Bmp2CreER, Osr1Cre and Gdf5Cre have distinct and partly complementary distributions of recombinase activity.

<p>Bmp2CreER activity after single dose tamoxifen (Tam) administration at 2 different times (E11.5, A-B or E13.5, C-D) compared to Osr1Cre (E-F) and Gdf5Cre (G-H) in E15.5 limbs. In each case, whole mount and adjacent longitudinal sections through dorsoventral axis of forelimb (FL) or hindlimb (HL) are oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Hatch marks in panels of longitudinal sections show the levels at which cross-sections were taken (rightmost panel sets; distal to proximal). Cross-sections are all oriented with dorsal at top and anterior-to-posterior (digit 1–5) from left-to-right. Note that Bmp2CreER labels LacZ-positive descendants mainly in interdigital mesenchyme, including collateral and joint ligaments, whereas Osr1Cre descendent cells reside in subcutaneous tissue and joint ligaments, and Gdf5Cre labels cells lining the joint regions and immediately adjacent subarticular and perichondrial cells at E15.5.</p

Tissue localization of Bmp2CreER activity during the course of limb development.

<p>Upper panels: 24-hour collection (A-J). Tissue localization of Bmp2CreER activity at 24 hours after tamoxifen (Tam) treatment on consecutive days from E10.5–14.5. Panels A,C,E,G,I (forelimb, FL) and B,D,F,H,J (hindlimb, HL) show longitudinal sections through limb dorsoventral axis oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Hatch marks in panels C-J show the levels at which cross-sections below panels C-J were taken (distal to proximal). Cross-sections are all oriented with dorsal at top and anterior-to-posterior (digit 1–5) from left-to-right. Lower panels: E13.5 collection (K-N). Tissue distribution of LacZ activity at E13.5 after tamoxifen (Tam) treatment at E10.5 (K,L) or E11.5 (M,N). For each treatment time, whole mount and adjacent longitudinal and cross-sections of forelimb (FL) or hindlimb (HL) are shown. Hatch marks in panels of longitudinal sections show the levels at which cross-sections through digit region were taken (distal to proximal). Longitudinal and cross-sections are oriented as for Upper Panels. Whole mount images are oriented the same as longitudinal sections.</p

Bmp2CreER activity 24 hours after tamoxifen (Tam) treatment surveyed at daily intervals during the course of limb development.

<p>A. LacZ activity at 48 hours after tamoxifen treatment at E9.5 with arrow pointing to weak activation in distal forelimb (FL inset). Only trace expression is seen in hindlimb (HL inset). (No appreciable LacZ staining was observed at 24 hours after tamoxifen treatment; data not shown). B. LacZ activity assayed at 24 hrs after tamoxifen treatment at times indicated above each panel. All insets to the right show forelimb (FL) and hindlimb (HL) buds oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Note that no LacZ staining is evident in tissues outside of the limb.</p

Comparison of Bmp2CreER activity levels and distribution assayed at E15.5 in LacZ-positive descendants, following tamoxifen (Tam) treatment between stages E11.25 to E11.75.

<p>For each tamoxifen (Tam) treatment time, whole mount and adjacent longitudinal sections through dorsoventral axis of forelimb (FL) or hindlimb (HL) are oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Hatch marks in panels of longitudinal sections show the levels at which cross-sections through digit region were taken (rightmost panel sets; distal to proximal). Cross-sections are all oriented with dorsal at top and anterior-to-posterior (digit 1–5) from left-to-right. Note that Cre activity transitions from digit condensations and interdigital mesenchyme to primarily interdigits and collateral ligaments during this time window of tamoxifen administration.</p

Tissue distribution of LacZ activity at E15.5 following tamoxifen (Tam) treatment at different stages from E10.5—E14.5.

