Journal: Developmental Biology

Article Title: Visualization of retinoic acid signaling in transgenic axolotls during limb development and regeneration

doi: 10.1016/j.ydbio.2012.05.015

Figure Lengend Snippet: (A–F) EGFP fluorescent images of transgenic axolotls through development were overlaid on top of brightfield images of the same animal to visualize anatomy and reporter activity. Embryonic stages are as indicated and rostral is to the right. (A–D) The pronephric duct (pnd) is expanding posteriorly along the flank of the animals. Somites 3–7 (s3–7) and the anterior neural tube (nt) is starting to fluoresce (C). (E,F) Several tissues showed EGFP fluorescence in the young larvae including developing forelimb buds (fl), gut, neuromast primordia (multiple arrows indicated by nm) that will develop into the posterior lateral line, the anterior (asc) and posterior (psc) region in the spinal cord, eyes (ey), and the postotic ganglionic complex (pgc). (G) IVIS Xenogen fluorescent image with a six month old transgenic axolotl on the left and wild-type animal on the right showing strong fluorescence in the adult RARE:EGFP transgenic animal.

Article Snippet: This fragment was cloned into a Gateway middle entry clone RARE:EGFP_ME with BP enzyme (Invitrogen, Carlsbad, CA, USA) and subsequently inserted along with p5E-Fse-Asc and p3E-polyA ( Kwan et al., 2007 ) into the multisite Gateway destination vector pDEST-iSce (Courtesy of J. Wittbrodt) with LR enzyme (Invitrogen, Carlsbad, CA, USA), which contains two 18 base pair I-SceI meganuclease target sequences that facilitate transgenic animal production.

Techniques: Transgenic Assay, Activity Assay, Fluorescence

Journal: Developmental Biology

Article Title: Visualization of retinoic acid signaling in transgenic axolotls during limb development and regeneration

doi: 10.1016/j.ydbio.2012.05.015

Figure Lengend Snippet: (A) Image of a stage 39 embryo showing the hind portion of the trunk and tail. Rostral is to the right. Notice the RARE:EGFP positive trunk, pronephric duct (pnd), and expanding posterior lateral line primordia (pll). (B–D) Histological sections of a ½ transgenic embryo. Sections move caudally from the hind portion of the head (B), to the forelimb portion of the trunk (C), to the caudal portion of the trunk (D). Images show that RARE:EGFP reports throughout the developing digestive tract including the pharynx (p), gut (g), and lower intestine (i), developing kidney including the pronephros (pn) (C) and pronephric duct (D), developing heart myocardium (hm) (B), forelimb bud mesenchyme (C), and lateral plate mesoderm (lpm) (C, D). (E–L) In situ hybridization showing raldh expression in similar regions to RARE:EGFP activity. (E) Transverse section through the mid trunk of a stage 41 embryo showing raldh2 expression in the pronephric duct. (F) Whole-mount in situ hybridization showing raldh1 expression in the developing heart. Rostral is to the right. (G) Transverse histological section of the embryo showed in F showing expression in the heart primordia (h). (H,I) Transverse histological sections through the developing aorta showing raldh2 expression (a). (J) Transverse histological section showing raldh2 expression in epicardium (ep) of a stage 43 embryo. (K) Image shows the exposed heart of a whole-mount in situ hybridized stage 43 embryo showing expression of raldh2. Rostral is upward. (L) Transverse histological section through the trunk of a stage 43 embryo showing mRNA expression in the lateral plate mesoderm (lpm). The epidermis was removed in this animal.

Article Snippet: This fragment was cloned into a Gateway middle entry clone RARE:EGFP_ME with BP enzyme (Invitrogen, Carlsbad, CA, USA) and subsequently inserted along with p5E-Fse-Asc and p3E-polyA ( Kwan et al., 2007 ) into the multisite Gateway destination vector pDEST-iSce (Courtesy of J. Wittbrodt) with LR enzyme (Invitrogen, Carlsbad, CA, USA), which contains two 18 base pair I-SceI meganuclease target sequences that facilitate transgenic animal production.

