persists into the anterior PSM, although expression is weaker in this region (I). When LacZ (purple) and Lfng RNA (orange) are viewed in a single embryo (J), LacZ RNA is observed in the gap between the caudal band of Lfng RNA (the line indicates this band's most anterior boundary) and the anterior band of Lfng RNA (bracket). The overlapping RNA expression patterns are schematized in (K).
Developmental Cell 2002 3, 75-84DOI: ( /S (02) )

8. Figure 7 Sequence and Mutation Analysis of FCE1
(A) Alignment of mouse and human FCE1 (block A of Morales et al., 2002, presented in this issue). Binding sites identified by MatInspector v2.2 are indicated.
(B) Mutation of E boxes alters expression in the caudal PSM. In construct 11, the two 6 bp E boxes have been removed, while in construct 12 the sequences have been altered with point mutations. The number of transgene lines that expressed LacZ at each site over the total number of integration sites identified are shown. Expression is seen in the hindbrain and neural tube (panel A) and anterior PSM (panel B), but expression in the posterior PSM is eliminated (panel B). The asterisk indicates that expression was very weak in this line.
(C) Summary model of the transcriptional regulation of the Lfng elements in the PSM. The genomic structure of the 5′ flank of Lfng is shown above a representation of the PSM and most caudal mature somite with anterior to the right. Downstream of Notch signaling, activation by bHLH binding to E boxes within FCE1 is required for cyclic expression of Lfng in the posterior PSM (shown as a circle with an arrow). Binding of cyclically expressed repressor molecules within FCE1 may also be necessary for dynamic expression driven by this regulatory region, and Hes7 is a candidate for this binding. Sequences in the 1.3 kb region containing the proximal promoter and conserved region 3 have two effects: refinement of cyclic expression (curved, dashed arrow) and activation of Lfng RNA expression in the anterior region II of the condensing somites (shaded boxes). In region I, both Lfng expression and Notch signaling are linked to the segmentation clock (dashed arrows), but the nature of this link is unknown at this time. In region II, Lfng expression and Notch signaling play roles in the establishment and maintenance of rostrocaudal patterning and may respond in part to the Fgf8 wavefront indicated by the blue triangle. An expanded view of somite −I is shown. In the caudal compartment, Notch acts through a presenilin-dependent pathway to activate Dll1 expression, and to repress Mesp2 expression. In the rostral compartment, Mesp2 acts through a Notch-dependent pathway to downregulate Dll1 expression (Takahashi et al., 2000). Lfng expression in this compartment may modulate Notch signaling via glycosylation of Notch and Dll ligands.
Developmental Cell 2002 3, 75-84DOI: ( /S (02) )