<p>Panels A,C,E,G (forelimb, FL) and B,D,F,H (hindlimb, HL) show longitudinal sections through limb dorsoventral axis oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Hatch marks in panels A-H show the levels at which cross-sections below panels A-H were taken (distal, proximal). Cross-sections are all oriented with dorsal at top and anterior-to-posterior (digit 1–5) from left-to-right. Note that tamoxifen treatment at E13.5 results in LacZ staining restricted to collateral ligament. Lower panel cartoon shows a diagram of an E15.5 digit cross-section with cartilage (red), tendon (blue) and ligament (green) structures annotated for reference.</p

A Near-IR Uncaging Strategy Based on Cyanine Photochemistry

The development of photocaging groups activated by near-IR light would enable new approaches for basic research and allow for spatial and temporal control of drug delivery. Here we report a near-IR light-initiated uncaging reaction sequence based on readily synthesized C4′-dialkylamine-substituted heptamethine cyanines. Phenol-containing small molecules are uncaged through sequential release of the C4′-amine and intramolecular cyclization. The release sequence is initiated by a previously unexploited photochemical reaction of the cyanine fluorophore scaffold. The uncaging process is compatible with biological milieu and is initiated with low intensity 690 nm light. We show that cell viability can be inhibited through light-dependent release of the estrogen receptor antagonist, 4-hydroxycyclofen. In addition, through uncaging of the same compound, gene expression is controlled with near-IR light in a ligand-dependent CreER^T/LoxP-reporter cell line derived from transgenic mice. These studies provide a chemical foundation that we expect will enable specific delivery of small molecules using cytocompatible, tissue penetrant near-IR light

Genotype analysis from timed-pregnant matings of <i>Snap23</i>^Δ/wt heterozygote mice.

<p>Embryo age, number of dissected embryos, and <i>Snap23</i> genotype results are summarized as indicated.</p

Oligonucleotide primers used in this study and estimated size of PCR products.

<p>The oligonucleotide sequences used in this study are listed in the upper table. The oligonucleotide primers were used to genotype the mice, to obtain genomic fragments of <i>Snap23</i>, and to generate probes for Southern blot analysis. The estimated sizes of PCR products obtained during genotyping the mice are indicated in the lower table.</p

Expression of SNAP-23 protein is reduced by half in <i>Snap23</i>^fl/wt and <i>Snap23</i>^Δ/wt mice.

<p>(<b>A</b>) <i>Snap23</i>^fl/wt heterozygous mice were mated and twelve two-week old pups from this mating were genotyped and analyzed for SNAP-23 protein expression. Genotyping of tail DNA was performed using PCR Primer set 2 from tail DNA. A single small PCR fragment (266 bp) is present in <i>Snap23</i>^wt/wt pups, and double fragments (266 and 400 bp) is present in <i>Snap23</i>^fl/wt pups. No homozygous <i>Snap23</i>^fl/fl pups were obtained when more than 50 pups were analyzed from <i>Snap23</i>^fl/wt heterozygous matings. For immunoblot analysis, whole brain was solubilized in modified RIPA lysis buffer and protein levels were analyzed by immunoblotting (WB) as indicated antibodies. (<b>B</b>) <i>Snap23</i>^Δ/wt heterozygous mice were mated and pups from this mating were genotyped and their brains were analyzed for expression of SNAP-23 and other SNARE proteins. Genotyping of tail DNA was performed using PCR Primer set 2. A single small PCR fragment (266 bp) is present in <i>Snap23</i>^wt/wt pups, and double fragments (266 and 492 bp) is present in <i>Snap23</i>^Δ/wt pups. No homozygous <i>Snap23</i>^Δ/Δ pups were ever obtained from <i>Snap23</i>^Δ/wt heterozygous matings. Whole brains were solubilized and analyzed by immunoblotting (WB) using the indicated antibodies.</p

<i>Snap23</i>^Δ/Δ blastocysts die prior to uterine implantation.

<p>(<b>A</b>) To evaluate the timing of embryonic lethality, embryos were collected from super-ovulated <i>Snap23</i>^Δ/wt females mated with <i>Snap23</i>^Δ/wt male mice by uterine flushing at E3.5. About 1/4 of the isolated blastocysts were morphologically abnormal and appeared to be degenerating; unlike sibling normal blastocysts they failed to develop any further during 24 hrs of culture (indicated by red arrows; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018444#pone-0018444-t002" target="_blank">Table 2</a>). (<b>B</b>) Representative example of genotyping analysis revealing that abnormal blastocysts are homozygous for the <i>Snap23</i> deleted allele (<i>Snap23</i>^Δ/Δ). Genomic DNA was isolated from individual blastocysts (shown in panel (A)) following 24 hr in culture, and genotyping was conducted using primers genoE2 SS, genoE2 AS, and genoE3 rev. PCR products for the <i>Snap23</i>^wt allele (266 bp) and for the <i>Snap23</i>^Δ allele (492 bp) are indicated.</p