Techniques: Transgenic Assay, In Situ Hybridization, Expressing, Activity Assay, In Situ

Journal: Developmental Biology

Article Title: Visualization of retinoic acid signaling in transgenic axolotls during limb development and regeneration

doi: 10.1016/j.ydbio.2012.05.015

Figure Lengend Snippet: (A,B,D,E) Images showing that RARE:EGFP reports in anterior (asc) and posterior (psc) regions of the spinal cord. (A) brachial nerve fibers (bn) can be seen emanating from the RARE:EGFP positive asc and innervating the forelimb. (B) Post-hatching larvae showing that RARE:EGFP expression intensifies in the asc and psc and the peripheral nerve fibers (pn) that innervate the trunk and posterior intestine (i) can be seen. (C) EGFP expression can also be seen in the eye and weak expression in the nasal canal (nc). (D) Transverse section of the psc in a ½ transgenic animal showing strong dorsal EGFP expression in the spinal cord as well as associated dorsal root ganglia (drg). (E) Close-up image of the hind portion of the trunk. Rostral is to the right. Peripheral nerves can be seen innervating the hindgut from the asc. The intestine is also EGFP positive, but the cloaca (cl) is EGFP negative. (F) Image of a transgenic animal’s tail showing descending EGFP positive spinal cord axons in the tail (sc) just below the EGFP positive developing neuromasts (nm and arrows) of the pll. Rostral is to the right. (G) Transverse section through the psc in a stage 43 embryo that was hybridized with a probe against raldh2. Raldh2 was the only raldh mRNA expressed in the developing central nervous system with expression in the roofplate of the spinal cord (rp).

Article Snippet: This fragment was cloned into a Gateway middle entry clone RARE:EGFP_ME with BP enzyme (Invitrogen, Carlsbad, CA, USA) and subsequently inserted along with p5E-Fse-Asc and p3E-polyA ( Kwan et al., 2007 ) into the multisite Gateway destination vector pDEST-iSce (Courtesy of J. Wittbrodt) with LR enzyme (Invitrogen, Carlsbad, CA, USA), which contains two 18 base pair I-SceI meganuclease target sequences that facilitate transgenic animal production.

Techniques: Expressing, Transgenic Assay

Journal: Developmental Biology

Article Title: Visualization of retinoic acid signaling in transgenic axolotls during limb development and regeneration

doi: 10.1016/j.ydbio.2012.05.015

Figure Lengend Snippet: (A-L) RARE:EGFP is strongly expressed in the forelimb mesenchyme and nerves (arrow) during limb development. EGFP expression becomes localized to the proximal region of the limb and decreases in intensity through development. Stars in A and C indicate expression in the proximal epidermis. (B’) raldh2 mRNA expression overlaps with RARE:EGFP expression in the early limb mesenchyme. (M-R) RARE:EGFP is absent in the hindlimb (hl) except for nerve fibers that innervate the hindlimb during development. (M–P) EGFP expression can be seen in the posterior spinal cord (psc) and peripheral nerve fibers (pn) originating from the psc are innervating the developing hindlimb (hl). (Q,R) Extensive peripheral nerve branching is obvious throughout the developing hindlimb, which continues into juvenile animals.

Article Snippet: This fragment was cloned into a Gateway middle entry clone RARE:EGFP_ME with BP enzyme (Invitrogen, Carlsbad, CA, USA) and subsequently inserted along with p5E-Fse-Asc and p3E-polyA ( Kwan et al., 2007 ) into the multisite Gateway destination vector pDEST-iSce (Courtesy of J. Wittbrodt) with LR enzyme (Invitrogen, Carlsbad, CA, USA), which contains two 18 base pair I-SceI meganuclease target sequences that facilitate transgenic animal production.

Techniques: Expressing

Journal: Developmental Biology

Article Title: Visualization of retinoic acid signaling in transgenic axolotls during limb development and regeneration

doi: 10.1016/j.ydbio.2012.05.015

Figure Lengend Snippet: Dorsal is up in images I-L. (A,B) RARE:EGFP is highly concentrated in the dorsal and ventral portion of the eye and perioptic mesenchyme (pm) during development. (C-F) Reporter activity in the eye overlaps with raldh1 and 3 mRNA expression in the dorsal and ventral eye where raldh1 is expressed exclusively in the dorsal portion of the eye (C,D) and raldh3 in both the dorsal and ventral regions (E,F). (A) The nasal canal (nc) also shows RARE:EGFP expression, which overlaps with raldh1, 2, and 3 mRNA expression. (G-J) Transverse-sections of embryos showing raldh expression in the nasal canal. (G,H) raldh3 expression is expressed in the lateral nasal canal and extends throughout the internal nare (in) that connects to the oral cavity. (I) Section through the nasal canal showing similar localization of raldh3 as raldh1. (J) Close-up image of an raldh2 positive nasal canal. Staining was localized primarily in the opening of the nasal canal and not throughout. (A,K,M) RARE:EGFP is expressed in the developing inner ear with strong expression in the otic vesicle (ov) through embryonic stages (A,K) and later in the postotic ganglia complex (pgc) during larval stages (M). This reporter activity in the inner ear corresponds to strong raldh3 expression throughout the medial and lateral regions of the otic vesicle (L). (N) The developing neuromasts (nm) of the posterior lateral line are EGFP positive and also express raldh2 (O,P). Notice the EGFP positive cells that resemble support cells of the developing neuromast.

Article Snippet: This fragment was cloned into a Gateway middle entry clone RARE:EGFP_ME with BP enzyme (Invitrogen, Carlsbad, CA, USA) and subsequently inserted along with p5E-Fse-Asc and p3E-polyA ( Kwan et al., 2007 ) into the multisite Gateway destination vector pDEST-iSce (Courtesy of J. Wittbrodt) with LR enzyme (Invitrogen, Carlsbad, CA, USA), which contains two 18 base pair I-SceI meganuclease target sequences that facilitate transgenic animal production.

Techniques: Activity Assay, Expressing, Staining

Journal: Developmental Biology

Article Title: Visualization of retinoic acid signaling in transgenic axolotls during limb development and regeneration

doi: 10.1016/j.ydbio.2012.05.015

Figure Lengend Snippet: (A) RARE:EGFP reports in the distal tip of the limb during the wound healing and the early limb bud stage of regeneration. The amputation plane is marked with a dashed line. Stars indicate the posterior region of the limb. (B) Top-down view of limb in A showing EGFP expression in the center of the wound epidermis. (C) Immunostaining of sections with a EGFP antibody showed reporter activity in the wound epidermis keratinocytes, peripheral nerve fibers (pn), and cells associated with the peripheral nerves. (D) At the mid-bud stage of regeneration, RARE:EGFP expression has become localized to the posterior portion of the regeneration blastema. Star indicates posterior. (E) At the late-bud and palette stages of regeneration, RARE:EGFP expression starts to decrease compared to earlier stages of regeneration. (F) Immunohistological section through a palette stage regeneration blastema. RARE:EGFP expression is exclusively found in the posterior epidermis indicated by a star. (G) RARE:EGFP expression in the distal tip of the regenerating hindlimb during the early limb bud stage of regeneration. (H) RARE:EGFP expression decreases at the late-bud and palette stages of hindlimb regeneration. (I) Supplemental RA induced stronger RARE:EGFP expression than during normal regeneration (as shown in A) distal to the amputation plane. EGFP expression was also expanded proximal to the amputation plane indicated with a dotted line. (J) Top-down view of two regenerating limbs treated with RA showing the increase in EGFP expression and expanded fluorescence compared to B. (K) Immunohistological section through the middle of an RA-treated limb showing an expansion of EGFP signal compared to normal regeneration shown in C. Strong up-regulation of RARE:EGFP was evident in blastema mesenchymal cells and basal keratinocytes (bk) although EGFP was also observed throughout the epidermal keratinocytes. (L–P) Confocal stacks of double-immunohistological stained sections showed that RARE:EGFP did not colocalize with substantial numbers of HNK-1 positive axons or schwann cells (red) (L) or 12/101 positive muscle fibers (red) (M). (N) Close-up image of a 12/101 positive muscle fiber near the amputation plane of an RA-treated limb showing that the RA-responding EGFP cells (Green) are associated with the muscle, but not the muscle fibers themselves. (O) Image of an RA-treated limb double-immunostained for isolectin B4 (red) and anti-GFP (Green) showing that there are many EGFP- isolectin stained cells throughout the blastema. (P) Close-up image of section in O shows that some RARE:EGFP positive cells were also positive for lectin indicated by an arrow. scale bars, 200μm. bk, basal keratinocytes.

Article Snippet: This fragment was cloned into a Gateway middle entry clone RARE:EGFP_ME with BP enzyme (Invitrogen, Carlsbad, CA, USA) and subsequently inserted along with p5E-Fse-Asc and p3E-polyA ( Kwan et al., 2007 ) into the multisite Gateway destination vector pDEST-iSce (Courtesy of J. Wittbrodt) with LR enzyme (Invitrogen, Carlsbad, CA, USA), which contains two 18 base pair I-SceI meganuclease target sequences that facilitate transgenic animal production.

Techniques: Expressing, Immunostaining, Activity Assay, Fluorescence, Staining